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STAT proteins have the function of signaling from the cell membrane into the nucleus, where they regulate gene transcription. Latent mammalian STAT proteins can form dimers in the cytoplasm even before receptor-mediated activation by specific tyrosine phosphorylation. Here we describe the 3.21-A crystal structure of an unphosphorylated STAT5a homodimer lacking the N-terminal domain as well as the C-terminal transactivation domain. The overall structure of this fragment is very similar to phosphorylated STATs. However, important differences exist in the dimerization mode. Although the interface between phosphorylated STATs is mediated by their Src-homology 2 domains, the unphosphorylated STAT5a fragment dimerizes in a completely different manner via interactions between their beta-barrel and four-helix bundle domains. The STAT4 N-terminal domain dimer can be docked onto this STAT5a core fragment dimer based on shape and charge complementarities. The separation of the dimeric arrangement, taking place upon activation and nuclear translocation of STAT5a, is demonstrated by fluorescence resonance energy transfer experiments in living cells.
Defects in podocyte signaling are the basis of many inherited glomerular diseases leading to glomerulosclerosis. CD2-associated protein (CD2AP) is highly expressed in podocytes and is considered to play an important role in the maintenance of the glomerular slit diaphragm. Mice deficient for CD2AP (CD2AP(-/-)) appear normal at birth but develop a rapid onset nephrotic syndrome at 3 weeks of age. We demonstrate that impaired intracellular signaling with subsequent podocyte damage is the reason for this delayed podocyte injury in CD2AP(-/-) mice. We document that CD2AP deficiency in podocytes leads to diminished signal initiation and termination of signaling pathways mediated by receptor tyrosine kinases (RTKs). In addition, we demonstrate that CIN85, a paralog of CD2AP, is involved in termination of RTK signaling in podocytes. CIN85 protein expression is increased in CD2AP(-/-) podocytes in vitro. Stimulation of CD2AP(-/-) podocytes with various growth factors, including insulin-like growth factor 1, vascular endothelial growth factor, and fibroblast growth factor, resulted in a significantly decreased phosphatidylinositol 3-kinase/AKT and ERK signaling response. Moreover, increased CIN85 protein is detectable in podocytes in diseased CD2AP(-/-) mice, leading to decreased base-line activation of ERK and decreased phosphorylation after growth factor stimulation in vivo. Because repression of CIN85 protein leads to a restored RTK signaling response, our results support an important role of CD2AP/CIN85 protein balance in the normal signaling response of podocytes.
Biological as well as advanced artificial intelligences (AIs) need to decide which goals to pursue. We review nature's solution to the time allocation problem, which is based on a continuously readjusted categorical weighting mechanism we experience introspectively as emotions. One observes phylogenetically that the available number of emotional states increases hand in hand with the cognitive capabilities of animals and that raising levels of intelligence entail ever larger sets of behavioral options. Our ability to experience a multitude of potentially conflicting feelings is in this view not a leftover of a more primitive heritage, but a generic mechanism for attributing values to behavioral options that can not be specified at birth. In this view, emotions are essential for understanding the mind. For concreteness, we propose and discuss a framework which mimics emotions on a functional level. Based on time allocation via emotional stationarity (TAES), emotions are implemented as abstract criteria, such as satisfaction, challenge and boredom, which serve to evaluate activities that have been carried out. The resulting timeline of experienced emotions is compared with the “character” of the agent, which is defined in terms of a preferred distribution of emotional states. The long-term goal of the agent, to align experience with character, is achieved by optimizing the frequency for selecting individual tasks. Upon optimization, the statistics of emotion experience becomes stationary.
Peracarid data were collected in the Southern Ocean and South Atlantic Ocean. Sampling was performed during nine different expeditions on board of RRS James Clark Ross and RV Polarstern, using epibenthic sledges (EBS) at depth ranging between 160–6348 m at 109 locations. The correlation between environmental variables and peracarid abundance was investigated. Abundance data comprise a total of 128570 peracarids (52366 were amphipods, 28516 were cumaceans, 36142 isopods, 5676 mysidaceans and 5870 were tanaidaceans). The presented data are useful to investigate the composition and abundance patterns of peracarid orders at a wide depth range and spatial scale in the Southern Ocean. They can also be reused to compare their abundance with that of other taxa in broader ecological surveys.
Insects with aquatic life stages can transfer sediment and water pollutants to terrestrial ecosystems, which has been described for metals, polyaromatic hydrocarbons, and polychlorinated chemicals. However, knowledge of the transfer of aquatic micropollutants released by wastewater treatment plants is scarce despite some preliminary studies on their occurrence in riparian spiders. In our study, we address a major analytical gap focusing on the transfer of the micropollutant carbamazepine from the larvae to the adult midges of Chironomus riparius using an optimized QuEChERS extraction method and HPLC–MS/MS applicable to both life stages down to the level of about three individuals. We show that the uptake of carbamazepine by larvae is concentration-dependent and reduces the emergence rate. Importantly, the body burden remained constant in adult midges. Using this information, we estimated the daily exposure of insectivorous tree swallows as terrestrial predators to carbamazepine using the energy demand of the predator and the energy content of the prey. Assuming environmentally relevant water concentrations of about 1 μg/L, the daily dose per kilogram of body weight for tree swallows was estimated to be 0.5 μg/kg/day. At places of high water contamination of 10 μg/L, the exposure may reach 5 μg/kg/day for this micropollutant of medium polarity. Considering body burden changes upon metamorphosis, this study fills the missing link between aquatic contamination and exposure in terrestrial habitats showing that wastewater pollutants can impact birds’ life. Clearly, further analytical methods for biota analysis in both habitats are urgently required to improve risk assessment.
Bird-mediated seed dispersal is crucial for the regeneration and viability of ecosystems, often resulting in complex mutualistic species networks. Yet, how this mutualism drives the evolution of seed dispersing birds is still poorly understood. In the present study we combine whole genome re-sequencing analyses and morphometric data to assess the evolutionary processes that shaped the diversification of the Eurasian nutcracker (Nucifraga), a seed disperser known for its mutualism with pines (Pinus). Our results show that the divergence and phylogeographic patterns of nutcrackers resemble those of other non-mutualistic passerine birds and suggest that their early diversification was shaped by similar biogeographic and climatic processes. The limited variation in foraging traits indicates that local adaptation to pines likely played a minor role. Our study shows that close mutualistic relationships between bird and plant species might not necessarily act as a primary driver of evolution and diversification in resource-specialized birds.
The E3 ubiquitin ligase MYCBP2 negatively regulates neuronal growth, synaptogenesis, and synaptic strength. More recently it was shown that MYCBP2 is also involved in receptor and ion channel internalization. We found that mice with a MYCBP2-deficiency in peripheral sensory neurons show prolonged thermal hyperalgesia. Loss of MYCBP2 constitutively activated p38 MAPK and increased expression of several proteins involved in receptor trafficking. Surprisingly, loss of MYCBP2 inhibited internalization of transient receptor potential vanilloid receptor 1 (TRPV1) and prevented desensitization of capsaicin-induced calcium increases. Lack of desensitization, TRPV internalization and prolonged hyperalgesia were reversed by inhibition of p38 MAPK. The effects were TRPV-specific, since neither mustard oil-induced desensitization nor behavioral responses to mechanical stimuli were affected. In summary, we show here for the first time that p38 MAPK activation can inhibit activity-induced ion channel internalization and that MYCBP2 regulates internalization of TRPV1 in peripheral sensory neurons as well as duration of thermal hyperalgesia through p38 MAPK.
To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an alpha-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15N and 2H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is approximately 15 degrees (+/- 7 degrees ). The alignment of both structured domains exhibits a mosaic spread of approximately 10 degrees . A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.
The majority of bacterial membrane-bound NiFe-hydrogenases and formate dehydrogenases have homologous membrane-integral cytochrome b subunits. The prototypic NiFe-hydrogenase of Wolinella succinogenes (HydABC complex) catalyzes H2 oxidation by menaquinone during anaerobic respiration and contains a membrane-integral cytochrome b subunit (HydC) that carries the menaquinone reduction site. Using the crystal structure of the homologous FdnI subunit of Escherichia coli formate dehydrogenase-N as a model, the HydC protein was modified to examine residues thought to be involved in menaquinone binding. Variant HydABC complexes were produced in W. succinogenes, and several conserved HydC residues were identified that are essential for growth with H2 as electron donor and for quinone reduction by H2. Modification of HydC with a C-terminal Strep-tag II enabled one-step purification of the HydABC complex by Strep-Tactin affinity chromatography. The tagged HydC, separated from HydAB by isoelectric focusing, was shown to contain 1.9 mol of heme b/mol of HydC demonstrating that HydC ligates both heme b groups. The four histidine residues predicted as axial heme b ligands were individually replaced by alanine in Strep-tagged HydC. Replacement of either histidine ligand of the heme b group proximal to HydAB led to HydABC preparations that contained only one heme b group. This remaining heme b could be completely reduced by quinone supporting the view that the menaquinone reduction site is located near the distal heme b group. The results indicate that both heme b groups are involved in electron transport and that the architecture of the menaquinone reduction site near the cytoplasmic side of the membrane is similar to that proposed for E. coli FdnI.
We analyzed a eukaryotically encoded rubredoxin from the cryptomonad Guillardia theta and identified additional domains at the N- and C-termini in comparison to known prokaryotic paralogous molecules. The cryptophytic N-terminal extension was shown to be a transit peptide for intracellular targeting of the protein to the plastid, whereas a C-terminal domain represents a membrane anchor. Rubredoxin was identified in all tested phototrophic eukaryotes. Presumably facilitated by its C-terminal extension, nucleomorph-encoded rubredoxin (nmRub) is associated with the thylakoid membrane. Association with photosystem II (PSII) was demonstrated by co-localization of nmRub and PSII membrane particles and PSII core complexes and confirmed by comparative electron paramagnetic resonance measurements. The midpoint potential of nmRub was determined as +125 mV, which is the highest redox potential of all known rubredoxins. Therefore, nmRub provides a striking example of the ability of the protein environment to tune the redox potentials of metal sites, allowing for evolutionary adaption in specific electron transport systems, as for example that coupled to the PSII pathway.
In Archaea, bacteria, and eukarya, ATP provides metabolic energy for energy-dependent processes. It is synthesized by enzymes known as A-type or F-type ATP synthase, which are the smallest rotatory engines in nature (Yoshida, M., Muneyuki, E., and Hisabori, T. (2001) Nat. Rev. Mol. Cell. Biol. 2, 669-677; Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312-2315). Here, we report the first projected structure of an intact A(1)A(0) ATP synthase from Methanococcus jannaschii as determined by electron microscopy and single particle analysis at a resolution of 1.8 nm. The enzyme with an overall length of 25.9 nm is organized in an A(1) headpiece (9.4 x 11.5 nm) and a membrane domain, A(0) (6.4 x 10.6 nm), which are linked by a central stalk with a length of approximately 8 nm. A part of the central stalk is surrounded by a horizontal-situated rodlike structure ("collar"), which interacts with a peripheral stalk extending from the A(0) domain up to the top of the A(1) portion, and a second structure connecting the collar structure with A(1). Superposition of the three-dimensional reconstruction and the solution structure of the A(1) complex from Methanosarcina mazei Gö1 have allowed the projections to be interpreted as the A(1) headpiece, a central and the peripheral stalk, and the integral A(0) domain. Finally, the structural organization of the A(1)A(0) complex is discussed in terms of the structural relationship to the related motors, F(1)F(0) ATP synthase and V(1)V(0) ATPases.
The cell division cycle protein 37 (Cdc37) and the 90-kDa heat shock protein (Hsp90) are molecular chaperones, which are crucial elements in the protein signaling pathway. The largest class of client proteins for Cdc37 and Hsp90 are protein kinases. The catalytic domains of these kinases are stabilized by Cdc37, and their proper folding and functioning is dependent on Hsp90. Here, we present the x-ray crystal structure of the 16-kDa middle domain of human Cdc37 at 1.88 angstroms resolution and the structure of this domain in complex with the 23-kDa N-terminal domain of human Hsp90 based on heteronuclear solution state NMR data and docking. Our results demonstrate that the middle domain of Cdc37 exists as a monomer. NMR and mutagenesis experiments reveal Leu-205 in Cdc37 as a key residue enabling complex formation. These findings can be very useful in the development of small molecule inhibitors against cancer.
Through its role in intron cleavage, tRNA splicing endonuclease (TSEN) plays a critical function in the maturation of intron-containing pre-tRNAs. The catalytic mechanism and core requirement for this process is conserved between archaea and eukaryotes, but for decades, it has been known that eukaryotic TSENs have evolved additional modes of RNA recognition, which have remained poorly understood. Recent research identified new roles for eukaryotic TSEN, including processing or degradation of additional RNA substrates, and determined the first structures of pre-tRNA-bound human TSEN complexes. These recent discoveries have changed our understanding of how the eukaryotic TSEN targets and recognizes substrates. Here, we review these recent discoveries, their implications, and the new questions raised by these findings.
Membrane-bound complex I (NADH:ubiquinone oxidoreductase) of the respiratory chain is considered the main site of mitochondrial radical formation and plays a major role in many mitochondrial pathologies. Structural information is scarce for complex I, and its molecular mechanism is not known. Recently, the 49-kDa subunit has been identified as part of the "catalytic core" conferring ubiquinone reduction by complex I. We found that the position of the 49-kDa subunit is clearly separated from the membrane part of complex I, suggesting an indirect mechanism of proton translocation. This contradicts all hypothetical mechanisms discussed in the field that link proton translocation directly to redox events and suggests an indirect mechanism of proton pumping by redox-driven conformational energy transfer.
We have isolated and characterized the cDNA encoding a Ca(2+)-dependent nucleoside diphosphatase (EC ) related to two secreted ATP- and ADP-hydrolyzing apyrases of the bloodsucking insects, Cimex lectularius and Phlebotomus papatasi. The rat brain-derived cDNA has an open reading frame of 1209 bp encoding a protein of 403 amino acids and a calculated molecular mass of 45.7 kDa. The mRNA was expressed in all tissues investigated, revealing two major transcripts with varying preponderance. The immunohistochemical analysis of the Myc-His-tagged enzyme expressed in Chinese hamster ovary cells revealed its association with the endoplasmic reticulum and also with pre-Golgi intermediates. Ca(2+)-dependent nucleoside diphosphatase is a membrane protein with its catalytic site facing the organelle lumen. It hydrolyzes nucleoside 5'-diphosphates in the order UDP >GDP = IDP >>>CDP but not ADP. Nucleoside 5'-triphosphates were hydrolyzed to a minor extent, and no hydrolysis of nucleoside 5'-monophosphates was observed. The enzyme was strongly activated by Ca(2+), insensitive to Mg(2+), and had a K(m) for UDP of 216 microm. Ca(2+)-dependent nucleoside diphosphatase may support glycosylation reactions related to quality control in the endoplasmic reticulum.
It is generally recognized that large-scale whaling in the 19th and 20th century led to a substantial reduction of the size of many cetacean populations, particularly those of the baleen whales (Mysticeti). The impact of these operations on genomic diversity of one of the most hunted whales, the fin whale (Balaenoptera physalus), has remained largely unaddressed because of the paucity of adequate samples and the limitation of applicable techniques. Here, we have examined the effect of whaling on the North Atlantic fin whale based on genomes of 51 individuals from Icelandic waters, representing three temporally separated intervals, 1989, 2009 and 2018 and provide a reference genome for the species. Demographic models suggest a noticeable drop of the effective population size of the North Atlantic fin whale around a century ago. The present results suggest that the genome-wide heterozygosity is not markedly reduced and has remained comparable with other baleen whale species. Similarly, there are no signs of apparent inbreeding, as measured by the proportion of long runs of homozygosity, or of a distinctively increased mutational load, as measured by the amount of putative deleterious mutations. Compared with other baleen whales, the North Atlantic fin whale appears to be less affected by anthropogenic influences than other whales such as the North Atlantic right whale, consistent with the presence of long runs of homozygosity and higher levels of mutational load in an otherwise more heterozygous genome. Thus, genome-wide assessments of other species and populations are essential for future, more specific, conservation efforts.
Excessive accumulation of the extracellular matrix is a hallmark of many inflammatory and fibrotic diseases, including those of the kidney. This study addresses the question whether NO, in addition to inhibiting the expression of MMP-9, a prominent metalloprotease expressed by mesangial cells, additionally modulates expression of its endogenous inhibitor TIMP-1. We demonstrate that exogenous NO has no modulatory effect on the extracellular TIMP-1 content but strongly amplifies the early increase in cytokine-induced TIMP-1 mRNA and protein levels. We examined whether transforming growth factor beta (TGFbeta), a potent profibrotic cytokine, is involved in the regulation of NO-dependent TIMP-1 expression. Experiments utilizing a pan-specific neutralizing TGFbeta antibody demonstrate that the NO-induced amplification of TIMP-1 is mediated by extracellular TGFbeta. Mechanistically, NO causes a rapid increase in Smad-2 phosphorylation, which is abrogated by the addition of neutralizing TGFbeta antisera. Similarly, the NO-dependent increase in Smad-2 phosphorylation is prevented in the presence of an inhibitor of TGFbeta-RI kinase, indicating that the NO-dependent activation of Smad-2 occurs via the TGFbeta-type I receptor. Furthermore, activation of the Smad signaling cascade by NO is corroborated by the NO-dependent increase in nuclear Smad-4 level and is paralleled by increased DNA binding of Smad-2/3 containing complexes to a TIMP-1-specific Smad-binding element (SBE). Reporter gene assays revealed that NO activates a 0.6-kb TIMP-1 gene promoter fragment as well as a TGFbeta-inducible and SBE-driven control promoter. Chromatin immunoprecipitation analysis also demonstrated DNA binding activity of Smad-3 and Smad-4 proteins to the TIMP-1-specific SBE. Finally, by enzyme-linked immunosorbent assay, we demonstrated that NO causes a rapid increase in TGFbeta(1) levels in cell supernatants. Together, these experiments demonstrate that NO by induction of the Smad signaling pathway modulates TIMP-1 expression.
Ubiquitin (Ub)-mediated regulation of plasmalemmal ion channel activity canonically occurs via stimulation of endocytosis. Whether ubiquitination can modulate channel activity by alternative mechanisms remains unknown. Here, we show that the transient receptor potential vanilloid 4 (TRPV4) cation channel is multiubiquitinated within its cytosolic N-terminal and C-terminal intrinsically disordered regions (IDRs). Mutagenizing select lysine residues to block ubiquitination of the N-terminal but not C-terminal IDR resulted in a marked elevation of TRPV4-mediated intracellular calcium influx, without increasing cell surface expression levels. Conversely, enhancing TRPV4 ubiquitination via expression of an E3 Ub ligase reduced TRPV4 channel activity but did not decrease plasma membrane abundance. These results demonstrate Ub-dependent regulation of TRPV4 channel function independent of effects on plasma membrane localization. Consistent with ubiquitination playing a key negative modulatory role of the channel, gain-of-function neuropathy-causing mutations in the TRPV4 gene led to reduced channel ubiquitination in both cellular and Drosophila models of TRPV4 neuropathy, whereas increasing mutant TRPV4 ubiquitination partially suppressed channel overactivity. Together, these data reveal a novel mechanism via which ubiquitination of an intracellular flexible IDR domain modulates ion channel function independently of endocytic trafficking and identify a contributory role for this pathway in the dysregulation of TRPV4 channel activity by neuropathy-causing mutations.
The signal transducer and activator of transcription (Stat) gene family comprises seven members with similarities in their domain structure and a common mode of activation. Members of this gene family mediate interferon induction of gene transcription and the response to a large number of growth factors and hormones. Extracellular ligand binding to transmembrane receptors causes the intracellular activation of associated tyrosine kinases, phosphorylation of Stat molecules, dimerization, and translocation to the nucleus. Prolactin-induced phosphorylation of Stat5 is a key event in the development and differentiation of mammary epithelial cells. In addition to the crucial phosphorylation at tyrosine 694, we have identified an O-linked N-acetylglucosamine (O-GlcNAc) as another secondary modification essential for the transcriptional induction by Stat5. This modification was only found on nuclear Stat5 after cytokine activation. Similar observations were made with Stat1, Stat3, and Stat6. Glycosylation of Stat5, however, does not seem to be a prerequisite for nuclear translocation. Mass spectrometric analysis revealed a glycosylated peptide in the N-terminal region of Stat5. Replacement of threonine 92 by an alanine residue (Stat5a-T92A) strongly reduced the prolactin induction of Stat5a glycosylation and abolished transactivation of a target gene promoter. Only the glycosylated form of Stat5 was able to bind the coactivator of transcription CBP, an essential interaction for Stat5-mediated gene transcription.
Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin 1beta (IL-1beta). We tested whether ligands of the peroxisome proliferator-activated receptor (PPARalpha) could influence the cytokine-induced expression of MMP-9. Different PPARalpha agonists dose-dependently inhibited the IL-1beta-triggered increase in gelatinolytic activity mainly by decreasing the MMP-9 steady-state mRNA levels. PPARalpha agonists on their own had no effects on MMP-9 mRNA levels and gelatinolytic activity. Surprisingly, the reduction of MMP-9 mRNA levels by PPARalpha activators contrasted with an amplification of cytokine-mediated MMP-9 gene promoter activity and mRNA expression. The potentiation of MMP-9 promoter activity functionally depends on an upstream peroxisome proliferator-responsive element-like binding site, which displayed an increased DNA binding of a PPARalpha immunopositive complex. In contrast, the IL-1beta-induced DNA-binding of nuclear factor kappaB was significantly impaired by PPARalpha agonists. Most interestingly, in the presence of an inducible nitric-oxide synthase (iNOS) inhibitor, the PPARalpha-mediated suppression switched to a strong amplification of IL-1beta-triggered MMP-9 mRNA expression. Concomitantly, activators of PPARalpha potentiated the cytokine-induced iNOS expression. Using actinomycin D, we found that NO, but not PPARalpha activators, strongly reduced the stability of MMP-9 mRNA. In contrast, the stability of MMP-9 protein was not affected by PPARalpha activators. In summary, our data suggest that the inhibitory effects of PPARalpha agonists on cytokine-induced MMP-9 expression are indirect and primarily due to a superinduction of iNOS with high levels of NO reducing the half-life of MMP-9 mRNA.
Type I interferons (IFNs) signal for their diverse biological effects by binding a common receptor on target cells, composed of the two transmembrane IFNAR1 and IFNAR2 proteins. We have previously differentially enhanced the antiproliferative activity of IFN by increasing the weak binding affinity of IFN to IFNAR1. In this study, we further explored the affinity interdependencies between the two receptor subunits and the role of IFNAR1 in differential IFN activity. For this purpose, we generated a panel of mutations targeting the IFNAR2 binding site on the background of the IFNalpha2 YNS mutant, which increases the affinity to IFNAR1 by 60-fold, resulting in IFNAR2-to-IFNAR1 binding affinity ratios ranging from 1000:1 to 1:1000. Both the antiproliferative and antiviral potencies of the interferon mutants clearly correlated to the in situ binding IC(50) values, independently of the relative contributions of the individual receptors, thus relating to the integral lifetime of the complex. However, the antiproliferative potency correlated throughout the entire range of affinities, as well as with prolonged IFNAR1 receptor down-regulation, whereas the antiviral potency reached a maximum at binding affinities equivalent to that of wild-type IFNalpha2. Our data suggest that (i) the specific activity of interferon is related to the ternary complex binding affinity and not to affinity toward individual receptor components and (ii) although the antiviral pathway is strongly dependent on pSTAT1 activity, the cytostatic effect requires additional mechanisms that may involve IFNAR1 down-regulation. This differential interferon response is ultimately mediated through distinct gene expression profiling.
The neuronal adaptor protein Fe65 is involved in brain development, Alzheimer disease amyloid precursor protein (APP) signaling, and proteolytic processing of APP. It contains three protein-protein interaction domains, one WW domain, and a unique tandem array of phosphotyrosine-binding (PTB) domains. The N-terminal PTB domain (Fe65-PTB1) was shown to interact with a variety of proteins, including the low density lipoprotein receptor-related protein (LRP-1), the ApoEr2 receptor, and the histone acetyltransferase Tip60. We have determined the crystal structures of human Fe65-PTB1 in its apo- and in a phosphate-bound form at 2.2 and 2.7A resolution, respectively. The overall fold shows a PTB-typical pleckstrin homology domain superfold. Although Fe65-PTB1 has been classified on an evolutionary basis as a Dab-like PTB domain, it contains attributes of other PTB domain subfamilies. The phosphotyrosine-binding pocket resembles IRS-like PTB domains, and the bound phosphate occupies the binding site of the phosphotyrosine (Tyr(P)) within the canonical NPXpY recognition motif. In addition Fe65-PTB1 contains a loop insertion between helix alpha2 and strand beta2(alpha2/beta2 loop) similar to members of the Shc-like PTB domain subfamily. The structural comparison with the Dab1-PTB domain reveals a putative phospholipid-binding site opposite the peptide binding pocket. We suggest Fe65-PTB1 to interact with its target proteins involved in translocation and signaling of APP in a phosphorylation-dependent manner.
Unlike other eukaryotes, plants possess a complex family of heat stress transcription factors (Hsfs) with usually more than 20 members. Among them, Hsfs A4 and A5 form a group distinguished from other Hsfs by structural features of their oligomerization domains and by a number of conserved signature sequences. We show that A4 Hsfs are potent activators of heat stress gene expression, whereas A5 Hsfs act as specific repressors of HsfA4 activity. The oligomerization domain of HsfA5 alone is necessary and sufficient to exert this effect. Due to the high specificity of the oligomerization domains, other class A Hsfs are not affected. Pull-down assay and yeast two-hybrid interaction tests demonstrate that the tendency to form HsfA4/A5 heterooligomers is stronger than the formation of homooligomers. The specificity of interaction between Hsfs A4 and A5 was confirmed by bimolecular fluorescence complementation experiments. The major role of the representatives of the HsfA4/A5 group, which are not involved in the conventional heat stress response, may reside in cell type-specific functions connected with the control of cell death triggered by pathogen infection and/or reactive oxygen species.
Host cell invasion by the facultative intracellular pathogen Listeria monocytogenes requires the invasion protein InlB in many cell types. InlB consists of an N-terminal internalin domain that binds the host cell receptor tyrosine kinase Met and C-terminal GW domains that bind to glycosaminoglycans (GAGs). Met binding and activation is required for host cell invasion, while the interaction between GW domains and GAGs enhances this effect. Soluble InlB elicits the same cellular phenotypes as the natural Met ligand hepatocyte growth factor/scatter factor (HGF/SF), e.g. cell scatter. So far, little is known about the central part of InlB, the B-repeat. Here we present a structural and functional characterization of the InlB B-repeat. The crystal structure reveals a variation of the β-grasp fold that is most similar to small ubiquitin-like modifiers (SUMOs). However, structural similarity also suggests a potential evolutionary relation to bacterial mucin-binding proteins. The B-repeat defines the prototype structure of a hitherto uncharacterized domain present in over a thousand bacterial proteins. Generally, this domain probably acts as a spacer or a receptor-binding domain in extracellular multi-domain proteins. In cellular assays the B-repeat acts synergistically with the internalin domain conferring to it the ability to stimulate cell motility. Thus, the B-repeat probably binds a further host cell receptor and thereby enhances signaling downstream of Met.
ADAM15 protein amplifies focal adhesion kinase phosphorylation under genotoxic stress conditions
(2012)
ADAM15, a disintegrin and metalloproteinase, is capable of counteracting genotoxic stress-induced apoptosis by the suppression of caspase-3 activation. A cell line expressing the membrane-bound ADAM15 without its cytoplasmic tail, however, lost this anti-apoptotic property, suggesting a crucial role of the intracellular domain as a scaffold for recruitment of survival signal-transducing kinases. Accordingly, an enhanced phosphorylation of FAK at Tyr-397, Tyr-576, and Tyr-861 was detected upon genotoxic stress by camptothecin in ADAM15-transfected T/C28a4 cells, but not in transfectants expressing an ADAM15 mutant without the cytoplasmic tail. Accordingly, a specific binding of the cytoplasmic ADAM15 domain to the C terminus of FAK could be shown by mammalian two-hybrid, pulldown, and far Western studies. In cells expressing full-length ADAM15, a concomitant activation of Src at Tyr-416 was detected upon camptothecin exposure. Cells transfected with a chimeric construct consisting of the extracellular IL-2 receptor α-chain and the cytoplasmic ADAM15 domain were IL-2-stimulated to prove that the ADAM15 tail can transduce a percepted extracellular signal to enhance FAK and Src phosphorylation. Our studies further demonstrate Src binding to FAK but not a direct Src interaction with ADAM15, suggesting FAK as a critical intracellular adaptor for ADAM15-dependent enhancement of FAK/Src activation. Moreover, the apoptosis induction elicited by specific inhibitors (PP2, FAK 14 inhibitor) of FAK/Src signaling was significantly reduced by ADAM15 expression. The newly uncovered counter-regulatory response to genotoxic stress in a chondrocytic survival pathway is potentially also relevant to apoptosis resistance in neoplastic growth.
The Arp2/3 complex nucleates and cross-links actin filaments at the leading edge of motile cells, and its activity is stimulated by C-terminal regions of WASP/Scar proteins, called VCA domains. VCA domains contain a verprolin homology sequence (V) that binds monomeric actin and central (C) and acidic sequences (A) that bind the Arp2/3 complex. Here we show that the C domain binds to monomeric actin with higher affinity (K(d) = 10 microm) than to the Arp2/3 complex (K(d) > 200 microm). Nuclear magnetic resonance spectroscopy reveals that actin binds to the N-terminal half of the C domain and that both the V and C domains can bind actin independently and simultaneously, indicating that they interact with different sites. Mutation of conserved hydrophobic residues in the actin-binding interface of the C domain disrupts activation of the Arp2/3 complex but does not alter affinity for the complex. By chemical cross-linking the C domain interacts with the p40 subunit of the Arp2/3 complex and, by fluorescence polarization anisotropy, the binding of actin and the Arp2/3 complex are mutually exclusive. Our results indicate that both actin and Arp2/3 binding are important for C domain function but that the C domain does not form a static bridge between the two. We propose a model for activation of the Arp2/3 complex in which the C domain first primes the complex by inducing a necessary conformational change and then initiates nucleus assembly by bringing an actin monomer into proximity of the primed complex.
The carnitine transporter CaiT from Escherichia coli belongs to the betaine, choline, and carnitine transporter family of secondary transporters. It acts as an L-carnitine/gamma-butyrobetaine exchanger and is predicted to span the membrane 12 times. Unlike the other members of this transporter family, it does not require an ion gradient and does not respond to osmotic stress (Jung, H., Buchholz, M., Clausen, J., Nietschke, M., Revermann, A., Schmid, R., and Jung, K. (2002) J. Biol. Chem. 277, 39251-39258). The structure and oligomeric state of the protein was examined in detergent and in lipid bilayers. Blue native gel electrophoresis indicated that CaiT was a trimer in detergent solution. This result was further supported by gel filtration and cross-linking studies. Electron microscopy and single particle analysis of the protein showed a triangular structure of three masses or two parallel elongated densities. Reconstitution of CaiT into lipid bilayers yielded two-dimensional crystals that indicated that CaiT was a trimer in the membrane, similar to its homologue BetP. The implications of the trimeric structure on the function of CaiT are discussed.
The subunit composition of the mitochondrial ATP synthase from Saccharomyces cerevisiae was analyzed using blue native gel electrophoresis and high resolution SDS-polyacrylamide gel electrophoresis. We report here the identification of a novel subunit of molecular mass of 6,687 Da, termed subunit j (Su j). An open reading frame of 127 base pairs (ATP18), which encodes for Su j, was identified on chromosome XIII. Su j does not display sequence similarity to ATP synthase subunits from other organisms. Data base searches, however, identified a potential homolog from Schizosaccharomyces pombe with 51% identity to Su j of S. cerevisiae. Su j, a small protein of 59 amino acid residues, has the characteristics of an integral inner membrane protein with a single transmembrane segment. Deletion of the ATP18 gene encoding Su j led to a strain (Deltasu j) completely deficient in oligomycin-sensitive ATPase activity and unable to grow on nonfermentable carbon sources. The presence of Su j is required for the stable expression of subunits 6 and f of the F0 membrane sector. In the absence of Su j, spontaneously arising rho- cells were observed that lacked also ubiquinol-cytochrome c reductase and cytochrome c oxidase activities. We conclude that Su j is a novel and essential subunit of yeast ATP synthase.
Cardiolipin stabilized supercomplexes of Saccharomyces cerevisiae respiratory chain complexes III and IV (ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase, respectively), but was not essential for their formation in the inner mitochondrial membrane because they were found also in a cardiolipin-deficient strain. Reconstitution with cardiolipin largely restored wild-type stability. The putative interface of complexes III and IV comprises transmembrane helices of cytochromes b and c1 and tightly bound cardiolipin. Subunits Rip1p, Qcr6p, Qcr9p, Qcr10p, Cox8p, Cox12p, and Cox13p and cytochrome c were not essential for the assembly of supercomplexes; and in the absence of Qcr6p, the formation of supercomplexes was even promoted. An additional marked effect of cardiolipin concerns cytochrome c oxidase. We show that a cardiolipin-deficient strain harbored almost inactive resting cytochrome c oxidase in the membrane. Transition to the fully active pulsed state occurred on a minute time scale.
SUMO proteins are ubiquitin-related modifiers implicated in the regulation of gene transcription, cell cycle, DNA repair, and protein localization. The molecular mechanisms by which the sumoylation of target proteins regulates diverse cellular functions remain poorly understood. Here we report isolation and characterization of SUMO1- and SUMO2-binding motifs. Using yeast two-hybrid system, bioinformatics, and NMR spectroscopy we define a common SUMO-interacting motif (SIM) and map its binding surfaces on SUMO1 and SUMO2. This motif forms a beta-strand that could bind in parallel or antiparallel orientation to the beta2-strand of SUMO due to the environment of the hydrophobic core. A negative charge imposed by a stretch of neighboring acidic amino acids and/or phosphorylated serine residues determines its specificity in binding to distinct SUMO paralogues and can modulate the spatial orientation of SUMO-SIM interactions.
The binding and activation of the discoidin domain receptor 1 by collagen has led to the conclusion that proteins from the extracellular matrix can directly induce receptor tyrosine kinase-mediated signaling cascades. A region in the extracellular domain of DDR1 homologous to the Dictyostelium discoideum protein discoidin-I is also present in the secreted human protein RS1. Mutations in RS1 cause retinoschisis, a genetic disorder characterized by ablation of the retina. By introducing point mutations into the discoidin domain of DDR1 at positions homologous to the retinoschisis mutations, ligand binding epitopes in the discoidin domain of DDR1 were mapped. Surprisingly, some residues only affected receptor phosphorylation, whereas others influenced both collagen-binding and receptor activation. Furthermore, two truncated DDR1 variants, lacking either the discoidin domain or the stalk region between the discoidin and transmembrane domain, were generated. We showed that (i) the discoidin domain was necessary and sufficient for collagen binding, (ii) only the region between discoidin and transmembrane domain was glycosylated, and (iii) the entire extracellular domain was essential for transmembrane signaling. Using these results, we were able to predict key sites in the collagen-binding epitope of DDR1 and to suggest a potential mechanism of signaling.
An efficient route for delivering specific proteins and peptides into neurons could greatly accelerate the development of therapies for various diseases, especially those involving intracellular defects such as Parkinson disease. Here we report the novel use of polybutylcyanoacrylate nanoparticles for delivery of intact, functional proteins into neurons and neuronal cell lines. Uptake of these particles is primarily dependent on endocytosis via the low density lipoprotein receptor. The nanoparticles are rapidly turned over and display minimal toxicity to cultured neurons. Delivery of three different functional cargo proteins is demonstrated. When primary neuronal cultures are treated with recombinant Escherichia coli beta-galactosidase as nanoparticle cargo, persistent enzyme activity is measured beyond the period of nanoparticle degradation. Delivery of the small GTPase rhoG induces neurite outgrowth and differentiation in PC12 cells. Finally, a monoclonal antibody directed against synuclein is capable of interacting with endogenous alpha-synuclein in cultured neurons following delivery via nanoparticles. Polybutylcyanoacrylate nanoparticles are thus useful for intracellular protein delivery in vitro and have potential as carriers of therapeutic proteins for treatment of neuronal disorders in vivo.
The Q80K polymorphism in the NS3-4A protease of the hepatitis C virus is associated with treatment failure of direct-acting antiviral agents. This polymorphism is highly prevalent in genotype 1a infections and stably transmitted between hosts. Here, we investigated the underlying molecular mechanisms of evolutionarily conserved coevolving amino acids in NS3-Q80K and revealed potential implications of epistatic interactions in immune escape and variants persistence. Using purified protein, we characterized the impact of epistatic amino acid substitutions on the physicochemical properties and peptide cleavage kinetics of the NS3-Q80K protease. We found that Q80K destabilized the protease protein fold (p < 0.0001). Although NS3-Q80K showed reduced peptide substrate turnover (p < 0.0002), replicative fitness in an H77S.3 cell culture model of infection was not significantly inferior to the WT virus. Epistatic substitutions at residues 91 and 174 in NS3-Q80K stabilized the protein fold (p < 0.0001) and leveraged the WT protease stability. However, changes in protease stability inversely correlated with enzymatic activity. In infectious cell culture, these secondary substitutions were not associated with a gain of replicative fitness in NS3-Q80K variants. Using molecular dynamics, we observed that the total number of residue contacts in NS3-Q80K mutants correlated with protein folding stability. Changes in the number of contacts reflected the compensatory effect on protein folding instability by epistatic substitutions. In summary, epistatic substitutions in NS3-Q80K contribute to viral fitness by mechanisms not directly related to RNA replication. By compensating for protein-folding instability, epistatic interactions likely protect NS3-Q80K variants from immune cell recognition.
Siglec-1 (sialoadhesin, CD169) is a surface receptor on human cells that mediates trans-enhancement of HIV-1 infection through recognition of sialic acid moieties in virus membrane gangliosides. Here, we demonstrate that mouse Siglec-1, expressed on the surface of primary macrophages in an interferon-α-responsive manner, captures murine leukemia virus (MLV) particles and mediates their transfer to proliferating lymphocytes. The MLV infection of primary B-cells was markedly more efficient than that of primary T-cells. The major structural protein of MLV particles, Gag, frequently co-localized with Siglec-1, and trans-infection, primarily of surface-bound MLV particles, efficiently occurred. To explore the role of sialic acid for MLV trans-infection at a submolecular level, we analyzed the potential of six sialic acid precursor analogs to modulate the sialylated ganglioside-dependent interaction of MLV particles with Siglec-1. Biosynthetically engineered sialic acids were detected in both the glycolipid and glycoprotein fractions of MLV producer cells. MLV released from cells carrying N-acyl-modified sialic acids displayed strikingly different capacities for Siglec-1-mediated capture and trans-infection; N-butanoyl, N-isobutanoyl, N-glycolyl, or N-pentanoyl side chain modifications resulted in up to 92 and 80% reduction of virus particle capture and trans-infection, respectively, whereas N-propanoyl or N-cyclopropylcarbamyl side chains had no effect. In agreement with these functional analyses, molecular modeling indicated reduced binding affinities for non-functional N-acyl modifications. Thus, Siglec-1 is a key receptor for macrophage/lymphocyte trans-infection of surface-bound virions, and the N-acyl side chain of sialic acid is a critical determinant for the Siglec-1/MLV interaction.
Upon infection of host cells, Legionella pneumophila releases a multitude of effector enzymes into the cells cytoplasm that hijack a plethora of cellular activities, including the hosts ubiquitination pathways. Effectors belonging to the SidE-family are involved in non-canonical serine phosphoribosyl ubiquitination of host substrate proteins contributing to the formation of a Legionella-containing vacuole that is crucial in the onset of Legionnaires’ disease. This dynamic process is reversed by effectors called Dups that hydrolyse the phosphodiester in the phosphoribosyl ubiquitinated protein. We installed reactive warheads on chemically prepared ribosylated ubiquitin to generate a set of probes targeting these Legionella enzymes. In vitro tests on recombinant DupA revealed that a vinyl sulfonate warhead was most efficient in covalent complex formation. Mutagenesis and x-ray crystallography approaches were used to identify the site of covalent crosslinking to be an allosteric cysteine residue. The subsequent application of this probe highlights the potential to selectively enrich the Dup enzymes from Legionella-infected cell lysates.
EF-P and its paralog EfpL (YeiP) differentially control translation of proline containing sequences
(2024)
Polyproline sequences (XPPX) stall ribosomes, thus being deleterious for all living organisms. In bacteria, translation elongation factor P (EF-P) plays a crucial role in overcoming such arrests. 12% of eubacteria possess an EF-P paralog – YeiP (EfpL) of unknown function. Here, we functionally and structurally characterize EfpL from Escherichia coli and demonstrate its yet unrecognized role in the translational stress response. Through ribosome profiling, we analyzed the EfpL arrest motif spectrum and discovered additional stalls beyond the canonical XPPX motifs at single-proline sequences (XPX), that both EF-P and EfpL can resolve. Notably, the two factors can also induce pauses. We further report that, contrary to the housekeeping EF-P, EfpL can sense the metabolic state of the cell, via lysine acylation. Together, our work uncovers a new player in ribosome rescue at proline-containing sequences, and provides evidence that co-occurrence of EF-P and EfpL is an evolutionary driver for higher bacterial growth rates.
Coarse-grained modeling has become an important tool to supplement experimental measurements, allowing access to spatio-temporal scales beyond all-atom based approaches. The GōMartini model combines structure- and physics-based coarse-grained approaches, balancing computational efficiency and accurate representation of protein dynamics with the capabilities of studying proteins in different biological environments. This paper introduces an enhanced GōMartini model, which combines a virtual-site implementation of Gō models with Martini 3. The implementation has been extensively tested by the community since the release of the new version of Martini. This work demonstrates the capabilities of the model in diverse case studies, ranging from protein-membrane binding to protein-ligand interactions and AFM force profile calculations. The model is also versatile, as it can address recent inaccuracies reported in the Martini protein model. Lastly, the paper discusses the advantages, limitations, and future perspectives of the Martini 3 protein model and its combination with Gō models.
The renin-angiotensin-aldosterone system plays a pivotal role in the regulation of salt and water homeostasis. Here, we demonstrate the expression and functional role of cGMP-dependent protein kinases (PKGs) in rat adrenal cortex. Expression of PKG II is restricted to adrenal zona glomerulosa (ZG) cells, whereas PKG I is localized to the adrenal capsule and blood vessels. Activation of the aldosterone system by a low sodium diet up-regulated the expression of PKG II, however, it did not change PKG I expression in adrenal cortex. Both, activation of PKG II in isolated ZG cell and adenoviral gene transfer of wild type PKG II into ZG cells enhanced aldosterone production. In contrast, inhibition of PKG II as well as infection with a PKG II catalytically inactive mutant had an inhibitory effect on aldosterone production. Steroidogenic acute regulatory (StAR) protein that regulates the rate-limiting step in steroidogenesis is a new substrate for PKG II and can be phosphorylated by PKG II in vitro at serine 55/56 and serine 99. Stimulation of aldosterone production by PKG II in contrast to stimulation by PKA did not activate StAR gene expression in ZG cells. The results presented indicate that PKG II activity in ZG cells is important for maintaining basal aldosterone production.
Atrial natriuretic peptide (ANP) plays a key regulatory role in arterial blood pressure homeostasis. We recently generated mice with selective deletion of the ANP receptor, guanylyl cyclase-A (GC-A), in vascular smooth muscle (SMC GC-A knockout (KO) mice) and reported that resting arterial blood pressure was completely normal in spite of clear abolition of the direct vasodilating effects of ANP (Holtwick, R., Gotthardt, M., Skryabin, B., Steinmetz, M., Potthast, R., Zetsche, B., Hammer, R. E., Herz, J., and Kuhn M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7142–7147). The purpose of this study was to clarify mechanisms compensating for the missing vasodilator responses to ANP. In particular, we analyzed the effect of the endothelial, cGMP-mediated vasodilators C-type natriuretic peptide and nitric oxide (NO). In isolated arteries from SMC GC-A KO mice, the vasorelaxing sensitivity to sodium nitroprusside and the endothelium-dependent vasodilator, acetylcholine, was significantly greater than in control mice. There was no difference in responses to C-type natriuretic peptide or to the activator of cGMP-dependent protein kinase I, 8-para-chlorophenylthio-cGMP. The aortic expression of soluble GC (sGC), but not of endothelial NO synthase or cGMP-dependent protein kinase I, was significantly increased in SMC GC-A KO mice. Chronic oral treatment with the NO synthase inhibitor Nw-nitro-l-arginine methyl ester increased arterial blood pressure, the effect being significantly enhanced in SMC GC-A KO mice. We conclude that SMC GC-A KO mice exhibit a higher vasodilating sensitivity to NO. This can be attributed to an enhanced expression of sGC, whereas the expression and/or activity levels of downstream cGMP-effector pathways are not involved. Increased vasodilating responsiveness to endothelial NO contributes to compensate for the missing vasodilating effect of ANP in SMC GC-A KO mice.
Phosphodiesterase type 2A (PDE2A) hydrolyzes cyclic nucleotides cAMP and cGMP, thus efficiently controlling cNMP-dependent signaling pathways. PDE2A is composed of an amino-terminal region, two regulatory GAF domains, and a catalytic domain. Cyclic nucleotide hydrolysis is known to be activated by cGMP binding to GAF-B; however, other mechanisms may operate to fine-tune local cyclic nucleotide levels. In a yeast two-hybrid screening we identified XAP2, a crucial component of the aryl hydrocarbon receptor (AhR) complex, as a major PDE2A-interacting protein. We mapped the XAP2 binding site to the GAF-B domain of PDE2A. PDE assays with purified proteins showed that XAP2 binding does not change the enzymatic activity of PDE2A. To analyze whether PDE2A could affect the function of XAP2, we studied nuclear translocation of AhR, i.e. the master transcription factor controlling the expression of multiple detoxification genes. Notably, regulation of AhR target gene expression is initiated by tetrachlorodibenzodioxin (TCDD) binding to AhR and by a poorly understood cAMP-dependent pathway followed by the translocation of AhR from the cytosol into the nucleus. Binding of PDE2A to XAP2 inhibited TCDD- and cAMP-induced nuclear translocation of AhR in Hepa1c1c7 hepatocytes. Furthermore, PDE2A attenuated TCDD-induced transcription in reporter gene assays. We conclude that XAP2 targets PDE2A to the AhR complex, thereby restricting AhR mobility, possibly by a local reduction of cAMP levels. Our results provide first insights into the elusive cAMP-dependent regulation of AhR.
Cyclic GMP-dependent protein kinases protein kinase G (PKG) Iα and PKGIβ are major mediators of cGMP signaling in the cardiovascular system. PKGIα is present in the heart, although its role in protection against ischemia/reperfusion injury is not known. We investigated the direct effect of PKGIα against necrosis and apoptosis following simulated ischemia (SI) and reoxygenation (RO) in cardiomyocytes. Adult rat cardiomyocytes were infected with adenoviral vectors containing hPKGIα or catalytically inactive mutant hPKGIαK390A. After 24 h, the cells were subjected to 90 min of SI and 2 h RO for necrosis (trypan blue exclusion and lactate dehydrogenase release) or 18 h RO for apoptosis studies. To evaluate the role of KATP channels, subgroups of cells were treated with 5-hydroxydecanoate (100 μm), HMR1098 (30 μm), or glibenclamide (50 μm), the respective blockers of mitochondrial, sarcolemmal, or both types of KATP channels prior to SI. The necrosis observed in 33.7 ± 1.6% of total myocytes in the SI-RO control group was reduced to 18.6 ± 0.8% by PKGIα (mean ± S.E., n = 7, p < 0.001). The apoptosis observed in 17.9 ± 1.3% of total myocytes in the SI-RO control group was reduced to 6.0 ± 0.6% by PKGIα (mean ± S.E., n = 7, p < 0.001). In addition, PKGIα inhibited the activation of caspase-3 after SI-RO in myocytes. Myocytes infected with the inactive PKGIαK390A mutant showed no protection. PKGIα enhanced phosphorylation of Akt, ERK1/2, and JNK, increased Bcl-2, inducible nitric-oxide synthase, endothelial nitric-oxide synthase, and decreased Bax expression. 5-Hydroxydecanoate and glibenclamide abolished PKGIα-mediated protection against necrosis and apoptosis. However, HMR1098, had no effect. A scavenger of reactive oxygen species, as well as inhibitors of phosphatidylinositol 3-kinase, ERK, JNK1, and NOS, also blocked PKGIα-mediated protection against necrosis and apoptosis. These results show that opening of mitochondrial KATP channels and generation of reactive oxygen species, in association with phosphorylation of Akt, ERK, and JNK, and increased expression of NOS and Bcl-2, play an essential role in the protective effect of PKGIα.
The social amoeba Dictyostelium discoideum is a widely used model organism for studying basic functions of protozoan and metazoan cells, such as osmoregulation and cell motility. There is evidence from other species that cellular water channels, aquaporins (AQP), are central to both processes. Yet, data on D. discoideum AQPs is almost absent. Despite cloning of two putative D. discoideum AQPs, WacA, and AqpA, water permeability has not been shown. Further, WacA and AqpA are expressed at the late multicellular stage and in spores but not in amoebae. We cloned a novel AQP, AqpB, from amoeboidal D. discoideum cells. Wild-type AqpB was impermeable to water, glycerol, and urea when expressed in Xenopus laevis oocytes. Neither stepwise truncation of the N terminus nor selected point mutations activated the water channel. However, mutational truncation by 12 amino acids of an extraordinary long intracellular loop induced water permeability of AqpB, hinting at a novel gating mechanism. This AqpB mutant was inhibited by mercuric chloride, confirming the presence of a cysteine residue in the selectivity filter as predicted by our structure model. We detected AqpB by Western blot analysis in a glycosylated and a non-glycosylated form throughout all developmental stages. When expressed in D. discoideum amoebae, AqpB-GFP fusion constructs localized to vacuolar structures, to the plasma membrane, and to lamellipodia-like membrane protrusions. We conclude that the localization pattern in conjunction with channel gating may be indicative of AqpB functions in osmoregulation as well as cell motility of D. discoideum.
The 2[4Fe-4S] ferredoxin from Chromatium vinosum arises as one prominent member of a recently defined family of proteins found in very diverse bacteria. The potentiometric circular dichroism titrations of the protein and of several molecular variants generated by site-directed mutagenesis have established that the reduction potentials of the two clusters differ widely by almost 200 mV. This large difference has been confirmed by electrochemical methods, and each redox transition has been assigned to one of the clusters. The unusually low potential center is surprisingly the one that displays a conventional CX1X2CX3X4C (Xn, variable amino acid) binding motif and a structural environment similar to that of clusters having less negative potentials. A comparison with other ferredoxins has highlighted factors contributing to the reduction potential of [4Fe-4S] clusters in proteins. (i) The loop between the coordinating cysteines 40 and 49 and the C terminus alpha-helix of C. vinosum ferredoxin cause a negative, but relatively moderate, shift of approximately 60 mV for the nearby cluster. (ii) Very negative potentials, below -600 mV, correlate with the presence of a bulky side chain in position X4 of the coordinating triad of cysteines. These findings set the framework in which previous observations on ferredoxins can be better understood. They also shed light onto the possible occurrence and properties of very low potential [4Fe-4S] clusters in less well characterized proteins.
Atovaquone is a substituted 2-hydroxynaphthoquinone that is used therapeutically to treat Plasmodium falciparum malaria, Pneumocystis carinii pneumonia, and Toxoplasma gondii toxoplasmosis. It is thought to act on these organisms by inhibiting the cytochrome bc1 complex. We have examined the interaction of atovaquone with the bc1 complex isolated from Saccharomyces cerevisiae, a surrogate, nonpathogenic fungus. Atovaquone inhibits the bc1 complex competitively with apparent Ki = 9 nm, raises the midpoint potential of the Rieske iron-sulfur protein from 285 to 385 mV, and shifts the g values in the EPR spectrum of the Rieske center. These results indicate that atovaquone binds to the ubiquinol oxidation pocket of the bc1 complex, where it interacts with the Rieske iron-sulfur protein. A computed energy-minimized structure for atovaquone liganded to the yeast bc1 complex suggests that a phenylalanine at position 275 of cytochrome b in the bovine bc1 complex, as opposed to leucine at the equivalent position in the yeast enzyme, is responsible for the decreased sensitivity of the bovine bc1 complex (Ki = 80 nm) to atovaquone. When a L275F mutation was introduced into the yeast cytochrome b, the sensitivity of the yeast enzyme to atovaquone decreased (Ki = 100 nm) with no loss in activity, confirming that the L275F exchange contributes to the differential sensitivity of these two species to atovaquone. These results provide the first molecular description of how atovaquone binds to the bc1 complex and explain the differential inhibition of the fungal versus mammalian enzymes.
The crystal structure of the bovine Rieske iron-sulfur protein indicates a sulfur atom (S-1) of the iron-sulfur cluster and the sulfur atom (Sgamma) of a cysteine residue that coordinates one of the iron atoms form hydrogen bonds with the hydroxyl groups of Ser-163 and Tyr-165, respectively. We have altered the equivalent Ser-183 and Tyr-185 in the Saccharomyces cerevisiae Rieske iron-sulfur protein by site-directed mutagenesis of the iron-sulfur protein gene to examine how these hydrogen bonds affect the midpoint potential of the iron-sulfur cluster and how changes in the midpoint potential affect the activity of the enzyme. Eliminating the hydrogen bond from the hydroxyl group of Ser-183 to S-1 of the cluster lowers the midpoint potential of the cluster by 130 mV, and eliminating the hydrogen bond from the hydroxyl group of Tyr-185 to Sgamma of Cys-159 lowers the midpoint potential by 65 mV. Eliminating both hydrogen bonds has an approximately additive effect, lowering the midpoint potential by 180 mV. Thus, these hydrogen bonds contribute significantly to the positive midpoint potential of the cluster but are not essential for its assembly. The activity of the bc1 complex decreases with the decrease in midpoint potential, confirming that oxidation of ubiquinol by the iron-sulfur protein is the rate-limiting partial reaction in the bc1 complex, and that the rate of this reaction is extensively influenced by the midpoint potential of the iron-sulfur cluster.
Mitochondria and chloroplasts are of endosymbiotic origin. Their integration into cells entailed the development of protein translocons, partially by recycling bacterial proteins. We demonstrate the evolutionary conservation of the translocon component Tic22 between cyanobacteria and chloroplasts. Tic22 in Anabaena sp. PCC 7120 is essential. The protein is localized in the thylakoids and in the periplasm and can be functionally replaced by a plant orthologue. Tic22 physically interacts with the outer envelope biogenesis factor Omp85 in vitro and in vivo, the latter exemplified by immunoprecipitation after chemical cross-linking. The physical interaction together with the phenotype of a tic22 mutant comparable with the one of the omp85 mutant indicates a concerted function of both proteins. The three-dimensional structure allows the definition of conserved hydrophobic pockets comparable with those of ClpS or BamB. The results presented suggest a function of Tic22 in outer membrane biogenesis.
Background: Although Tic22 is involved in protein import into chloroplasts, the function in cyanobacteria is unknown.
Results: Cyanobacterial Tic22 is required for OM biogenesis, shares structural features with chaperones, and can be substituted by plant Tic22.
Conclusion: Tic22, involved in outer membrane biogenesis, is functionally conserved in cyanobacteria and plants.
Significance: The findings are important for the understanding of periplasmic protein transport.
Proteins of the Omp85 family are conserved in all kingdoms of life. They mediate protein transport across or protein insertion into membranes and reside in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Omp85 proteins contain a C-terminal transmembrane β-barrel and a soluble N terminus with a varying number of polypeptide-transport-associated or POTRA domains. Here we investigate Omp85 from the cyanobacterium Anabaena sp. PCC 7120. The crystallographic three-dimensional structure of the N-terminal region shows three POTRA domains, here named P1 to P3 from the N terminus. Molecular dynamics simulations revealed a hinge between P1 and P2 but in contrast show that P2 and P3 are fixed in orientation. The P2-P3 arrangement is identical as seen for the POTRA domains from proteobacterial FhaC, suggesting this orientation is a conserved feature. Furthermore, we define interfaces for protein-protein interaction in P1 and P2. P3 possesses an extended loop unique to cyanobacteria and plantae, which influences pore properties as shown by deletion. It now becomes clear how variations in structure of individual POTRA domains, as well as the different number of POTRA domains with both rigid and flexible connections make the N termini of Omp85 proteins versatile adaptors for a plentitude of functions.
Precursor protein translocation across the outer chloroplast membrane depends on the action of the Toc complex, containing GTPases as recognizing receptor components. The G domains of the GTPases are known to dimerize. In the dimeric conformation an arginine contacts the phosphate moieties of bound nucleotide in trans. Kinetic studies suggested that the arginine in itself does not act as an arginine finger of a reciprocal GTPase-activating protein (GAP). Here we investigate the specific function of the residue in two GTPase homologues. Arginine to alanine replacement variants have significantly reduced affinities for dimerization compared with wild-type GTPases. The amino acid exchange does not impact on the overall fold and nucleotide binding, as seen in the monomeric x-ray crystallographic structure of the Arabidopsis Toc33 arginine-alanine replacement variant at 2.0A. We probed the catalytic center with the transition state analogue GDP/AlF(x) using NMR and analytical ultracentrifugation. AlF(x) binding depends on the arginine, suggesting the residue can play a role in catalysis despite the non-GAP nature of the homodimer. Two non-exclusive functional models are discussed: 1) the coGAP hypothesis, in which an additional factor activates the GTPase in homodimeric form; and 2) the switch hypothesis, in which a protein, presumably the large Toc159 GTPase, exchanges with one of the homodimeric subunits, leading to activation.
In high light, the antenna system in oxygenic photosynthetic organisms switches to a photoprotective mode, dissipating excess energy in a process called non-photochemical quenching (NPQ). Diatoms exhibit very efficient NPQ, accompanied by a xanthophyll cycle in which diadinoxanthin is de-epoxidized into diatoxanthin. Diatoms accumulate pigments from this cycle in high light, and exhibit faster and more pronounced NPQ. The mechanisms underlying NPQ in diatoms remain unclear, but it can be mimicked by aggregation of their isolated light-harvesting complexes, FCP (fucoxanthin chlorophyll-a/c protein). We assess this model system by resonance Raman measurements of two peripheral FCPs, trimeric FCPa and nonameric FCPb, isolated from high- and low-light-adapted cells (LL, HL). Quenching is associated with a reorganisation of these proteins, affecting the conformation of their bound carotenoids, and in a manner which is highly dependent on the protein considered. FCPa from LL diatoms exhibits significant changes in diadinoxanthin structure, together with a smaller conformational change of at least one fucoxanthin. For these LL-FCPa, quenching is associated with consecutive events, displaying distinct spectral signatures, and its amplitude correlates with the planarity of the diadinoxanthin structure. HL-FCPa aggregation is associated with a change in planarity of a 515-nm-absorbing fucoxanthin, and, to a lesser extent, of diadinoxanthin. Finally, in FCPb, a blue-absorbing fucoxanthin is primarily affected. FCPs thus possess a plastic structure, undergoing several conformational changes upon aggregation, dependent upon their precise composition and structure. NPQ in diatoms may therefore arise from a combination of structural changes, dependent on the environment the cells are adapted to.
Microbial rhodopsins are omnipresent on Earth, however the vast majority of them remain uncharacterized. Here we describe a new rhodopsin clade from cold-adapted organisms and cold environments, such as glaciers, denoted as CryoRhodopsins (CryoRs). Our data suggest that CryoRs have photosensory activity. A distinguishing feature of the clade is the presence of a buried arginine residue close to the cytoplasmic face of its members. Combining single-particle cryo-electron microscopy and X-ray crystallography with the rhodopsin activation by light, we demonstrate that the arginine stabilizes a strongly blue-shifted intermediate of an extremely slow CryoRhodopsin photocycle. Together with extensive spectroscopic characterization, our investigations on CryoR1 and CryoR2 proteins reveal mechanisms of photoswitching in the newly identified clade and demonstrate principles of the adaptation of these rhodopsins to low temperatures.
Microbial rhodopsins are omnipresent on Earth, however the vast majority of them remain uncharacterized. Here we describe a new rhodopsin group from cold-adapted organisms and cold environments, such as glaciers, denoted as CryoRhodopsins (CryoRs). Our data suggest that CryoRs have dual functionality switching between inward transmembrane proton translocation and photosensory activity, both of which can be modulated with UV light. CryoR1 exhibits two subpopulations in the ground state, which upon light activation lead to transient photocurrents of opposing polarities. A distinguishing feature of the group is the presence of a buried arginine residue close to the cytoplasmic face of its members. Combining single-particle cryo-electron microscopy and X-ray crystallography with the rhodopsin activation by lit, we demonstrate that the arginine stabilizes a UV-absorbing intermediate of an extremely slow CryoRhodopsin photocycle. Together with extensive spectroscopic characterization, our investigations on CryoR1 and CryoR2 proteins reveal mechanisms of photoswitching in the newly identified group and demonstrate principles of the adaptation of these rhodopsins to low temperatures.Microbial rhodopsins are omnipresent on Earth, however the vast majority of them remain uncharacterized. Here we describe a new rhodopsin group from cold-adapted organisms and cold environments, such as glaciers, denoted as CryoRhodopsins (CryoRs). Our data suggest that CryoRs have dual functionality switching between inward transmembrane proton translocation and photosensory activity, both of which can be modulated with UV light. CryoR1 exhibits two subpopulations in the ground state, which upon light activation lead to transient photocurrents of opposing polarities. A distinguishing feature of the group is the presence of a buried arginine residue close to the cytoplasmic face of its members. Combining single-particle cryo-electron microscopy and X-ray crystallography with the rhodopsin activation by light, we demonstrate that the arginine stabilizes a UV-absorbing intermediate of an extremely slow CryoRhodopsin photocycle. Together with extensive spectroscopic characterization, our investigations on CryoR1 and CryoR2 proteins reveal mechanisms of photoswitching in the newly identified group and demonstrate principles of the adaptation of these rhodopsins to low temperatures.
Methods using environmental DNA to explore and analyze biodiversity from previously unexplored habitats and ecosystems have become increasingly popular in recent years. This is particularly due to the potential reduction in necessary taxonomic expertise, the opportunity to assess microorganismal communities, and decreased time investments required to cover large spatial extents. In forests, the surface of tree bark is an important habitat for epiphytic diversity. Because of the large surface area rich in micro-niches, the seasonal stability of the substrate, and the longevity of trees, tree bark surfaces provide an ideal habitat for many species. Yet, we lack a comprehensive understanding of their communities and the environmental drivers behind the community assembly. These missing links hinder the exploration of the forest microbiome as a whole and limits our understanding of functions of a large forest habitat and its connections to other forest microbiomes. With a holistic eDNA metabarcoding approach, encompassing samples of three major taxonomic groups (e.g. bacteria, fungi, and green algae), as well as simultaneous collections from multiple forest habitats we can contribute to closing these gaps and increase our knowledge of the forest microbiome.
My dissertation is set within the framework of the Biodiversity Exploratories and was conducted in four parts: I. the establishment of an eDNA metabarcoding workflow to reveal the local diversity of the bark surface microbiome; II. the upscaling of the method to large geographic and environmental gradients to uncover the drivers of the microbiome; III. the integration of soil and bark samples to investigate compositional differences in two important forest habitats; IV. the evaluation of eDNA metabarcoding as a tool for biodiversity assessments of lichen diversity in forests.
In the first part, I developed a simple, cost-effective and fast sampling strategy to acquire eDNA samples from the bark of trees in forest ecosystems. Using readily available medical-specimen-collection swabs I sampled bark surfaces of individual trees in Central German forests and used metabarcoding to amplify marker genes of green algae, fungi and bacteria. From the sequencing reads I calculated the first diversity estimates of the major organismal groups of bark surface microbiomes from Central European forests. Overall the methodology produced reliable results, allowing for an expanded sampling in the second part.
In the second part of the dissertation, I expanded the sampling based on the results of part one. I collected bark surface samples from the three regions of the Biodiversity Exploratories covering large spatial and environmental gradients representative for Central European forests. The collection included composite samples from 150 plots and over 750 trees. Utilizing measurements of climatic and forest structure variables provided by the Biodiversity Exploratories, as well as my own community data, I identified the biotic and abiotic drivers behind alpha and beta diversity of the bark surface microbiome.
In the third part, I studied the differences between the bark surface as an unexplored and the soil as an example of a well characterized forest microbiome. Using only the fungal part of the large sampling campaign and soil samples obtained from the same plots at the same time, I assessed the commonalities and differences of the micro-communities of these distinct forest niches. Furthermore, I included two coniferous and one deciduous tree species to examine, if the effect of tree species, previously shown for soil microbiomes, also holds true for the bark surface.
In the last part of my dissertation, I used eDNA in a more applied way as a tool in biodiversity assessments of lichenized fungi. I compared the results from eDNA metabarcoding to an expert floristic mapping conducted in the same plots in 2007/2008. I assigned functional guilds to the fungal taxa obtained in the large sampling campaign and used a subset that was assigned as lichenized fungi.
In conclusion, I showed that eDNA metabarcoding is a valuable tool to reveal the unknown diversity of microorganisms in forest ecosystems. In particular, my results advance our understanding of the bark surface microbiome, an underexplored habitat within forests. The tightly linked interactions of the three major microbial groups underline that studies need to take holistic approaches across multiple taxonomic groups to deepen our understanding of processes governing the assembly of microbiomes. Results from my dissertation may serve as a foundation to inform hypotheses addressing the functions of forest microbiomes. The massive diversity data collected may also contribute to closing the gap in our understanding of macro-organisms and micro-organisms with respect to diversity distributions and patterns of richness, and serve as a baseline for predictions of biodiversity responses under future anthropogenic change.
Biotechnological processes offer better production conditions for a wide variety of goods of industrial interest. The production of aromatic compounds, for example, involves molecules of great value for cosmetic, plastic, agrochemical and pharmaceutic industries. However, the yield of such processes frequently prevents a proper implementtation that would allow the replacement of traditional production processes.
Numerous rational engineering approaches have been attempted to enhance metabolic pathways associated with desired products. Unfortunately, genetic modifications and heterologous pathway expression often lead to a higher metabolic burden on the producing organisms, ultimately leading to reduced production levels and fitness.
This project utilised adaptive laboratory evolution to better understand the development of synthetic cooperative consortia, using S. cerevisiae as a model organism. Specifically, a synthetic cooperative consortium was developed around the exchange of lysine and tyrosine, which was subjected to adaptive laboratory evolution aiming to induce mutations that would improve the system’s fitness either by enhanced production or upgraded stress resistance. Consequently, the mutant strains isolated after the evolution rounds were sequenced to identify relevant variations that could be related to the growth and production phenotypes observed.
The insights derived from this project are expected to contribute to further developing synthetic cooperative consortia with utilitarian purposes.
IHMCIF: an extension of the PDBx/mmCIF data standard for integrative structure determination methods
(2024)
IHMCIF (github.com/ihmwg/IHMCIF) is a data information framework that supports archiving and disseminating macromolecular structures determined by integrative or hybrid modeling (IHM), and making them Findable, Accessible, Interoperable, and Reusable (FAIR). IHMCIF is an extension of the Protein Data Bank Exchange/macromolecular Crystallographic Information Framework (PDBx/mmCIF) that serves as the framework for the Protein Data Bank (PDB) to archive experimentally determined atomic structures of biological macromolecules and their complexes with one another and small molecule ligands (e.g., enzyme cofactors and drugs). IHMCIF serves as the foundational data standard for the PDB-Dev prototype system, developed for archiving and disseminating integrative structures. It utilizes a flexible data representation to describe integrative structures that span multiple spatiotemporal scales and structural states with definitions for restraints from a variety of experimental methods contributing to integrative structural biology. The IHMCIF extension was created with the benefit of considerable community input and recommendations gathered by the Worldwide Protein Data Bank (wwPDB) Task Force for Integrative or Hybrid Methods (wwpdb.org/task/hybrid). Herein, we describe the development of IHMCIF to support evolving methodologies and ongoing advancements in integrative structural biology. Ultimately, IHMCIF will facilitate the unification of PDB-Dev data and tools with the PDB archive so that integrative structures can be archived and disseminated through PDB.
The spike protein of SARS-CoV-2 is a highly flexible membrane receptor that triggers the translocation of the virus into cells by attaching to the human receptors. Like other type I membrane receptors, this protein has several extracellular domains connected by flexible hinges. The presence of these hinges results in high flexibility, which consequently results in challenges in defining the conformation of the protein. Here, We developed a new method to define the conformational space based on a few variables inspired by the robotic field’s methods to determine a robotic arm’s forward kinematics. Using newly performed atomistic molecular dynamics (MD) simulations and publicly available data, we found that the Denavit-Hartenberg (DH) parameters can reliably show the changes in the local conformation. Furthermore, the rotational and translational components of the homogenous transformation matrix constructed based on the DH parameters can identify the changes in the global conformation of the spike and also differentiate between the conformation with a similar position of the spike head, which other types of parameters, such as spherical coordinates, fail to distinguish between such conformations. Finally, the new method will be beneficial for looking at the conformational heterogeneity in all other type I membrane receptors.
Highlights
• Sampling the large conformational space of disordered proteins requires extensive molecular dynamics (MD) simulations.
• Fragment assembly complements MD simulations to produce extensive ensembles of disordered proteins with atomic detail.
• Hierarchical chain growth (HCG) ensembles capture key experimental descriptors “out of the box”.
• HCG has revealed local structural characteristics associated with protein dysfunction in neurodegeneration.
Abstract
Disordered proteins and nucleic acids play key roles in cellular function and disease. Here, we review recent advances in the computational exploration of the conformational dynamics of flexible biomolecules. While atomistic molecular dynamics (MD) simulation has seen a lot of improvement in recent years, large-scale computing resources and careful validation are required to simulate full-length disordered biopolymers in solution. As a computationally efficient alternative, hierarchical chain growth (HCG) combines pre-sampled chain fragments in a statistically reproducible manner into ensembles of full-length atomically detailed biomolecular structures. Experimental data can be integrated during and after chain assembly. Applications to the neurodegeneration-linked proteins α-synuclein, tau, and TDP-43, including as condensate, illustrate the use of HCG. We conclude by highlighting the emerging connections to AI-based structural modeling including AlphaFold2.
From hunting and foraging to clearing land for agriculture, humans modify forest biodiversity, landscapes, and climate. Forests constantly undergo disturbance–recovery dynamics and understanding them is a major objective of ecologists and conservationists. Chronosequences are a useful tool for understanding global restoration efforts. They represent a space-for-time substitution approach suited for the quantification of the resistance of ecosystem properties to withstand disturbance and the resilience of these properties until reaching pre-disturbance levels. Here we introduce a newly established chronosequence with 62 plots (50 ⍰ 50 m) in active cacao plantations and pastures, early and late regeneration, and mature old-growth forests, across a 200 km2 area in the extremely wet Chocó rainforest. Our chronosequence covers by far the largest total area of plots compared to others in the Neotropics. Plots ranged from 159–615 masl in a forested landscape with 74 ± 2.8 % forest cover within a 1-km radius including substantial old-growth forest cover. Land-use legacy and regeneration time were not confounded by elevation. We tested how six forest structure variables (maximum tree height and DBH, basal area, number of stems, vertical vegetation heterogeneity, and light availability), aboveground biomass (AGB), and rarefied tree species richness change along our chronosequence. Forest structure variables, AGB, and tree species richness increased with regeneration time and are predicted to reach similar levels to those in old-growth forests after ca. 30–116, 202, and 108 yrs, respectively. Compared to previous work in the Neotropics, old-growth forests in Canandé accumulate high AGB that takes one of the largest time spans reported until total recovery. Our chronosequence comprises one of the largest tree species pools, covers the largest total area of regenerating and old-growth forests, and has higher forest cover than other Neotropical chronosequences. Hence, our chronosequence can be used to determine the time for recovery and stability (resistance and resilience) of different taxa and ecosystem functions, including species interaction networks. This integrative effort will ultimately help to understand how one of the most diverse forests on the planet recovers from large-scale disturbances.
Pin1 is a peptidyl-prolyl cis/trans isomerase (PPIase) essential for cell cycle regulation. Pin1-catalyzed peptidyl-prolyl isomerization provides a key conformational switch to activate phosphorylation sites with the common phospho-Ser/Thr-Pro sequence motif. This motif is ubiquitously exploited in cellular response to a variety of signals. Pin1 is able to bind phospho-Ser/Thr-Pro-containing sequences at two different sites that compete for the same substrate. One binding site is located within the N-terminal WW domain, which is essential for protein targeting and localization. The other binding site is located in the C-terminal catalytic domain, which is structural homologous to the FK506-binding protein (FKBP) class of PPIases. A flexible linker of 12 residues connects the WW and catalytic domain. To characterize the structure and dynamics of full-length Pin1 in solution, high resolution NMR methods have been used to map the nature of interactions between the two domains of Pin1. In addition, the influence of target peptides on domain interactions has been investigated. The studies reveal a dynamic picture of the domain interactions. 15N spin relaxation data, differential chemical shift mapping, and residual dipolar coupling data indicate that Pin1 can either behave as two independent domains connected by the flexible linker or as a single intact domain with some amount of hinge bending motion depending on the sequence of the bound peptide. The functional importance of the modulation of relative domain flexibility in light of the multitude of interaction partners of Pin1 is discussed.
Inhibition of the soluble epoxide hydrolase (sEH) has beneficial effects on vascular inflammation and hypertension indicating that the enzyme may be a promising target for drug development. As the enzymatic core of the hydrolase domain of the human sEH contains two tyrosine residues (Tyr383 and Tyr466) that are theoretically crucial for enzymatic activity, we addressed the hypothesis that the activity of the sEH may be affected by nitrosative stress. Epoxide hydrolase activity was detected in human and murine endothelial cells as well in HEK293 cells and could be inhibited by either authentic peroxynitrite (ONOO−) or the ONOO− generator 3-morpholino-sydnonimine (SIN-1). Protection of the enzymatic core with 1-adamantyl-3-cyclohexylurea in vitro decreased sensitivity to SIN-1. Both ONOO− and SIN-1 elicited the tyrosine nitration of the sEH protein and mass spectrometry analysis of tryptic fragments revealed nitration on several tyrosine residues including Tyr383 and Tyr466. Mutation of the latter residues to phenylalanine was sufficient to abrogate epoxide hydrolase activity. In vivo, streptozotocin-induced diabetes resulted in the tyrosine nitration of the sEH in murine lungs and a significant decrease in its activity. Taken together, these data indicate that the activity of the sEH can be regulated by the tyrosine nitration of the protein. Moreover, nitrosative stress would be expected to potentiate the physiological actions of arachidonic acid epoxides by preventing their metabolism to the corresponding diols.
The lipid content of skin plays a determinant role in its barrier function with a particularly important role attributed to linoleic acid and its derivatives. Here we explored the consequences of interfering with the soluble epoxide hydrolase (sEH) on skin homeostasis. sEH; which converts fatty acid epoxides generated by cytochrome P450 enzymes to their corresponding diols, was largely restricted to the epidermis which was enriched in sEH-generated diols. Global deletion of the sEH increased levels of epoxides, including the linoleic acid-derived epoxide; 12,13-epoxyoctadecenoic acid (12,13-EpOME), and increased basal keratinocyte proliferation. sEH deletion (sEH-/- mice) resulted in thicker differentiated spinous and corneocyte layers compared to wild-type mice, a hyperkeratosis phenotype that was reproduced in wild-type mice treated with a sEH inhibitor. sEH deletion made the skin sensitive to inflammation and sEH-/- mice developed thicker imiquimod-induced psoriasis plaques than the control group and were more prone to inflammation triggered by mechanical stress with pronounced infiltration and activation of neutrophils as well as vascular leak and increased 12,13-EpOME and leukotriene (LT) B4 levels. Topical treatment of LTB4 antagonist after stripping successfully inhibited inflammation and neutrophil infiltration both in wild type and sEH-/- skin. While 12,13-EpoME had no effect on the trans-endothelial migration of neutrophils, like LTB4, it effectively induced neutrophil adhesion and activation. These observations indicate that while the increased accumulation of neutrophils in sEH-deficient skin could be attributed to the increase in LTB4 levels, both 12,13-EpOME and LTB4 contribute to neutrophil activation. Our observations identify a protective role of the sEH in the skin and should be taken into account when designing future clinical trials with sEH inhibitors.
Highlights
• TAM polarization induces CP RNA.
• CP RNA expression is regulated by HIF-2 and STAT1.
• CP RNA is transferred from TAMs to HT1080 cells.
• CP RNA is translated by HT1080 cells and protects from ferroptosis.
• Co-cultured HT1080 cells decrease iron and lipid peroxidation.
Abstract
Solid tumors are characterized by hypoxic areas, which are prone for macrophage infiltration. Once infiltrated, macrophages polarize to tumor associated macrophages (TAM) to support tumor progression. Therefore, the crosstalk between TAMs and tumor cells is of current interest for the development of novel therapeutic strategies. These may comprise induction of an iron- and lipid peroxidation-dependent form of cell death, known as ferroptosis. To study the macrophage - tumor cell crosstalk we polarized primary human macrophages towards a TAM-like phenotype, co-cultured them with HT1080 fibrosarcoma cells, and analyzed the tumor cell response to ferroptosis induction. In TAMs the expression of ceruloplasmin mRNA increased, which was driven by hypoxia inducible factor 2 and signal transducer and activator of transcription 1. Subsequently, ceruloplasmin mRNA was transferred from TAMs to HT1080 cells via extracellular vesicles. In tumor cells, mRNA was translated into protein to protect HT1080 cells from RSL3-induced ferroptosis. Mechanistically this was based on reduced iron abundance and lipid peroxidation. Interestingly, in naïve macrophages also hypoxia induced ceruloplasmin under hypoxia and a co-culture of HT1080 cells with hypoxic macrophages recapitulated the protective effect observed in TAM co-cultures. In conclusion, TAMs provoke tumor cells to release iron and thereby protect them from lipid peroxidation/ferroptosis.
Nicotinamide adenine dinucleotide (NAD) serves as a cap-like structure on cellular RNAs (NAD-RNAs) in all domains of life including the bacterium Escherichia coli. NAD also acts as a key molecule in phage-host interactions, where bacterial immune systems deplete NAD to abort phage infection. Nevertheless, NAD-RNAs have not yet been identified during phage infections of bacteria and the mechanisms of their synthesis and degradation are unknown in this context. The T4 phage that specifically infects E. coli presents an important model to study phage infections, but a systematic analysis of the presence and dynamics of NAD-RNAs during T4 phage infection is lacking. Here, we investigate the presence of NAD-RNAs during T4 phage infection in a dual manner. By applying time-resolved NAD captureSeq, we identify NAD-capped host and phage transcripts and their dynamic regulation during phage infection. We provide evidence that NAD-RNAs are – as reported earlier – generated by the host RNA polymerase by initiating transcription with NAD at canonical transcription start sites. In addition, we characterize NudE.1 – a T4 phage-encoded Nudix hydrolase – as the first phage-encoded NAD-RNA decapping enzyme. T4 phages carrying inactive NudE.1 display a delayed lysis phenotype. This study investigates for the first time the dual epitranscriptome of a phage and its host, thereby introducing epitranscriptomics as an important field of phage research.
Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain, and the conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins with a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a literal central role in ISGylation, underscoring it as an important drug target for boosting innate antiviral immunity. Depending on the type of conjugated protein and the ultimate target protein, E2 enzymes have been shown to function as monomers, dimers, or both. UbcH8 has been crystalized in both monomeric and dimeric forms, but the functional state is unclear. Here, we used a combined approach of small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy to characterize UbcH8′s oligomeric state in solution. SAXS revealed a dimeric UbcH8 structure that could be dissociated when fused with an N-terminal glutathione S-transferase molecule. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a backside dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at ISG15 and E3 interfaces - providing hypotheses for the protein′s functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques in providing structural information about proteins in solution.
Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain, and the conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins with a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a literal central role in ISGylation, underscoring it as an important drug target for boosting innate antiviral immunity. Depending on the type of conjugated protein and the ultimate target protein, E2 enzymes have been shown to function as monomers, dimers, or both. UbcH8 has been crystalized in both monomeric and dimeric forms, but the functional state is unclear. Here, we used a combined approach of small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy to characterize UbcH8′s oligomeric state in solution. SAXS revealed a dimeric UbcH8 structure that could be dissociated when fused with an N-terminal glutathione S-transferase molecule. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a backside dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at ISG15 and E3 interfaces - providing hypotheses for the protein′s functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques in providing structural information about proteins in solution.
Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain, and the conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins with a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a literal central role in ISGylation, underscoring it as an important drug target for boosting innate antiviral immunity. Depending on the type of conjugated protein and the ultimate target protein, E2 enzymes have been shown to function as monomers, dimers, or both. UbcH8 has been crystalized in both monomeric and dimeric forms, but the functional state is unclear. Here, we used a combined approach of small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy to characterize UbcH8’s oligomeric state in solution. SAXS revealed a dimeric UbcH8 structure that could be dissociated when fused with an N-terminal glutathione S-transferase molecule. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a backside dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at ISG15 and E3 interfaces - providing hypotheses for the protein’s functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques in providing structural information about proteins in solution.
Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain, and the conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins with a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a literal central role in ISGylation, underscoring it as an important drug target for boosting innate antiviral immunity. Depending on the type of conjugated protein and the ultimate target protein, E2 enzymes have been shown to function as monomers, dimers, or both. UbcH8 has been crystalized in both monomeric and dimeric forms, but the functional state is unclear. Here, we used a combined approach of small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy to characterize UbcH8’s oligomeric state in solution. SAXS revealed a dimeric UbcH8 structure that could be dissociated when fused with an N-terminal glutathione S-transferase molecule. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a backside dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at ISG15 and E3 interfaces - providing hypotheses for the protein’s functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques in providing structural information about proteins in solution.
Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain, and the conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins with a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a literal central role in ISGylation, underscoring it as an important drug target for boosting innate antiviral immunity. Depending on the type of conjugated protein and the ultimate target protein, E2 enzymes have been shown to function as monomers, dimers, or both. UbcH8 has been crystalized in both monomeric and dimeric forms, but the functional state is unclear. Here, we used a combined approach of small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy to characterize UbcH8’s oligomeric state in solution. SAXS revealed a dimeric UbcH8 structure that could be dissociated when fused with an N-terminal glutathione S-transferase molecule. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a backside dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at ISG15 and E3 interfaces - providing hypotheses for the protein’s functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques in providing structural information about proteins in solution.
Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain, and the conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins with a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a literal central role in ISGylation, underscoring it as an important drug target for boosting innate antiviral immunity. Depending on the type of conjugated protein and the ultimate target protein, E2 enzymes have been shown to function as monomers, dimers, or both. UbcH8 has been crystalized in both monomeric and dimeric forms, but the functional state is unclear. Here, we used a combined approach of small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy to characterize UbcH8’s oligomeric state in solution. SAXS revealed a dimeric UbcH8 structure that could be dissociated when fused with an N-terminal glutathione S-transferase molecule. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a backside dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at ISG15 and E3 interfaces - providing hypotheses for the protein’s functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques in providing structural information about proteins in solution.
Targeted protein degradation (TPD) has recently emerged as an exciting new drug modality. However, the strategy of developing small molecule-based protein degraders has evolved over the past two decades and has now established molecular tags that are already in clinical use, as well as chimeric molecules, PROteolysis TArgeting Chimeras (PROTACs), based mainly on ligand systems developed for the two E3 ligases CRBN and VHL. The large size of the human E3 ligase family suggests that PROTACs can be developed by targeting a large diversity of E3 ligases, some of which have restricted expression patterns with the potential to design disease- or tissue-specific degraders. Indeed, many new E3 ligands have been published recently, confirming the druggability of E3 ligases. This review summarises recent data on E3 ligases and highlights the challenges in developing these molecules into efficient PROTACs rivalling the established degrader systems.
MicroRNAs (miRNAs) are critical post-transcriptional regulators in many biological processes. They act by guiding RNA-induced silencing complexes to miRNA response elements (MREs) in target mRNAs, inducing translational inhibition and/or mRNA degradation. Functional MREs are expected to predominantly occur in the 3’ untranslated region and involve perfect base-pairing of the miRNA seed. Here, we generate a high-resolution map of miR-181a/b-1 (miR-181) MREs to define the targeting rules of miR-181 in developing murine T-cells. By combining a multi-omics approach with computational high-resolution analyses, we uncover novel miR-181 targets and demonstrate that miR-181 acts predominantly through RNA destabilization. Importantly, we discover an alternative seed match and identify a distinct set of targets with repeat elements in the coding sequence which are targeted by miR-181 and mediate translational inhibition. In conclusion, deep profiling of MREs in primary cells is critical to expand physiologically relevant targetomes and establish context-dependent miRNA targeting rules.
Key Points:
* Deep profiling identifies novel targets of miR-181 associated with global gene regulation.
* miR-181 MREs in repeat elements in the coding sequence act through translational inhibition.
* High-resolution analysis reveals an alternative seed match in functional MREs.
Meliolales (black mildews) is an order of plant parasitic ascomycetous fungi in the tropics and subtropics. They are frequently overgrown and parasitized by other fungi, known as hyperparasites. During the last few years, species of hyperparasitic fungi on Meliolales have been collected in Benin and Panama. A new species of Paranectria and seven new reports of hyperparasites of different systematic groups are presented here with detailed descriptions and illustrations, together with new data concerning fungal hosts and host plants. The new species is called Paranectria longiappendiculata, characterized by exceptionally long appendages carried by the ascospores. New records for Benin and Panama are Calloriopsis herpotricha, Dimerosporiella cephalosporii, Isthmospora glabra, Isthmospora trichophila, Malacaria meliolicola, Paranectriella hemileiae, and Paranectriella minuta. Calloriopsis herpotricha is recorded for Africa and D. cephalosporii and P. hemileiae for America for the first time, suggesting an apparently pantropical distribution. Findings show a blatant lack of investigation on hyperparasitic fungi in the tropics. The phylogenetic positions of three of these newly reported species, C. herpotricha, D. cephalosporii, and P. minuta, are shown based on the analysis of internal transcribed spacer (ITS), large subunit (LSU), and small subunit (SSU) rDNA sequences. These sequences were generated in the context of the present study for the first time.
Background and purpose: In patients with epilepsies of structural origin, brain atrophy and pathological alterations of the tissue microstructure extending beyond the putative epileptogenic lesion have been reported. However, in patients without any evidence of epileptogenic lesions on diagnostic magnetic resonance imaging (MRI), impairment of the brain microstructure has been scarcely elucidated. Using multiparametric quantitative (q) magnetic resonance imaging MRI, we aimed to investigate diffuse impairment of the microstructural tissue integrity in MRI-negative focal epilepsy patients.
Methods: 27 MRI-negative patients with focal epilepsy (mean age 33.1 ± 14.2 years) and 27 matched healthy control subjects underwent multiparametric qMRI including T1, T2, and PD mapping at 3 T. After tissue segmentation based on synthetic anatomies, mean qMRI parameter values were extracted from the cerebral cortex, the white matter (WM) and the deep gray matter (GM) and compared between patients and control subjects. Apart from calculating mean values for the qMRI parameters across the respective compartments, voxel-wise analyses were performed for each tissue class.
Results: There were no significant differences for mean values of quantitative T1, T2, and PD obtained from the cortex, the WM and the deep GM between the groups. Furthermore, the voxel-wise analyses did not reveal any clusters indicating significant differences between patients and control subjects for the qMRI parameters in the respective compartments.
Conclusions: Based on the employed methodology, no indication for an impairment of the cerebral microstructural tissue integrity in MRI-negative patients with focal epilepsy was found in this study. Further research will be necessary to identify relevant factors and mechanisms contributing to microstructural brain tissue damage in various subgroups of patients with epilepsy.
Inhibitors against the NS3-4A protease of hepatitis C virus (HCV) have proven to be useful drugs in the treatment of HCV infection. Although variants have been identified with mutations that confer resistance to these inhibitors, the mutations do not restore replicative fitness and no secondary mutations that rescue fitness have been found. To gain insight into the molecular mechanisms underlying the lack of fitness compensation, we screened known resistance mutations in infectious HCV cell culture with different genomic backgrounds. We observed that the Q41R mutation of NS3-4A efficiently rescues the replicative fitness in cell culture for virus variants containing mutations at NS3-Asp168. To understand how the Q41R mutation rescues activity, we performed protease activity assays complemented by molecular dynamics simulations, which showed that protease-peptide interactions far outside the targeted peptide cleavage sites mediate substrate recognition by NS3-4A and support protease cleavage kinetics. These interactions shed new light on the mechanisms by which NS3-4A cleaves its substrates, viral polyproteins and a prime cellular antiviral adaptor protein, the mitochondrial antiviral signaling protein MAVS. Peptide binding is mediated by an extended hydrogen-bond network in NS3-4A that was effectively optimized for protease-MAVS binding in Asp168 variants with rescued replicative fitness from NS3-Q41R. In the protease harboring NS3-Q41R, the N-terminal cleavage products of MAVS retained high affinity to the active site, rendering the protease susceptible for potential product inhibition. Our findings reveal delicately balanced protease-peptide interactions in viral replication and immune escape that likely restrict the protease adaptive capability and narrow the virus evolutionary space.
Earliella scabrosa is a pantropical species of Polyporales (Basidiomycota) and well-studied concerning its morphology and taxonomy. However, its pantropical intraspecific genetic diversity and population differentiation is unknown. We initiated this study to better understand the genetic variation within E. scabrosa and to test if cryptic species are present. Sequences of three DNA regions, the nuclear ribosomal internal transcribed spacer (ITS), the large subunit ribosomal DNA (LSU), and the translation elongation factor (EF1α) were analysed for 66 samples from 15 geographical locations. We found a high level of genetic diversity (haplotype diversity, Hd = 0.88) and low nucleotide diversity (π = 0.006) across the known geographical range of E. scabrosa based on ITS sequences. The analysis of molecular variance (AMOVA) indicates that the genetic variability is mainly found among geographical populations. The results of Mantel tests confirmed that the genetic distance among populations of E. scabrosa is positively correlated with the geographical distance, which indicates that geographical isolation is an important factor for the observed genetic differentiation. Based on phylogenetic analyses of combined dataset ITS-LSU-EF1α, the low intraspecific divergences (0–0.3%), and the Automated Barcode Gap Discovery (ABGD) analysis, E. scabrosa can be considered as a single species with five different geographical populations. Each population might be in the process of allopatric divergence and in the long-term they may evolve and become distinct species.
Hyperparasitic fungi on black mildews (Meliolales, Ascomycota) : hidden diversity in the tropics
(2023)
Meliolales (Sordariomycetes, Ascomycota) is a group of obligate plant parasitic microfungi mainly distributed in the tropics and subtropics. Meliolalean fungi are commonly known as “black mildews”, as they form black, superficial hyphae on the surface of vegetative and reproductive organs of vascular plants. They are considered biotrophic parasites, and the infections caused by black mildews can lead to a decrease in the photosynthetic activity of plants, as well as to an increase in the temperature and respiration rate of their leaves.
Meliolales are frequently parasitized by hyperparasitic fungi, i.e., parasitic fungi that have parasitic hosts. These hyperparasites are all Ascomycota and belong mainly to the Dothideomycetes and Sordariomycetes. Although hyperparasites represent a megadiverse group, species were only described by morphology until 1980, and the systematic position of more than 60 % of known species is still unclear. In addition, there are no DNA reference sequences available in public databases for any of the species of hyperparasites of Meliolales, and no ecological studies have been done up to now.
Before this study, no exact number of hyperparasitic fungi growing on colonies of black mildews existed. Here, we present a checklist including 189 species of fungi known to be hyperparasitic on Meliolales, but the number of existing species is likely to be even higher. The elaboration of this species checklist laid the foundations for this investigation, as it helped to understand the present state of knowledge of hyperparasitic fungi on Meliolales worldwide.
For the present study, fresh specimens of leaves infected with colonies of Meliolales and hyperparasites were opportunistically collected at 32 collection sites in Western Panama and Benin, West Africa, in 2020 and 2022, respectively. In total, 100 samples of plant specimens infected with black mildews were collected, of which 58 samples were parasitized by hyperparasitic fungi. 31 species and morphospecies of hyperparasitic fungi were identified. In addition, 35 historical specimens, including 12 type specimens, were examined for the present work.
DNA of hyperparasitic fungi was isolated directly from conidia, synnemata, apothecia, perithecia or pseudothecia of fresh and dried specimens. The main challenges faced by scientists in doing molecular studies of hyperparasitic fungi are related to the fact that the hyperparasitic fungi are intermingled with tissues of the meliolalean hosts and other organisms present in a given sample. This makes the isolation of DNA exclusively from the hyperparasite difficult. Moreover, hyperparasitic fungi on Meliolales are biotrophs and cannot be grown axenically. The hosts themselves are also biotrophic, further complicating DNA isolation from either partner. These factors have contributed to a lack of reference sequences in public databases. After more than 100 attempts, DNA of 20 specimens of hyperparasitic fungi, representing seven species, has been isolated in the context of the present investigation. Three partial nuclear gene regions were amplified and sequenced: nrLSU, nrSSU and nrITS. The datasets were assembled for phylogenetic analyses applying Maximum Likelihood (ML) and Bayesian inference (BI) methods. DNA sequences of hyperparasitic fungi on Meliolales were generated for the first time in the context of the present investigation.
Hyperparasitic fungi on Meliolales do not represent a single systematic group, but a polyphyletic ecological guild of fungi. Because of this huge diversity, only the systematics of species of perithecioid hyperparasites, as well as of the species of the genera Atractilina and Spiropes known to be hyperparasitic on black mildews was discussed in this thesis, as they represented the most common groups of fungi found in Benin and Panama. The results indicated, for example, the systematic position of Dimerosporiella cephalosporii and Paranectriella minuta in the Sordariomycetes and Dothideomycetes, respectively. In addition, the first record of a hyperparasitic fungus of black mildews in the Lecanoromycetes, namely Calloriopsis herpotricha, is reported here. The systematics of Atractilina parasitica and of some species of Spiropes is also discussed here.
In the context of the present investigation, four species new to science were described. They are presented with detailed descriptions, photos and scientific illustrations. Taxonomic studies of this thesis also generated seven new synonyms, nine new records for Benin, seven for Panama, one for Africa and two for mainland America, as well as the confirmation of one anamorph-teleomorph connection by molecular sequence data.
The ecology of hyperparasitic fungi on Meliolales is complex and far from being completely understood. The hypothesis of host specificity between hyperparasitic fungi, their meliolalean hosts and their plant hosts was tested for the first time, through a tritrophic network analysis. Results indicate that hyperparasites of Meliolales are generalists concerning genera of Meliolales, but apparently specialists at the level of order. In addition, hyperparasitic fungi tend to be found alongside their meliolalean hosts, suggesting a pantropical distribution.
Cryo-electron tomography combined with subtomogram averaging (StA) has yielded high-resolution structures of macromolecules in their native context. However, high-resolution StA is not commonplace due to beam-induced sample drift, images with poor signal-to-noise ratios (SNR), challenges in CTF correction, and limited particle number. Here we address these issues by collecting tilt series with a higher electron dose at the zero-degree tilt. Particles of interest are then located within reconstructed tomograms, processed by conventional StA, and then re-extracted from the high-dose images in 2D. Single particle analysis tools are then applied to refine the 2D particle alignment and generate a reconstruction. Use of our hybrid StA (hStA) workflow improved the resolution for tobacco mosaic virus from 7.2 to 4.4 Å and for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. These resolution gains make hStA a promising approach for other StA projects aimed at achieving subnanometer resolution.
Cryo electron tomography (cryo-ET) combined with subtomogram averaging (StA) enables structural determination of macromolecules in their native context. A few structures were reported by StA at resolution higher than 4.5 Å, however all of these are from viral structural proteins or vesicle coats. Reaching high resolution for a broader range of samples is uncommon due to beam-induced sample drift, poor signal-to-noise ratio (SNR) of images, challenges in CTF correction, limited number of particles. Here we propose a strategy to address these issues, which consists of a tomographic data collection scheme and a processing workflow. Tilt series are collected with higher electron dose at zero-degree tilt in order to increase SNR. Next, after performing StA conventionally, we extract 2D projections of the particles of interest from the higher SNR images and use the single particle analysis tools to refine the particle alignment and generate a reconstruction. We benchmarked our proposed hybrid StA (hStA) workflow and improved the resolution for tobacco mosaic virus from 7.2 to 5.2 Å and the resolution for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. We demonstrate that hStA can improve the resolution obtained by conventional StA and promises to be a useful tool for StA projects aiming at subnanometer resolution or higher.
Classical molecular dynamics (MD) simulations provide unmatched spatial and time resolution of protein structure and function. However, accuracy of MD simulations often depends on the quality of force field parameters and the time scale of sampling. Another limitation of conventional MD simulations is that the protonation states of titratable amino acid residues remain fixed during simulations, even though protonation state changes coupled to conformational dynamics are central to protein function. Due to the uncertainty in selecting protonation states, classical MD simulations are sometimes performed with all amino acids modeled in their standard charged states at pH 7. Here we performed and analyzed classical MD simulations on high-resolution cryo-EM structures of two membrane proteins that transfer protons by catalyzing protonation/deprotonation reactions. In simulations performed with amino acids modeled in their standard protonation state the structure diverges far from its starting conformation. In comparison, MD simulations performed with pre-determined protonation states of amino acid residues reproduce the structural conformation, protein hydration, and protein-water and protein-protein interactions of the structure much better. The results suggest it is crucial to perform basic protonation state calculations, especially on structures where protonation changes play an important functional role, prior to launching any MD simulations. Furthermore, the combined approach of protonation state prediction and MD simulations can provide valuable information on the charge states of amino acids in the cryo-EM sample. Even though accurate prediction of protonation states currently remains a challenge, we introduce an approach of combining pKa prediction with cryo-EM density map analysis that helps in improving not only the protonation state predictions, but also the atomic modeling of density data.
The development of resistance to chemotherapeutic agents, such as Doxorubicin (DOX) and cytarabine (AraC), is one of the greatest challenges to the successful treatment of Acute Myeloid Leukemia (AML). Such acquisition is often underlined by a metabolic reprogramming that can provide a therapeutic opportunity, as it can lead to the emergence of vulnerabilities and dependencies to be exploited as targets against the resistant cells. In this regard, genome-scale metabolic models (GSMMs) have emerged as powerful tools to integrate multiple layers of data to build cancer-specific models and identify putative metabolic vulnerabilities. Here, we use genome-scale metabolic modelling to reconstruct a GSMM of the THP1 AML cell line and two derivative cell lines, one with acquired resistance to AraC and the second with acquired resistance to DOX. We also explore how, adding to the transcriptomic layer, the metabolomic layer enhances the selectivity of the resulting condition specific reconstructions. The resulting models enabled us to identify and experimentally validate that drug-resistant THP1 cells are sensitive to the FDA-approved antifolate methotrexate. Moreover, we discovered and validated that the resistant cell lines could be selectively targeted by inhibiting squalene synthase, providing a new and promising strategy to directly inhibit cholesterol synthesis in AML drug resistant cells.
The Kinase Chemogenomic Set (KCGS): An open science resource for kinase vulnerability identification
(2019)
We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS) is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.
Die Zahl der gramnegativen Bakterien auf der WHO-Liste der Antibiotikaresistenzen hat in den letzten Jahrzehnten erheblich zugenommen. Schätzungen zufolge wird die Antibiotikaresistenz bis 2050 tödlicher sein als Krebs. Die äußere Membran gramnegativer Bakterien ist aufgrund ihres wichtigsten Strukturbestandteils, des Lipopolysaccharids (LPS), sehr anpassungsfähig an Umweltveränderungen. Das LPS macht gramnegative Bakterien von Natur aus resistent gegen viele Antibiotika und führt somit zu Antibiotikaresistenz. Der bakterielle ATP-bindende Kassettentransporter (ABC-Transporter) MsbA spielt eine entscheidende Rolle bei der Regulierung der bakteriellen Außenmembran, indem er das Kern-LPS durch ATP-Hydrolyse über die Innenmembran von gramnegativen Bakterien flockt. Darüber hinaus fungiert diese Floppase als Efflux-Pumpe, indem sie Medikamente durch die innere Membran transportiert, was sie zu einem interessanten Ziel für Medikamente macht. Vor kurzem wurden zwei verschiedene Klassen von MsbA-Inhibitoren entdeckt: (1) Tetrahydrobenzothiophene (TBT), die den LPS-Transport aufheben, und (2) Chinolinderivate, die sowohl die ATP-Hydrolyse als auch die LPS-Translokation blockieren. Darüber hinaus hat die Bestimmung der 3D-Struktur von MsbA durch Rontgen- und Kryo-EM mehrere interessante Zustände der Floppase ergeben. Die Kernspinresonanzspektroskopie ist eine hervorragende biophysikalische Methode zur Ergänzung der vorhandenen 3D-Strukturdaten. Insbesondere ermöglicht die Festkörper-NMR die Untersuchung von Membranproteinen in einer nativen Umgebung (z. B. in einer Lipiddoppelschicht). In der Vergangenheit hat unser Labor mithilfe der Festkörper-NMR einige detaillierte Mechanismen von MsbA aufgedeckt. Trotz der zahlreichen Fortschritte bei der Untersuchung der ABC-Transporterprotein-Superfamilie ist der spezifische Prozess der Substrattranslokation von MsbA noch immer unbekannt. Es wird angenommen, dass dieser Translokationsprozess über die Kopplungshelices (CHs) erfolgt, die sich zwischen der Transmembranregion (TMD) und der Nukleotidbindungsdomäne (NBD) befinden. Nukleotid-Bindungsdomäne (NBD). Zu diesem Zweck wird dem Zusammenspiel zwischen der TMD und der NBD über die CHs besondere Aufmerksamkeit gewidmet, mit dem Ziel, den Prozess der Substrattranslokation mithilfe von funktionellen Assays und Festkörper-NMR zu verstehen. Bei letzterem wurden spezifische Reporter in die CHs eingeführt, um Konformationsänderungen in 2D-spektroskopischen Daten zu verfolgen. Darüber hinaus wurde zeitaufgelöste NMR eingesetzt, um die Auswirkungen verschiedener Substrate in der TMD während der ATP-Hydrolyse in der NBD sichtbar zu machen. Die einzigartigen Reporter in den CHs haben Konformationsänderungen in bestimmten katalytischen Zuständen gezeigt. Darüber hinaus scheinen verschiedene Substrate die Kinetik der ATP-Hydrolyse zu beeinflussen. Die Ergebnisse zeigten, dass einige Substrate einen bevorzugten katalytischen Zustand innerhalb des ATP-Hydrolyse Zyklus aufweisen, der möglicherweise einen gekoppelten oder ungekoppelten Kinasemechanismus hat. Diese Ergebnisse könnten verschiedene Einblicke in die molekulare Struktur potenzieller neuer Antibiotika liefern.
Substantial progress in the field of neuroscience has been made from anaesthetized preparations. Ketamine is one of the most used drugs in electrophysiology studies, but how ketamine affects neuronal responses is poorly understood. Here, we used in vivo electrophysiology and computational modelling to study how the auditory cortex of bats responds to vocalisations under anaesthesia and in wakefulness. In wakefulness, acoustic context increases neuronal discrimination of natural sounds. Neuron models predicted that ketamine affects the contextual discrimination of sounds regardless of the type of context heard by the animals (echolocation or communication sounds). However, empirical evidence showed that the predicted effect of ketamine occurs only if the acoustic context consists of low-pitched sounds (e.g., communication calls in bats). Using the empirical data, we updated the naïve models to show that differential effects of ketamine on cortical responses can be mediated by unbalanced changes in the firing rate of feedforward inputs to cortex, and changes in the depression of thalamo-cortical synaptic receptors. Combined, our findings obtained in vivo and in silico reveal the effects and mechanisms by which ketamine affects cortical responses to vocalisations.
Discrepancies between knockdown and knockout animal model phenotypes have long stood as a perplexing phenomenon. Several mechanisms explaining such observations have been proposed, namely the toxicity or the off-target effects of the knockdown reagents, as well as, in certain cases, genetic robustness – an organism's ability to maintain its phenotype despite genetic perturbations. In addition to these explanations, transcriptional adaptation (TA), a phenomenon defined as an event whereby a mutation in one gene leads to transcriptional upregulation or downregulation of another, adapting, gene or genes expression, has been recently proposed as an alternative explanation for the conflicting knockdown and knockout phenotype paradox.
Since its discovery in 2015, TA's precise mechanism remains a subject of ongoing research. Majority of evidence suggests that mutant mRNA degradation plays a central in TA. Epigenetic remodeling is also thought to play a role, as evidenced by an increase in active histone marks at the transcription start sites of the adapting genes. Whether mRNA degradation is indeed the key player in TA remains debated. Furthermore, it is still unknown how exactly TA develops, what adapting genes it targets, and whether genomic mutations that render mutant mRNA sensitive to degradation are required for TA to occur.
Throughout the experiments described in this Dissertation, I have designed an inducible TA system where TA can be triggered on demand and its effects on the cell’s transcriptome followed through time. I have demonstrated that degradation-prone transgenes, once induced and expressed, can be efficiently degraded, resulting in the protein loss-independent upregulation of adapting genes via TA. Adapting genes with higher degree of sequence similarity become upregulated faster than genes with lower degree of sequence similarity. Further functionality of this approach to study TA is limited by the leakiness of the inducible gene expression system; however, constitutively expressed degradation-prone transgenes were used to demonstrate TA in human cells.
In addition, I have developed an approach to target wild-type cytoplasmic mRNAs without altering the cell’s genome and reported a TA-like phenomenon, which manifested as adapting gene upregulation not relying on mutations in other genes. Cytoplasmic mRNA cleavage with CRISPR-Cas13d triggered a TA-like response in three different gene models: Actg1 knockdown, Ctnna1 knockdown, and Nckap1 knockdown. After comparing two different modes of triggering TA, CRISPR-Cas9 knockout versus CRISPR-Cas13d knockdown, I reported little overlap between the dysregulated genes and suggested that diverse mRNA degradation modes led to distinct TA responses. In addition, the transcriptional increase of Actg2 caused by CRISPR-Cas13d-mediated Actg1 mRNA cleavage did not require chromatin accessibility changes.
Experiments and genetic tools described in this dissertation investigated how TA develops from its earliest onset, how it affects the global transcriptome of the cell, as well as provided compelling evidence for an mRNA degradation-central TA mechanism. I have created tools to study both direct and indirect TA gene targets and unveiled important insights into the temporal dynamics of TA. Genes with higher sequence similarity were found to be upregulated more rapidly than those with lower similarity. Furthermore, it was revealed that the epigenetic properties of TA responses vary depending on the triggering mechanism. Cas13d-mediated degradation of wild-type mRNAs led to immediate transcriptional enhancement independent of epigenetic changes, which stood in contrast to previously measured alterations in chromatin accessibility in CRISPR-Cas9 mutants. This research has thus significantly advanced our knowledge of TA and provided valuable tools and findings that contribute to the broader understanding of gene expression regulation in response to mRNA degradation.
RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large ribonucleoprotein particles (RNPs) these proteins function in. Using UV-induced RNA-protein crosslinking of entire cells, protein complex purification and mass spectrometric analysis, we identified >100 in vivo RNA crosslinks in 16 nuclear mRNP components in Saccharomyces cerevisiae. For functional analysis, we chose Npl3, which displayed crosslinks in its two RNA recognition motifs (RRMs) and in the connecting flexible linker region. Both RRM domains and the linker uniquely contribute to RNA recognition as revealed by NMR and structural analyses. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains. Notably, an npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of other mRNP components into nuclear mRNPs, establishing a so far unknown function of Npl3 in nuclear mRNP assembly. Taken together, our integrative analysis uncovers a specific function of the RNA-binding activity of the nuclear mRNP component Npl3. This approach can be readily applied to RBPs in any RNA metabolic process.
RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large messenger ribonucleoprotein particle (mRNP) assemblies, these proteins often function in. Using UV-induced RNA-protein crosslinking and subsequent mass spectrometric analysis, we first identified more than 100 in vivo RNA crosslinks in 16 nuclear mRNP components in S. cerevisiae. For functional analysis, we chose Npl3, for which we determined crosslinks in its two RNA recognition motifs (RRM) and in the flexible linker region connecting the two. Using NMR and structural analyses, we show that both RRM domains and the linker uniquely contribute to RNA recognition. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains of Npl3. Notably, the npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of further mRNP components into nuclear mRNPs, establishing a function of Npl3 in nuclear mRNP assembly. Taken together, we determined the specific function of the RNA-binding activity of the nuclear mRNP component Npl3, an approach that can be applied to many RBPs in any RNA metabolic process.
Zinc finger (ZnF) domains appear in a pool of structural contexts and despite their small size achieve varying target specificities, covering single-stranded and double-stranded DNA and RNA as well as proteins. Combined with other RNA-binding domains, ZnFs enhance affinity and specificity of RNA-binding proteins (RBPs). The ZnF-containing immunoregulatory RBP Roquin initiates mRNA decay, thereby controlling the adaptive immune system. Its unique ROQ domain shape-specifically recognizes stem-looped cis-elements in mRNA 3’-untranslated regions (UTR). The N-terminus of Roquin contains a RING domain for protein-protein interactions and a ZnF, which was suggested to play an essential role in RNA decay by Roquin. The ZnF domain boundaries, its RNA motif preference and its interplay with the ROQ domain have remained elusive, also driven by the lack of high-resolution data of the challenging protein. We provide the solution structure of the Roquin-1 ZnF and use an RBNS-NMR pipeline to show that the ZnF recognizes AU-rich elements (ARE). We systematically refine the contributions of adenines in a poly(U)-background to specific complex formation. With the simultaneous binding of ROQ and ZnF to a natural target transcript of Roquin, our study for the first time suggests how Roquin integrates RNA shape and sequence specificity through the ROQ-ZnF tandem.
Highlights
• Seed size mediates seedling recruitment in tropical forests and pastures.
• Large-seeded species recruited better than small-seeded species in the forest.
• Recruitment of large-seeded species in pastures was limited by surface temperature.
• Large-seeded species should be protected against drought in regenerating pastures.
Abstract
Seedling recruitment is a key process of plant regeneration that often depends on plant functional traits, such as seed size. To optimize forest restoration efforts, we need to better understand how seedling recruitment of different seed sizes varies along environmental gradients with strong variation in abiotic and biotic factors. To understand these interacting effects, we conducted a sowing experiment with different-sized seeds in forests and pastures in the tropical mountains of southern Ecuador. We quantified seedling recruitment in relation to temperature, soil moisture and biotic pressures. We sowed seeds of five tree species of varying seed size at three elevations (1000, 2000 and 3000 m a.s.l.) in primary forest and pastures. We tested (1) how habitat type influences the recruitment of seedlings belonging to three small- and two large-seeded species, and (2) how abiotic and biotic factors limit seedling recruitment of species with different seed sizes. We found that seedlings of the two large-seeded species recruited better than seedlings of the three small-seeded species, but only in the forest habitat. Seedling recruitment of large seeds was primarily limited by high surface temperature, which explains lower recruitment of large seeds in pastures compared to forests. Our study shows that seed size can be a key trait mediating variability in seedling recruitment in tropical ecosystems. We conclude that restoration measures should aim to mitigate extreme temperatures in tropical pastures to aid the natural regeneration of large-seeded tree species.
G protein-coupled receptors (GPCRs) play a crucial role in modulating physiological responses and serve as the main drug target. Specifically, salmeterol and salbutamol which are used for the treatment of pulmonary diseases, exert their effects by activating the GPCR β2-adrenergic receptor (β2AR). In our study, we employed coarse-grained molecular dynamics simulations with the Martini 3 force field to investigate the dynamics of drug molecules in membranes in presence and absence of β2AR. Our simulations reveal that in more than 50% of the flip-flop events the drug molecules use the β2AR surface to permeate the membrane. The pathway along the GPCR surface is significantly more energetically favorable for the drug molecules, which was revealed by umbrella sampling simulations along spontaneous flip-flop pathways. Furthermore, we assessed the behavior of drugs with intracellular targets, such as kinase inhibitors, whose therapeutic efficacy could benefit from this observation. In summary, our results show that β2AR surface interactions can significantly enhance membrane permeation of drugs, emphasizing their potential for consideration in future drug development strategies.
The tremendous diversity of life in the ocean has proven to be a rich source of inspiration for drug discovery, with success rates for marine natural products up to 4 times higher than other naturally derived compounds. Yet the marine biodiscovery pipeline is characterized by chronic underfunding, bottlenecks and, ultimately, untapped potential. For instance, a lack of taxonomic capacity means that, on average, 20 years pass between the discovery of new organisms and the formal publication of scientific names, a prerequisite to proceed with detecting and isolating promising bioactive metabolites. The need for “edge” research that can spur novel lines of discovery and lengthy high-risk drug discovery processes, are poorly matched with research grant cycles. Here we propose five concrete pathways to broaden the biodiscovery pipeline and open the social and economic potential of the ocean genome for global benefit: (1) investing in fundamental research, even when the links to industry are not immediately apparent; (2) cultivating equitable collaborations between academia and industry that share both risks and benefits for these foundational research stages; (3) providing new opportunities for early-career researchers and under-represented groups to engage in high-risk research without risking their careers; (4) sharing data with global networks; and (5) protecting genetic diversity at its source through strong conservation efforts. The treasures of the ocean have provided fundamental breakthroughs in human health and still remain under-utilised for human benefit, yet that potential may be lost if we allow the biodiscovery pipeline to become blocked in a search for quick-fix solutions.
The transporter associated with antigen processing (TAP) is an essential machine of the adaptive immune system that translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen for loading of major histocompatibility class I molecules. To examine this ABC transport complex in mechanistic detail, we have established, after extensive screening and optimization, the solubilization, purification, and reconstitution for TAP to preserve its function in each step. This allowed us to determine the substrate-binding stoichiometry of the TAP complex by fluorescence cross-correlation spectroscopy. In addition, the TAP complex shows strict coupling between peptide binding and ATP hydrolysis, revealing no basal ATPase activity in the absence of peptides. These results represent an optimal starting point for detailed mechanistic studies of the transport cycle of TAP by single molecule experiments to analyze single steps of peptide translocation and the stoichiometry between peptide transport and ATP hydrolysis.
RBFOX1 is a highly pleiotropic gene that contributes to several psychiatric and neurodevelopmental disorders. Both rare and common variants in RBFOX1 have been associated with several psychiatric conditions, but the mechanisms underlying the pleiotropic effects of RBFOX1 are not yet understood. Here we found that, in zebrafish, rbfox1 is expressed in spinal cord, mid- and hindbrain during developmental stages. In adults, expression is restricted to specific areas of the brain, including telencephalic and diencephalic regions with an important role in receiving and processing sensory information and in directing behaviour. To investigate the effect of rbfox1 deficiency on behaviour, we used rbfox1sa15940, a rbfox1 loss-of-function line. We found that rbfox1sa15940 mutants present hyperactivity, thigmotaxis, decreased freezing behaviour and altered social behaviour. We repeated these behavioural tests in a second rbfox1 loss-of-function line with a different genetic background, rbfox1del19, and found that rbfox1 deficiency affects behaviour similarly in this line, although there were some differences. rbfox1del19 mutants present similar thigmotaxis, but stronger alterations in social behaviour and lower levels of hyperactivity than rbfox1sa15940 fish. Taken together, these results suggest that rbfox1 deficiency leads to multiple behavioural changes in zebrafish that might be modulated by environmental, epigenetic and genetic background effects, and that resemble phenotypic alterations present in Rbfox1-deficient mice and in patients with different psychiatric conditions. Our study thus highlights the evolutionary conservation of rbfox1 function in behaviour and paves the way to further investigate the mechanisms underlying rbfox1 pleiotropy on the onset of neurodevelopmental and psychiatric disorders.
RBFOX1 is a highly pleiotropic gene that contributes to several psychiatric and neurodevelopmental disorders. Both rare and common variants in RBFOX1 have been associated with several psychiatric conditions, but the mechanisms underlying the pleiotropic effects of RBFOX1 are not yet understood. Here we found that, in zebrafish, rbfox1 is expressed in spinal cord, mid- and hindbrain during developmental stages. In adults, expression is restricted to specific areas of the brain, including telencephalic and diencephalic regions with an important role in receiving and processing sensory information and in directing behaviour. To investigate the effect of rbfox1 deficiency on behaviour, we used rbfox1sa15940, a rbfox1 loss-of-function line. We found that rbfox1sa15940 mutants present hyperactivity, thigmotaxis, decreased freezing behaviour and altered social behaviour. We repeated these behavioural tests in a second rbfox1 loss-of-function line with a different genetic background, rbfox1del19, and found that rbfox1 deficiency affects behaviour similarly in this line, although there were some differences. rbfox1del19 mutants present similar thigmotaxis, but stronger alterations in social behaviour and lower levels of hyperactivity than rbfox1sa15940 fish. Taken together, these results suggest that rbfox1 deficiency leads to multiple behavioural changes in zebrafish that might be modulated by environmental, epigenetic and genetic background effects, and that resemble phenotypic alterations present in Rbfox1-deficient mice and in patients with different psychiatric conditions. Our study thus highlights the evolutionary conservation of rbfox1 function in behaviour and paves the way to further investigate the mechanisms underlying rbfox1 pleiotropy on the onset of neurodevelopmental and psychiatric disorders.
Parallel multisite recordings in the visual cortex of trained monkeys revealed that the responses of spatially distributed neurons to natural scenes are ordered in sequences. The rank order of these sequences is stimulus-specific and maintained even if the absolute timing of the responses is modified by manipulating stimulus parameters. The stimulus specificity of these sequences was highest when they were evoked by natural stimuli and deteriorated for stimulus versions in which certain statistical regularities were removed. This suggests that the response sequences result from a matching operation between sensory evidence and priors stored in the cortical network. Decoders trained on sequence order performed as well as decoders trained on rate vectors but the former could decode stimulus identity from considerably shorter response intervals than the latter. A simulated recurrent network reproduced similarly structured stimulus-specific response sequences, particularly once it was familiarized with the stimuli through non-supervised Hebbian learning. We propose that recurrent processing transforms signals from stationary visual scenes into sequential responses whose rank order is the result of a Bayesian matching operation. If this temporal code were used by the visual system it would allow for ultrafast processing of visual scenes.
Prenatal and postnatal experiences associated with epigenetic changes in the adult mouse brain
(2018)
To analyze the influences of early-life history on the brain epigenome, the offspring of mouse dams kept in an enriched or standard environment were exposed postnatally to enriched, standard, or adverse conditions. The methylation patterns of 7 candidate genes (9 loci) involved in developmental programming of stress vulnerability/resilience and psychiatric disease were analyzed in 6 brain regions of adult male and female mice. Exposure to an enriched prenatal environment was associated with widespread epigenetic changes (all of small effect size), affecting 29 of 324 (9%) gene/region-specific methylation patterns. The effects of either adverse or enriched postnatal conditions were tested separately in the two prenatal cohorts. Significant changes were observed in 2 of 324 (0.6%) loci in offspring of dams in a standard environment and 6 of 324 (1.9%) loci in animals that were exposed prenatally to an enriched environment. Prenatal life experiences appear to have a bigger effect on the adult brain epigenome than postnatal experiences. Positive prenatal life experiences may increase epigenetic plasticity of the brain later in life. All observed between-group differences were sex-specific, consistent with largely different developmental trajectories of the male and female brain. Multiple changes of small effect size are consistent with a multifactorial model of developmental programming of adult behavior and disease susceptibility.
In order to investigate the involvement of primary visual cortex (V1) in working memory (WM), parallel, multisite recordings of multiunit activity were obtained from monkey V1 while the animals performed a delayed match-to-sample (DMS) task. During the delay period, V1 population firing rate vectors maintained a lingering trace of the sample stimulus that could be reactivated by intervening impulse stimuli that enhanced neuronal firing. This fading trace of the sample did not require active engagement of the monkeys in the DMS task and likely reflects the intrinsic dynamics of recurrent cortical networks in lower visual areas. This renders an active, attention-dependent involvement of V1 in the maintenance of working memory contents unlikely. By contrast, population responses to the test stimulus depended on the probabilistic contingencies between sample and test stimuli. Responses to tests that matched expectations were reduced which agrees with concepts of predictive coding.
The simultaneous inhibition of HDACs and BET proteins has shown promising anti-proliferative effects against different cancer types, including the difficult to treat pancreatic cancer. In this work, the strategy of concurrently targeting HDACs and BET proteins was pursued by developing different types of dual inhibitors.
By developing a novel scaffold that selectively inhibits HDAC1/2 together with BET proteins in cells, an effective tool for the investigation of pancreatic cancer, and other diseases which are sensitive to epigenetic processes, was created. The compound’s small size further gives the opportunity to further develop the inhibitor towards optimized pharmacokinetic properties, potentially resulting in a drug for cancer treatment.
A second novel approach that was pursued, was the development of a small-molecule degrader, targeting HDACs and BET proteins. Through synthesizing a variety of different molecules, a compound that was capable of lowering BRD4 levels and, at the same time, increasing histone acetylation was developed. While additional mechanistic investigations are needed to verify the degradation, the potent antiproliferative effects in pancreatic cancer cells encourage further studies following this alternative new strategy.
The enzyme acetyl-CoA carboxylase (ACC) plays a crucial role in fatty acid metabolism. In recent years, ACC has been recognized as a promising drug target for treating different diseases. However, the role of ACC in vascular endothelial cells (ECs) has been neglected so far. To characterize the role of ACC, we used the ACC inhibitor, soraphen A, as a chemical tool, and also a gene silencing approach. We found that ACC1 was the predominant isoform in human umbilical vein ECs as well as in human microvascular ECs and that soraphen A reduced the levels of malonyl-CoA. We revealed that ACC inhibition shifted the lipid composition of EC membranes. Accordingly, membrane fluidity, filopodia formation, and migratory capacity were reduced. The antimigratory action of soraphen A depended on an increase in the cellular proportion of PUFAs and, most importantly, on a decreased level of phosphatidylglycerol. Our study provides a causal link between ACC, membrane lipid composition, and cell migration in ECs. Soraphen A represents a useful chemical tool to investigate the role of fatty acid metabolism in ECs and ACC inhibition offers a new and valuable therapeutic perspective for the treatment of EC migration-related diseases.