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"Ästhetisch ist, was hilft"
(2017)
We present here a set of 13C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose 13C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C40 nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C10,H10 ribose signals. The experiments were applied to two RNA hairpin structures. The current set of 13C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, 13C-direct detected NMR methods constitute useful complements to the conventional 1H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs. Keywords: NMR spectroscopy , Direct carbon , detection , RNA
50 years of amino acid hydrophobicity scales : revisiting the capacity for peptide classification
(2016)
Background: Physicochemical properties are frequently analyzed to characterize protein-sequences of known and unknown function. Especially the hydrophobicity of amino acids is often used for structural prediction or for the detection of membrane associated or embedded β-sheets and α-helices. For this purpose many scales classifying amino acids according to their physicochemical properties have been defined over the past decades. In parallel, several hydrophobicity parameters have been defined for calculation of peptide properties. We analyzed the performance of separating sequence pools using 98 hydrophobicity scales and five different hydrophobicity parameters, namely the overall hydrophobicity, the hydrophobic moment for detection of the α-helical and β-sheet membrane segments, the alternating hydrophobicity and the exact ß-strand score.
Results: Most of the scales are capable of discriminating between transmembrane α-helices and transmembrane β-sheets, but assignment of peptides to pools of soluble peptides of different secondary structures is not achieved at the same quality. The separation capacity as measure of the discrimination between different structural elements is best by using the five different hydrophobicity parameters, but addition of the alternating hydrophobicity does not provide a large benefit. An in silico evolutionary approach shows that scales have limitation in separation capacity with a maximal threshold of 0.6 in general. We observed that scales derived from the evolutionary approach performed best in separating the different peptide pools when values for arginine and tyrosine were largely distinct from the value of glutamate. Finally, the separation of secondary structure pools via hydrophobicity can be supported by specific detectable patterns of four amino acids.
Conclusion: It could be assumed that the quality of separation capacity of a certain scale depends on the spacing of the hydrophobicity value of certain amino acids. Irrespective of the wealth of hydrophobicity scales a scale separating all different kinds of secondary structures or between soluble and transmembrane peptides does not exist reflecting that properties other than hydrophobicity affect secondary structure formation as well. Nevertheless, application of hydrophobicity scales allows distinguishing between peptides with transmembrane α-helices and β-sheets. Furthermore, the overall separation capacity score of 0.6 using different hydrophobicity parameters could be assisted by pattern search on the protein sequence level for specific peptides with a length of four amino acids.
Bromodomains (BRDs) are conserved protein interaction modules which recognize (read) acetyl-lysine modifications, however their role(s) in regulating cellular states and their potential as targets for the development of targeted treatment strategies is poorly understood. Here we present a set of 25 chemical probes, selective small molecule inhibitors, covering 29 human bromodomain targets. We comprehensively evaluate the selectivity of this probe-set using BROMOscan and demonstrate the utility of the set identifying roles of BRDs in cellular processes and potential translational applications. For instance, we discovered crosstalk between histone acetylation and the glycolytic pathway resulting in a vulnerability of breast cancer cell lines under conditions of glucose deprivation or GLUT1 inhibition to inhibition of BRPF2/3 BRDs. This chemical probe-set will serve as a resource for future applications in the discovery of new physiological roles of bromodomain proteins in normal and disease states, and as a toolset for bromodomain target validation.
Analysis of whole cell lipid extracts of bacteria by means of ultra-performance (UP)LC-MS allows a comprehensive determination of the lipid molecular species present in the respective organism. The data allow conclusions on its metabolic potential as well as the creation of lipid profiles, which visualize the organism's response to changes in internal and external conditions. Herein, we describe: i) a fast reversed phase UPLC-ESI-MS method suitable for detection and determination of individual lipids from whole cell lipid extracts of all polarities ranging from monoacylglycerophosphoethanolamines to TGs; ii) the first overview of a wide range of lipid molecular species in vegetative Myxococcus xanthus DK1622 cells; iii) changes in their relative composition in selected mutants impaired in the biosynthesis of α-hydroxylated FAs, sphingolipids, and ether lipids; and iv) the first report of ceramide phosphoinositols in M. xanthus, a lipid species previously found only in eukaryotes.
Aging of biological systems is controlled by various processes which have a potential impact on gene expression. Here we report a genome-wide transcriptome analysis of the fungal aging model Podospora anserina. Total RNA of three individuals of defined age were pooled and analyzed by SuperSAGE (serial analysis of gene expression). A bioinformatics analysis identified different molecular pathways to be affected during aging. While the abundance of transcripts linked to ribosomes and to the proteasome quality control system were found to decrease during aging, those associated with autophagy increase, suggesting that autophagy may act as a compensatory quality control pathway. Transcript profiles associated with the energy metabolism including mitochondrial functions were identified to fluctuate during aging. Comparison of wild-type transcripts, which are continuously down-regulated during aging, with those down-regulated in the long-lived, copper-uptake mutant grisea, validated the relevance of age-related changes in cellular copper metabolism. Overall, we (i) present a unique age-related data set of a longitudinal study of the experimental aging model P. anserina which represents a reference resource for future investigations in a variety of organisms, (ii) suggest autophagy to be a key quality control pathway that becomes active once other pathways fail, and (iii) present testable predictions for subsequent experimental investigations.
Coevolution of viruses and their hosts represents a dynamic molecular battle between the immune system and viral factors that mediate immune evasion. After the abandonment of smallpox vaccination, cowpox virus infections are an emerging zoonotic health threat, especially for immunocompromised patients. Here we delineate the mechanistic basis of how cowpox viral CPXV012 interferes with MHC class I antigen processing. This type II membrane protein inhibits the coreTAP complex at the step after peptide binding and peptide-induced conformational change, in blocking ATP binding and hydrolysis. Distinct from other immune evasion mechanisms, TAP inhibition is mediated by a short ER-lumenal fragment of CPXV012, which results from a frameshift in the cowpox virus genome. Tethered to the ER membrane, this fragment mimics a high ER-lumenal peptide concentration, thus provoking a trans-inhibition of antigen translocation as supply for MHC I loading. These findings illuminate the evolution of viral immune modulators and the basis of a fine-balanced regulation of antigen processing.
A new type of Na+-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif
(2013)
Abstract: The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na+. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F1Fo-ATP synthase with a novel Na+ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na+ specificity in physiological settings. Consistently, activity measurements showed Na+ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na+ ionophore monensin. Furthermore, Na+ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na+ coupling is provided by two identical crystal structures of the c11 ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na+ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na+ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.
Author Summary: Essential cellular processes such as biosynthesis, transport, and motility are sustained by the energy released in the hydrolysis of ATP, the universal energy carrier in living cells. Most ATP in the cell is produced by a membrane-bound enzyme, the ATP synthase, through a rotary mechanism that is coupled to the translocation of ions across the membrane. The majority of ATP synthases are energized by transmembrane electrochemical gradients of protons (proton-motive force), but a number of organisms, including some important human pathogens, use gradients of sodium ions instead (sodium-motive force). The ion specificity of ATP synthases is determined by a membrane-embedded sub-complex, the c-ring, which is the smallest known biological rotor. The functional mechanism of the rotor ring and its variations among different organisms are of wide interest, because of this enzyme's impact on metabolism and disease, and because of its potential for nanotechnology applications. Here, we characterize a previously unrecognized type of Na+-driven ATP synthase from the opportunistic human pathogen Fusobacterium nucleatum, which is implicated in periodontal diseases. We analyzed this ATP synthase and its rotor ring through a multi-disciplinary approach, combining cell-growth and biochemical assays, X-ray crystallography and computer-simulation methods. Two crystal structures of the membrane rotor were solved, at low and high pH, revealing an atypical ion-recognition motif mediated by two carboxylate side-chains. This motif is shared by other human pathogens, such as Mycobacterium tuberculosis or Streptococcus pneumonia, whose ATP synthases are targets of novel antibiotic drugs. The implications of this ion-recognition mode on the mechanism of the ATP synthase and the cellular bioenergetics of F. nucleatum were thus examined. Our results provide the basis for future pharmacological efforts against this important pathogen.
The ubiquitin-binding zinc finger (UBZ) is a type of zinc-coordinating β-β-α fold domain found mainly in proteins involved in DNA repair and transcriptional regulation. Here, we report the crystal structure of the UBZ domain of Y-family DNA polymerase (pol) η and the crystal structure of the complex between the UBZ domain of Werner helicase-interacting protein 1 (WRNIP1) and ubiquitin, crystallized using the GFP fusion technique. In contrast to the pol η UBZ, which has been proposed to bind ubiquitin via its C-terminal α-helix, ubiquitin binds to a novel surface of WRNIP1 UBZ composed of the first β-strand and the C-terminal α-helix. In addition, we report the structure of the tandem UBZ domains of Tax1-binding protein 1 (TAX1BP1) and show that the second UBZ of TAX1BP1 binds ubiquitin, presumably in a manner similar to that of WRNIP1 UBZ. We propose that UBZ domains can be divided into at least two different types in terms of the ubiquitin-binding surfaces: the pol η type and the WRNIP1 type.
Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the “foot” region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis.
Autophagy is a cytosolic quality control process that recognizes substrates through receptor‐mediated mechanisms. Procollagens, the most abundant gene products in Metazoa, are synthesized in the endoplasmic reticulum (ER), and a fraction that fails to attain the native structure is cleared by autophagy. However, how autophagy selectively recognizes misfolded procollagens in the ER lumen is still unknown. We performed siRNA interference, CRISPR‐Cas9 or knockout‐mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells. We found that the ER‐resident lectin chaperone Calnexin (CANX) and the ER‐phagy receptor FAM134B are required for autophagy‐mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co‐receptor that recognizes ER luminal misfolded procollagens and interacts with the ER‐phagy receptor FAM134B. In turn, FAM134B binds the autophagosome membrane‐associated protein LC3 and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation. Thus, a crosstalk between the ER quality control machinery and the autophagy pathway selectively disposes of proteasome‐resistant misfolded clients from the ER.
Diploid transgenic organisms are either hemi- or homozygous. Genetic assays are, therefore, required to identify the genotype. Our AGameOfClones vector concept uses two clearly distinguishable transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination leads to hemizygous individuals that carry only one marker. In the following generation, heterozygous descendants are identified by the presence of both markers and produce homozygous progeny that are selected by the lack of one marker. We prove our concept in Tribolium castaneum by systematically creating multiple functional homozygous transgenic lines suitable for long-term fluorescence live imaging. Our approach saves resources and simplifies transgenic organism handling. Since the concept relies on the universal Cre-Lox system, it is expected to work in all diploid model organisms, for example, insects, zebrafish, rodents and plants. With appropriate adaptions, it can be used in knock-out assays to preselect homozygous individuals and thus minimize the number of wasted animals.
The use of cardiac troponins (cTn) is the gold standard for diagnosing myocardial infarction. Independent of myocardial infarction (MI), however, sex, age and kidney function affect cTn levels. Here we developed a method to adjust cTnI levels for age, sex, and renal function, maintaining a unified cut-off value such as the 99th percentile. A total of 4587 individuals enrolled in a prospective longitudinal study were used to develop a model for adjustment of cTn. cTnI levels correlated with age and estimated glomerular filtration rate (eGFR) in males/females with rage = 0.436/0.518 and with reGFR = −0.142/−0.207. For adjustment, these variables served as covariates in a linear regression model with cTnI as dependent variable. This adjustment model was then applied to a real-world cohort of 1789 patients with suspected acute MI (AMI) (N = 407). Adjusting cTnI showed no relevant loss of diagnostic information, as evidenced by comparable areas under the receiver operator characteristic curves, to identify AMI in males and females for adjusted and unadjusted cTnI. In specific patients groups such as in elderly females, adjusting cTnI improved specificity for AMI compared with unadjusted cTnI. Specificity was also improved in patients with renal dysfunction by using the adjusted cTnI values. Thus, the adjustments improved the diagnostic ability of cTnI to identify AMI in elderly patients and in patients with renal dysfunction. Interpretation of cTnI values in complex emergency cases is facilitated by our method, which maintains a single diagnostic cut-off value in all patients.
Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.
Shrew-1, also called AJAP1, is a transmembrane protein associated with E-cadherin-mediated adherence junctions and a putative tumor suppressor. Apart from its interaction with β-catenin and involvement in E-cadherin internalization, little structure or function information exists. Here we explored shrew-1 expression during postnatal differentiation of mammary gland as a model system. Immunohistological analyses with antibodies against either the extracellular or the cytoplasmic domains of shrew-1 consistently revealed the expression of full-length shrew-1 in myoepithelial cells, but only part of it in luminal cells. While shrew-1 localization remained unaltered in myoepithelial cells, nuclear localization occurred in luminal cells during lactation. Based on these observations, we identified two unknown shrew-1 transcript variants encoding N-terminally truncated proteins. The smallest shrew-1 protein lacks the extracellular domain and is most likely the only variant present in luminal cells. RNA analyses of human tissues confirmed that the novel transcript variants of shrew-1 exist in vivo and exhibit a differential tissue expression profile. We conclude that our findings are essential for the understanding and interpretation of future functional and interactome analyses of shrew-1 variants.
Background: The ideal biofuel should not only be a regenerative fuel from renewable feedstocks, but should also be compatible with the existing fuel distribution infrastructure and with normal car engines. As the so-called drop-in biofuel, the fatty alcohol 1-octanol has been described as a valuable substitute for diesel and jet fuels and has already been produced fermentatively from sugars in small amounts with engineered bacteria via reduction of thioesterase-mediated premature release of octanoic acid from fatty acid synthase or via a reversal of the β-oxidation pathway.
Results: The previously engineered short-chain acyl-CoA producing yeast Fas1R1834K/Fas2 fatty acid synthase variant was expressed together with carboxylic acid reductase from Mycobacterium marinum and phosphopantetheinyl transferase Sfp from Bacillus subtilis in a Saccharomyces cerevisiae Δfas1 Δfas2 Δfaa2 mutant strain. With the involvement of endogenous thioesterases, alcohol dehydrogenases, and aldehyde reductases, the synthesized octanoyl-CoA was converted to 1-octanol up to a titer of 26.0 mg L−1 in a 72-h fermentation. The additional accumulation of 90 mg L−1 octanoic acid in the medium indicated a bottleneck in 1-octanol production. When octanoic acid was supplied externally to the yeast cells, it could be efficiently converted to 1-octanol indicating that re-uptake of octanoic acid across the plasma membrane is not limiting. Additional overexpression of aldehyde reductase Ahr from Escherichia coli nearly completely prevented accumulation of octanoic acid and increased 1-octanol titers up to 49.5 mg L−1. However, in growth tests concentrations even lower than 50.0 mg L−1 turned out to be inhibitory to yeast growth. In situ extraction in a two-phase fermentation with dodecane as second phase did not improve growth, indicating that 1-octanol acts inhibitive before secretion. Furthermore, 1-octanol production was even reduced, which results from extraction of the intermediate octanoic acid to the organic phase, preventing its re-uptake.
Conclusions: By providing chain length control via an engineered octanoyl-CoA producing fatty acid synthase, we were able to specifically produce 1-octanol with S. cerevisiae. Before metabolic engineering can be used to further increase product titers and yields, strategies must be developed that cope with the toxic effects of 1-octanol on the yeast cells.
Targeting self-renewal and tumorigenicity has been proposed as a potential strategy against cancer stem cells (CSCs). Epigenetic proteins are key modulators of gene expression and cancer development contributing to regulation and maintenance of self-renewal and tumorigenicity. Here, we have screened a small-molecule epigenetic inhibitor library using 3D in vitro models in order to determine potential epigenetic targets associated with self-renewal and tumorigenicity in Canine Mammary Cancer (CMC) cells. We identified inhibition of BET proteins as a promising strategy to inhibit CMC colonies and tumorspheres formation. Low doses of (+)-JQ1 were able to downregulate important genes associated to self-renewal pathways such as WNT, NOTCH, Hedgehog, PI3K/AKT/mTOR, EGF receptor and FGF receptor in CMC tumorspheres. In addition, we observed downregulation of ZEB2, a transcription factor important for the maintenance of self-renewal in canine mammary cancer cells. Furthermore, low doses of (+)-JQ1 were not cytotoxic in CMC cells cultured in 2D in vitro models but induced G2/M cell cycle arrest accompanied by upregulation of G2/M checkpoint-associated genes including BTG2 and CCNG2. Our work indicates the BET inhibition as a new strategy for canine mammary cancers by modulating the self-renewal phenotype in tumorigenic cells such as CSCs.
Background The EGF receptor has been shown to internalize via clathrin-independent endocytosis (CIE) in a ligand concentration dependent manner. From a modeling point of view, this resembles an ultrasensitive response, which is the ability of signaling networks to suppress a response for low input values and to increase to a pre-defined level for inputs exceeding a certain threshold. Several mechanisms to generate this behaviour have been described theoretically, the underlying assumptions of which, however, have not been experimentally demonstrated for the EGF receptor internalization network. Results Here, we present a mathematical model of receptor sorting into alternative pathways that explains the EGF-concentration dependent response of CIE. The described mechanism involves a saturation effect of the dominant clathrin-dependent endocytosis pathway and implies distinct steady-states into which the system is forced for low vs high EGF stimulations. The model is minimal since no experimentally unjustified reactions or parameter assumptions are imposed. We demonstrate the robustness of the sorting effect for large parameter variations and give an analytic derivation for alternative steady-states that are reached. Further, we describe extensibility of the model to more than two pathways which might play a role in contexts other than receptor internalization. Conclusions Our main result is that a scenario where different endocytosis routes consume the same form of receptor corroborates the observation of a clear-cut, stimulus dependent sorting. This is especially important since a receptor modification discriminating between the pathways has not been found. The model is not restricted to EGF receptor internalization and might account for ultrasensitivity in other cellular contexts.
Membrane proteins frequently assemble into higher order homo- or hetero-oligomers within their natural lipid environment. This complex formation can modulate their folding, activity as well as substrate selectivity. Non-disruptive methods avoiding critical steps, such as membrane disintegration, transfer into artificial environments or chemical modifications are therefore essential to analyze molecular mechanisms of native membrane protein assemblies. The combination of cell-free synthetic biology, nanodisc-technology and non-covalent mass spectrometry provides excellent synergies for the analysis of membrane protein oligomerization within defined membranes. We exemplify our strategy by oligomeric state characterization of various membrane proteins including ion channels, transporters and membrane-integrated enzymes assembling up to hexameric complexes. We further indicate a lipid-dependent dimer formation of MraY translocase correlating with the enzymatic activity. The detergent-free synthesis of membrane protein/nanodisc samples and the analysis by LILBID mass spectrometry provide a versatile platform for the analysis of membrane proteins in a native environment.
ANGIOGENES : knowledge database for protein-coding and noncoding RNA genes in endothelial cells
(2016)
Increasing evidence indicates the presence of long noncoding RNAs (lncRNAs) is specific to various cell types. Although lncRNAs are speculated to be more numerous than protein-coding genes, the annotations of lncRNAs remain primitive due to the lack of well-structured schemes for their identification and description. Here, we introduce a new knowledge database “ANGIOGENES” (http://angiogenes.uni-frankfurt.de) to allow for in silico screening of protein-coding genes and lncRNAs expressed in various types of endothelial cells, which are present in all tissues. Using the latest annotations of protein-coding genes and lncRNAs, publicly-available RNA-seq data was analyzed to identify transcripts that are expressed in endothelial cells of human, mouse and zebrafish. The analyzed data were incorporated into ANGIOGENES to provide a one-stop-shop for transcriptomics data to facilitate further biological validation. ANGIOGENES is an intuitive and easy-to-use database to allow in silico screening of expressed, enriched and/or specific endothelial transcripts under various conditions. We anticipate that ANGIOGENES serves as a starting point for functional studies to elucidate the roles of protein-coding genes and lncRNAs in angiogenesis.
Alternative polyadenylation (APA) is a widespread mechanism that contributes to the sophisticated dynamics of gene regulation. Approximately 50% of all protein-coding human genes harbor multiple polyadenylation (PA) sites; their selective and combinatorial use gives rise to transcript variants with differing length of their 3' untranslated region (3'UTR). Shortened variants escape UTR-mediated regulation by microRNAs (miRNAs), especially in cancer, where global 3'UTR shortening accelerates disease progression, dedifferentiation and proliferation. Here we present APADB, a database of vertebrate PA sites determined by 3' end sequencing, using massive analysis of complementary DNA ends. APADB provides (A)PA sites for coding and non-coding transcripts of human, mouse and chicken genes. For human and mouse, several tissue types, including different cancer specimens, are available. APADB records the loss of predicted miRNA binding sites and visualizes next-generation sequencing reads that support each PA site in a genome browser. The database tables can either be browsed according to organism and tissue or alternatively searched for a gene of interest. APADB is the largest database of APA in human, chicken and mouse. The stored information provides experimental evidence for thousands of PA sites and APA events. APADB combines 3' end sequencing data with prediction algorithms of miRNA binding sites, allowing to further improve prediction algorithms. Current databases lack correct information about 3'UTR lengths, especially for chicken, and APADB provides necessary information to close this gap. Database URL: http://tools.genxpro.net/apadb/
Synaptic release sites are characterized by exocytosis-competent synaptic vesicles tightly anchored to the presynaptic active zone (PAZ) whose proteome orchestrates the fast signaling events involved in synaptic vesicle cycle and plasticity. Allocation of the amyloid precursor protein (APP) to the PAZ proteome implicated a functional impact of APP in neuronal communication. In this study, we combined state-of-the-art proteomics, electrophysiology and bioinformatics to address protein abundance and functional changes at the native hippocampal PAZ in young and old APP-KO mice. We evaluated if APP deletion has an impact on the metabolic activity of presynaptic mitochondria. Furthermore, we quantified differences in the phosphorylation status after long-term-potentiation (LTP) induction at the purified native PAZ. We observed an increase in the phosphorylation of the signaling enzyme calmodulin-dependent kinase II (CaMKII) only in old APP-KO mice. During aging APP deletion is accompanied by a severe decrease in metabolic activity and hyperphosphorylation of CaMKII. This attributes an essential functional role to APP at hippocampal PAZ and putative molecular mechanisms underlying the age-dependent impairments in learning and memory in APP-KO mice.
Abstract: The hallmarks of Alzheimer’s disease (AD) are characterized by cognitive decline and behavioral changes. The most prominent brain region affected by the progression of AD is the hippocampal formation. The pathogenesis involves a successive loss of hippocampal neurons accompanied by a decline in learning and memory consolidation mainly attributed to an accumulation of senile plaques. The amyloid precursor protein (APP) has been identified as precursor of Aβ-peptides, the main constituents of senile plaques. Until now, little is known about the physiological function of APP within the central nervous system. The allocation of APP to the proteome of the highly dynamic presynaptic active zone (PAZ) highlights APP as a yet unknown player in neuronal communication and signaling. In this study, we analyze the impact of APP deletion on the hippocampal PAZ proteome. The native hippocampal PAZ derived from APP mouse mutants (APP-KOs and NexCreAPP/APLP2-cDKOs) was isolated by subcellular fractionation and immunopurification. Subsequently, an isobaric labeling was performed using TMT6 for protein identification and quantification by high-resolution mass spectrometry. We combine bioinformatics tools and biochemical approaches to address the proteomics dataset and to understand the role of individual proteins. The impact of APP deletion on the hippocampal PAZ proteome was visualized by creating protein-protein interaction (PPI) networks that incorporated APP into the synaptic vesicle cycle, cytoskeletal organization, and calcium-homeostasis. The combination of subcellular fractionation, immunopurification, proteomic analysis, and bioinformatics allowed us to identify APP as structural and functional regulator in a context-sensitive manner within the hippocampal active zone network.
Author Summary: More than 20 years ago, the amyloid precursor protein (APP) was identified as the precursor protein of the Aβ peptide, the main component of senile plaques in brains affected by Alzheimer’s disease. However, little is known about the physiological function of amyloid precursor protein. Allocating APP to the proteome of the structurally and functionally dynamic presynaptic active zone highlights APP as a hitherto unknown player within the presynaptic network. The hippocampus is the most prominent brain region for learning and memory consolidation, and a vulnerable target for neurodegenerative disease, e. g. Alzheimer’s disease. Therefore, our experimental design is focused on the hippocampal neurotransmitter release site. Currently, the underlying mechanism of how APP acts within presynaptic networks is still elusive. Within the scope of this research article, we constructed a network of APP within the presynaptic active zone and how deletion of APP affects these individual networks. We combine bioinformatics tools and biochemical approaches to address the dataset provided by proteomics. Furthermore, we could unravel that APP executes regulatory functions within the synaptic vesicle cycle, cytoskeletal rearrangements and Ca2+-homeostasis. Taken together, our findings offer a new perspective on the physiological function of APP in the central nervous system and may provide a molecular link to the pathogenesis of Alzheimer’s disease.
Cardiac arrhythmias are often associated with mutations in ion channels or other proteins. To enable drug development for distinct arrhythmias, model systems are required that allow implementing patient-specific mutations. We assessed a muscular pump in Caenorhabditis elegans. The pharynx utilizes homologues of most of the ion channels, pumps and transporters defining human cardiac physiology. To yield precise rhythmicity, we optically paced the pharynx using channelrhodopsin-2. We assessed pharynx pumping by extracellular recordings (electropharyngeograms--EPGs), and by a novel video-microscopy based method we developed, which allows analyzing multiple animals simultaneously. Mutations in the L-type VGCC (voltage-gated Ca(2+)-channel) EGL-19 caused prolonged pump duration, as found for analogous mutations in the Cav1.2 channel, associated with long QT syndrome. egl-19 mutations affected ability to pump at high frequency and induced arrhythmicity. The pharyngeal neurons did not influence these effects. We tested whether drugs could ameliorate arrhythmia in the optogenetically paced pharynx. The dihydropyridine analog Nemadipine A prolonged pump duration in wild type, and reduced or prolonged pump duration of distinct egl-19 alleles, thus indicating allele-specific effects. In sum, our model may allow screening of drug candidates affecting specific VGCCs mutations, and permit to better understand the effects of distinct mutations on a macroscopic level.
Glioblastoma is one of the deadliest malignancies and is virtually incurable. Accumulating evidence indicates that a small population of cells with a stem-like phenotype is the major culprit of tumor recurrence. Enhanced DNA repair capacity and expression of stemness marker genes are the main characteristics of these cells. Elimination of this population might delay or prevent tumor recurrence following radiochemotherapy. The aim of this study was to analyze whether interference with the Hedgehog signaling (Hh) pathway or combined Hh/Notch blockade using small-molecule inhibitors can efficiently target these cancer stem cells and sensitize them to therapy. Using tumor sphere lines and primary patient-derived glioma cultures we demonstrate that the Hh pathway inhibitor GANT61 (GANT) and the arsenic trioxide (ATO)-mediated Hh/Notch inhibition are capable to synergistically induce cell death in combination with the natural anticancer agent (−)-Gossypol (Gos). Only ATO in combination with Gos also strongly decreased stemness marker expression and prevented sphere formation and recovery. These synergistic effects were associated with distinct proteomic changes indicating diminished DNA repair and markedly reduced stemness. Finally, using an organotypic brain slice transplantation model, we show that combined ATO/Gos treatment elicits strong growth inhibition or even complete elimination of tumors. Collectively, our data show for the first time that ATO and Gos, two drugs that can be used in the clinic, represent a promising targeted therapy approach for the synergistic elimination of glioma stem-like cells.
Antigen presentation to cytotoxic T lymphocytes via major histocompatibility complex class I (MHC I) molecules depends on the heterodimeric transporter associated with antigen processing (TAP). For efficient antigen supply to MHC I molecules in the ER, TAP assembles a macromolecular peptide-loading complex (PLC) by recruiting tapasin. In evolution, TAP appeared together with effector cells of adaptive immunity at the transition from jawless to jawed vertebrates and diversified further within the jawed vertebrates. Here, we compared TAP function and interaction with tapasin of a range of species within two classes of jawed vertebrates. We found that avian and mammalian TAP1 and TAP2 form heterodimeric complexes across taxa. Moreover, the extra N-terminal domain TMD0 of mammalian TAP1 and TAP2 as well as avian TAP2 recruits tapasin. Strikingly, however, only TAP1 and TAP2 from the same taxon can form a functional heterodimeric translocation complex. These data demonstrate that the dimerization interface between TAP1 and TAP2 and the tapasin docking sites for PLC assembly are conserved in evolution, whereas elements of antigen translocation diverged later in evolution and are thus taxon specific.
Background: Histone lysine demethylases (KDMs) are of interest as drug targets due to their regulatory roles in chromatin organization and their tight associations with diseases including cancer and mental disorders. The first KDM inhibitors for KDM1 have entered clinical trials, and efforts are ongoing to develop potent, selective and cell-active ‘probe’ molecules for this target class. Robust cellular assays to assess the specific engagement of KDM inhibitors in cells as well as their cellular selectivity are a prerequisite for the development of high-quality inhibitors. Here we describe the use of a high-content cellular immunofluorescence assay as a method for demonstrating target engagement in cells.
Results: A panel of assays for the Jumonji C subfamily of KDMs was developed to encompass all major branches of the JmjC phylogenetic tree. These assays compare compound activity against wild-type KDM proteins to a catalytically inactive version of the KDM, in which residues involved in the active-site iron coordination are mutated to inactivate the enzyme activity. These mutants are critical for assessing the specific effect of KDM inhibitors and for revealing indirect effects on histone methylation status. The reported assays make use of ectopically expressed demethylases, and we demonstrate their use to profile several recently identified classes of KDM inhibitors and their structurally matched inactive controls. The generated data correlate well with assay results assessing endogenous KDM inhibition and confirm the selectivity observed in biochemical assays with isolated enzymes. We find that both cellular permeability and competition with 2-oxoglutarate affect the translation of biochemical activity to cellular inhibition.
Conclusions: High-content-based immunofluorescence assays have been established for eight KDM members of the 2-oxoglutarate-dependent oxygenases covering all major branches of the JmjC-KDM phylogenetic tree. The usage of both full-length, wild-type and catalytically inactive mutant ectopically expressed protein, as well as structure-matched inactive control compounds, allowed for detection of nonspecific effects causing changes in histone methylation as a result of compound toxicity. The developed assays offer a histone lysine demethylase family-wide tool for assessing KDM inhibitors for cell activity and on-target efficacy. In addition, the presented data may inform further studies to assess the cell-based activity of histone lysine methylation inhibitors.
ATP-binding cassette (ABC) systems translocate a wide range of solutes across cellular membranes. The thermophilic Gram-negative eubacterium Thermus thermophilus, a model organism for structural genomics and systems biology, discloses ∼46 ABC proteins, which are largely uncharacterized. Here, we functionally analyzed the first two and only ABC half-transporters of the hyperthermophilic bacterium, TmrA and TmrB. The ABC system mediates uptake of the drug Hoechst 33342 in inside-out oriented vesicles that is inhibited by verapamil. TmrA and TmrB form a stable heterodimeric complex hydrolyzing ATP with a Km of 0.9 mm and kcat of 9 s−1 at 68 °C. Two nucleotides can be trapped in the heterodimeric ABC complex either by vanadate or by mutation inhibiting ATP hydrolysis. Nucleotide trapping requires permissive temperatures, at which a conformational ATP switch is possible. We further demonstrate that the canonic glutamate 523 of TmrA is essential for rapid conversion of the ATP/ATP-bound complex into its ADP/ATP state, whereas the corresponding aspartate in TmrB (Asp-500) has only a regulatory role. Notably, exchange of this single noncanonic residue into a catalytic glutamate cannot rescue the function of the E523Q/D500E complex, implicating a built-in asymmetry of the complex. However, slow ATP hydrolysis in the newly generated canonic site (D500E) strictly depends on the formation of a posthydrolysis state in the consensus site, indicating an allosteric coupling of both active sites.
We have investigated the mechanism responsible for half-of-the-sites activity in the dimeric cytochrome bc(1) complex from Paracoccus denitrificans by characterizing the kinetics of inhibitor binding to the ubiquinol oxidation site at center P. Both myxothiazol and stigmatellin induced a 2-3 nm shift of the visible absorbance spectrum of the b(L) heme. The shift generated by myxothiazol was symmetric, with monophasic kinetics that indicate equal binding of this inhibitor to both center P sites. In contrast, stigmatellin generated an asymmetric shift in the b(L) spectrum, with biphasic kinetics in which each phase contributed approximately half of the total magnitude of the spectral change. The faster binding phase corresponded to a more symmetrical shift of the b(L) spectrum relative to the slower binding phase, indicating that approximately half of the center P sites bound stigmatellin more slowly and in a different position relative to the b(L) heme, generating a different effect on its electronic environment. Significantly, the slow stigmatellin binding phase was lost as the inhibitor concentration was increased. This implies that a conformational change is transmitted from one center P site in the dimer to the other upon stigmatellin binding to one monomer, rendering the second site less accessible to the inhibitor. Because the position that stigmatellin occupies at center P is considered to be analogous to that of the quinol substrate at the moment of electron transfer, these results indicate that the productive enzyme-substrate configuration is prevented from occurring in both monomers simultaneously.
The degradation of nonfunctional mitochondrial proteins is of fundamental relevance for maintenance of cellular homeostasis. The heteromeric CLPXP protein complex in the mitochondrial matrix is part of this process. In the fungal aging model Podospora anserina, ablation of CLPXP leads to an increase in healthy lifespan. Here, we report that this counterintuitive increase depends on a functional autophagy machinery. In PaClpXP mutants, autophagy is involved in energy conservation and the compensation of impairments in respiration. Strikingly, despite the impact on mitochondrial function, it is not mitophagy but general autophagy that is constitutively induced and required for longevity. In contrast, in another long-lived mutant ablated for the mitochondrial PaIAP protease, autophagy is neither induced nor required for lifespan extension. Our data provide novel mechanistic insights into the capacity of different forms of autophagy to compensate impairments of specific components of the complex mitochondrial quality control network and about the biological role of mitochondrial CLPXP in the control of cellular energy metabolism.
Target-specific treatment modalities are currently not available for triple-negative breast cancer (TNBC), and acquired chemotherapy resistance is a primary obstacle for the treatment of these tumors. Here we employed derivatives of BT-549 and MDA-MB-468 TNBC cell lines that were adapted to grow in the presence of either 5-Fluorouracil, Doxorubicin or Docetaxel in an aim to identify molecular pathways involved in the adaptation to drug-induced cell killing. All six drug-adapted BT-549 and MDA-MB-468 cell lines displayed cross resistance to chemotherapy and decreased apoptosis sensitivity. Expression of the anti-apoptotic co-chaperone BAG3 was notably enhanced in two thirds (4/6) of the six resistant lines simultaneously with higher expression of HSP70 in comparison to parental controls. Doxorubicin-resistant BT-549 (BT-549rDOX20) and 5-Fluorouracil-resistant MDA-MB-468 (MDA-MB-468r5-FU2000) cells were chosen for further analysis with the autophagy inhibitor Bafilomycin A1 and lentiviral depletion of ATG5, indicating that enhanced cytoprotective autophagy partially contributes to increased drug resistance and cell survival. Stable lentiviral BAG3 depletion was associated with a robust down-regulation of Mcl-1, Bcl-2 and Bcl-xL, restoration of drug-induced apoptosis and reduced cell adhesion in these cells, and these death-sensitizing effects could be mimicked with the BAG3/Hsp70 interaction inhibitor YM-1 and by KRIBB11, a selective transcriptional inhibitor of HSF-1. Furthermore, BAG3 depletion was able to revert the EMT-like transcriptional changes observed in BT-549rDOX20 and MDA-MB-468r5-FU2000 cells. In summary, genetic and pharmacological interference with BAG3 is capable to resensitize TNBC cells to treatment, underscoring its relevance for cell death resistance and as a target to overcome therapy resistance of breast cancer.
Isolated complex I deficiency is a common biochemical phenotype observed in pediatric mitochondrial disease and often arises as a consequence of pathogenic variants affecting one of the ∼65 genes encoding the complex I structural subunits or assembly factors. Such genetic heterogeneity means that application of next-generation sequencing technologies to undiagnosed cohorts has been a catalyst for genetic diagnosis and gene-disease associations. We describe the clinical and molecular genetic investigations of four unrelated children who presented with neuroradiological findings and/or elevated lactate levels, highly suggestive of an underlying mitochondrial diagnosis. Next-generation sequencing identified bi-allelic variants in NDUFA6, encoding a 15 kDa LYR-motif-containing complex I subunit that forms part of the Q-module. Functional investigations using subjects’ fibroblast cell lines demonstrated complex I assembly defects, which were characterized in detail by mass-spectrometry-based complexome profiling. This confirmed a marked reduction in incorporated NDUFA6 and a concomitant reduction in other Q-module subunits, including NDUFAB1, NDUFA7, and NDUFA12. Lentiviral transduction of subjects’ fibroblasts showed normalization of complex I. These data also support supercomplex formation, whereby the ∼830 kDa complex I intermediate (consisting of the P- and Q-modules) is in complex with assembled complex III and IV holoenzymes despite lacking the N-module. Interestingly, RNA-sequencing data provided evidence that the consensus RefSeq accession number does not correspond to the predominant transcript in clinically relevant tissues, prompting revision of the NDUFA6 RefSeq transcript and highlighting not only the importance of thorough variant interpretation but also the assessment of appropriate transcripts for analysis.
Biallelic mutations in TMEM126B cause severe complex i deficiency with a variable clinical phenotype
(2016)
Complex I deficiency is the most common biochemical phenotype observed in individuals with mitochondrial disease. With 44 structural subunits and over 10 assembly factors, it is unsurprising that complex I deficiency is associated with clinical and genetic heterogeneity. Massively parallel sequencing (MPS) technologies including custom, targeted gene panels or unbiased whole-exome sequencing (WES) are hugely powerful in identifying the underlying genetic defect in a clinical diagnostic setting, yet many individuals remain without a genetic diagnosis. These individuals might harbor mutations in poorly understood or uncharacterized genes, and their diagnosis relies upon characterization of these orphan genes. Complexome profiling recently identified TMEM126B as a component of the mitochondrial complex I assembly complex alongside proteins ACAD9, ECSIT, NDUFAF1, and TIMMDC1. Here, we describe the clinical, biochemical, and molecular findings in six cases of mitochondrial disease from four unrelated families affected by biallelic (c.635G>T [p.Gly212Val] and/or c.401delA [p.Asn134Ilefs∗2]) TMEM126B variants. We provide functional evidence to support the pathogenicity of these TMEM126B variants, including evidence of founder effects for both variants, and establish defects within this gene as a cause of complex I deficiency in association with either pure myopathy in adulthood or, in one individual, a severe multisystem presentation (chronic renal failure and cardiomyopathy) in infancy. Functional experimentation including viral rescue and complexome profiling of subject cell lines has confirmed TMEM126B as the tenth complex I assembly factor associated with human disease and validates the importance of both genome-wide sequencing and proteomic approaches in characterizing disease-associated genes whose physiological roles have been previously undetermined.
Rpn13 is an intrinsic ubiquitin receptor of the 26S proteasome regulatory subunit that facilitates substrate capture prior to degradation. Here we show that the C-terminal region of Rpn13 binds to the tetratricopeptide repeat (TPR) domain of SGTA, a cytosolic factor implicated in the quality control of mislocalised membrane proteins (MLPs). The overexpression of SGTA results in a substantial increase in steady-state MLP levels, consistent with an effect on proteasomal degradation. However, this effect is strongly dependent upon the interaction of SGTA with the proteasomal component Rpn13. Hence, overexpression of the SGTA-binding region of Rpn13 or point mutations within the SGTA TPR domain both inhibit SGTA binding to the proteasome and substantially reduce MLP levels. These findings suggest that SGTA can regulate the access of MLPs to the proteolytic core of the proteasome, implying that a protein quality control cycle that involves SGTA and the BAG6 complex can operate at the 19S regulatory particle. We speculate that the binding of SGTA to Rpn13 enables specific polypeptides to escape proteasomal degradation and/or selectively modulates substrate degradation.
Bone marrow and plasma FGF‐23 in heart failure patients : novel insights into the heart–bone axis
(2019)
Aims: Fibroblast growth factor 23 (FGF‐23) is known to be elevated in patients with congestive heart failure (CHF). As FGF‐23 is expressed in the bone but can also be expressed in the myocardium, the origin of serum FGF‐23 in CHF remains unclear. It is also unclear if FGF‐23 expressed in the bone is associated with outcome in CHF. The aim of the present study was to investigate FGF‐23 levels measured in bone marrow plasma (FGF‐23‐BM) and in peripheral blood (FGF‐23‐P) in CHF patients to gain further insights into the heart–bone axis of FGF‐23 expression. We also investigated possible associations between FGF‐23‐BM as well as FGF‐23‐P and outcome in CHF patients.
Methods and results: We determined FGF‐23‐P and FGF‐23‐BM levels in 203 CHF patients (85% male, mean age 61.3 years) with a left ventricular ejection fraction (LVEF) ≤45% and compared them with those of 48 healthy controls (48% male, mean age 39.2 years). We investigated the association between FGF‐23‐BM and FGF‐23‐P with all‐cause mortality in CHF patients, 32 events, median follow‐up 1673 days, interquartile range [923, 1828]. FGF‐23‐P (median 60.3 vs. 22.0 RU/mL, P < 0.001) and FGF‐23‐BM (median 130.7 vs. 93.1 RU/mL, P < 0.001) levels were higher in CHF patients compared with healthy controls. FGF‐23‐BM levels were significantly higher than FGF‐23‐P levels in both CHF patients and in healthy controls (P < 0.001). FGF‐23‐P and FGF‐23‐BM correlated significantly with LVEF (r = −0.37 and r = −0.33, respectively), N terminal pro brain natriuretic peptide levels (r = 0.57 and r = 0.6, respectively), New York Heart Association status (r = 0.28 and r = 0.25, respectively), and estimated glomerular filtration rate (r = −0.43 and r = −0.41, respectively) (P for all <0.001) and were independently associated with all‐cause mortality in CHF patients after adjustment for LVEF, estimated glomerular filtration rate, New York Heart Association status, and N terminal pro brain natriuretic peptide, hazard ratio 2.71 [confidence interval: 1.18–6.20], P = 0.018, and hazard ratio 2.80 [confidence interval: 1.19–6.57], P = 0.018, respectively.
Conclusions: In CHF patients, FGF‐23 is elevated in bone marrow plasma and is independently associated with heart failure severity and all‐cause mortality. The failing heart seems to interact via FGF‐23 within a heart–bone axis.
Broad AOX expression in a genetically tractable mouse model does not disturb normal physiology
(2017)
Plants and many lower organisms, but not mammals, express alternative oxidases (AOXs) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell lines, Drosophila disease models and, most recently, in the mouse, where multiple lentivector-AOX transgenes conferred substantial expression in specific tissues. Here, we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The AOXRosa26 mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello; moreover, animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOXRosa26 mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo.
Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.
Endangered species of hosts are coupled with endangered species of parasites, which share the risk of co-extinction. Conservation efforts sometimes include breeding of rare species in captivity. Data on parasites of captive populations of endangered species is scarce and the ability of small numbers of captive host individuals to support the biodiversity of native parasites is limited. Examination of ectosymbionts of the critically endangered Philippine eagles and the endangered Mindanao Hawk-Eagle kept at the Philippine Eagle Center, Philippines, revealed three feather mite species despite regular treatment with insecticide powder. No other ectosymbiont taxa were detected. Studies in morphology and molecular phylogeny of these feather mites based on mitochondrial and nuclear DNA markers indicate that species found were typical for Accipitridae. Three new pterolichoid feather mite species (Acari: Pterolichoidea) were described from two species of eagles (Accipitriformes: Accipitridae) endemic to the Philippines: Hieracolichus philippinensis sp. n. (Gabuciniidae) and Pseudalloptinus pithecophagae sp. n. (Pterolichidae) from the Great Philippine Eagle Pithecophaga jefferyi Ogilvie-Grant, 1896, and Pseudogabucinia nisaeti sp. n. (Kramerellidae) from the Mindanao Hawk-Eagle Nisaetus pinskeri Gould, 1863. The presence of H. philippinensis on P. jefferyi supports the recent finding that the Great Philippine Eagle belongs to the lineage of serpent eagles (Circaetinae) rather than to the Harpy and other eagles.
Heart and vessels form a highly complex organ system in which extremely diverse cells have to work together correctly to provide all organs with blood. In past decades, heart biology placed its focus on whole tissues or cell isolates. Now, however, new technologies allow the tracing of a diversity of cell types and their individual responses to signals down to the level of proteins and genes. Researchers hope this will help them better support the regeneration of diseased hearts.
SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining individual-resolution cross-linking and immunoprecipitation (iCLIP) and mass spectrometry, we show that elevated arginine methylation of SRSF5 and lower phosphorylation levels of cobound SRSF2 enhance shuttling of SRSF5 in P19 cells by modulating protein-protein and protein-RNA interactions. Moreover, SRSF5 is bound to pluripotency-specific transcripts such as Lin28a and Pou5f1/Oct4 in the cytoplasm. SRSF5 depletion reduces and overexpression increases their cytoplasmic mRNA levels, suggesting that enhanced mRNA export by SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19 cells leads to dramatically reduced SRSF5 shuttling. Our findings indicate that posttranslational modification of SR proteins underlies the regulation of their mRNA export activities and distinguishes pluripotent from differentiated cells.
Mechanisms regulating protein degradation ensure the correct and timely expression of transcription factors such as hypoxia inducible factor (HIF). Under normal O2 tension, HIFα subunits are targeted for proteasomal degradation, mainly through vHL-dependent ubiquitylation. Deubiquitylases are responsible for reversing this process. Although the mechanism and regulation of HIFα by ubiquitin-dependent proteasomal degradation has been the object of many studies, little is known about the role of deubiquitylases. Here, we show that expression of HIF2α (encoded by EPAS1) is regulated by the deubiquitylase Cezanne (also known as OTUD7B) in an E2F1-dependent manner. Knockdown of Cezanne downregulates HIF2α mRNA, protein and activity independently of hypoxia and proteasomal degradation. Mechanistically, expression of the HIF2α gene is controlled directly by E2F1, and Cezanne regulates the stability of E2F1. Exogenous E2F1 can rescue HIF2α transcript and protein expression when Cezanne is depleted. Taken together, these data reveal a novel mechanism for the regulation of the expression of HIF2α, demonstrating that the HIF2α promoter is regulated by E2F1 directly and that Cezanne regulates HIF2α expression through control of E2F1 levels. Our results thus suggest that HIF2α is controlled transcriptionally in a cell-cycle-dependent manner and in response to oncogenic signalling.
Cells respond to protein misfolding and aggregation in the cytosol by adjusting gene transcription and a number of post-transcriptional processes. In parallel to functional reactions, cellular structure changes as well; however, the mechanisms underlying the early adaptation of cellular compartments to cytosolic protein misfolding are less clear. Here we show that the mammalian ubiquitin ligase C-terminal Hsp70-interacting protein (CHIP), if freed from chaperones during acute stress, can dock on cellular membranes thus performing a proteostasis sensor function. We reconstituted this process in vitro and found that mainly phosphatidic acid and phosphatidylinositol-4-phosphate enhance association of chaperone-free CHIP with liposomes. HSP70 and membranes compete for mutually exclusive binding to the tetratricopeptide repeat domain of CHIP. At new cellular locations, access to compartment-specific substrates would enable CHIP to participate in the reorganization of the respective organelles, as exemplified by the fragmentation of the Golgi apparatus (effector function).
Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.
We demonstrate high-density labelling of cellular DNA and RNA using click chemistry and perform confocal and super-resolution microscopy. We visualize the crescent and ring-like structure of densely packed RNA in nucleoli. We further demonstrate click chemistry with unnatural amino acids for super-resolution imaging of outer-membrane proteins of E. coli.
The yeast bc1 complex (complex III) and cytochrome oxidase (complex IV) are mosaics of core subunits encoded by the mitochondrial genome and additional nuclear-encoded proteins imported from the cytosol. Both complexes build in the mitochondrial inner membrane various supramolecular assemblies. The formation of the individual complexes and their supercomplexes depends on the activity of dedicated assembly factors. We identified a so far uncharacterized mitochondrial protein (open reading frame YDR381C-A) as an important assembly factor for complex III, complex IV, and their supercomplexes. Therefore, we named this protein Cox interacting (Coi) 1. Deletion of COI1 results in decreased respiratory growth, reduced membrane potential, and hampered respiration, as well as slow fermentative growth at low temperature. In addition, coi1Δ cells harbour reduced steady-state levels of subunits of complexes III and IV as well as of the assembled complexes and supercomplexes. Interaction of Coi1 with respiratory chain subunits seems transient, as it appears to be a stoichiometric subunit neither of complex III nor of complex IV. Collectively, this work identifies a novel protein that plays a role in the assembly of the mitochondrial respiratory chain.
Motivation: Complexome profiling combines native gel electrophoresis with mass spectrometry to obtain the inventory, composition and abundance of multiprotein assemblies in an organelle. Applying complexome profiling to determine the effect of a mutation on protein complexes requires separating technical and biological variations from the variations caused by that mutation.
Results: We have developed the COmplexome Profiling ALignment (COPAL) tool that aligns multiple complexome profiles with each other. It includes the abundance profiles of all proteins on two gels, using a multi-dimensional implementation of the dynamic time warping algorithm to align the gels. Subsequent progressive alignment allows us to align multiple profiles with each other. We tested COPAL on complexome profiles from control mitochondria and from Barth syndrome (BTHS) mitochondria, which have a mutation in tafazzin gene that is involved in remodeling the inner mitochondrial membrane phospholipid cardiolipin. By comparing the variation between BTHS mitochondria and controls with the variation among either, we assessed the effects of BTHS on the abundance profiles of individual proteins. Combining those profiles with gene set enrichment analysis allows detecting significantly affected protein complexes. Most of the significantly affected protein complexes are located in the inner mitochondrial membrane (mitochondrial contact site and cristae organizing system, prohibitins), or are attached to it (the large ribosomal subunit).
Availability and implementation: COPAL is written in python and is available from http://github.com/cmbi/copal.
Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3′-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level.
The inner boundary and the cristae membrane are connected by pore-like structures termed crista junctions (CJs). The MICOS complex is required for CJ formation and enriched at CJs. Here, we address the roles of the MICOS subunits Mic27 and Mic10. We observe a positive genetic interaction between Mic27 and Mic60 and deletion of Mic27 results in impaired formation of CJs and altered cristae membrane curvature. Mic27 acts in an antagonistic manner to Mic60 as it promotes oligomerization of the F1FO-ATP synthase and partially restores CJ formation in cells lacking Mic60. Mic10 impairs oligomerization of the F1FO-ATP synthase similar to Mic60. Applying complexome profiling, we observed that deletion of Mic27 destabilizes the MICOS complex but does not impair formation of a high molecular weight Mic10 subcomplex. Moreover, this Mic10 subcomplex comigrates with the dimeric F1FO-ATP synthase in a Mic27-independent manner. Further, we observed a chemical crosslink of Mic10 to Mic27 and of Mic10 to the F1FO-ATP synthase subunit e. We corroborate the physical interaction of the MICOS complex and the F1FO-ATP synthase. We propose a model in which part of the F1FO-ATP synthase is linked to the MICOS complex via Mic10 and Mic27 and by that is regulating CJ formation.
Mitochondrial complex I has a key role in cellular energy metabolism, generating a major portion of the proton motive force that drives aerobic ATP synthesis. The hydrophilic arm of the L-shaped ~1 MDa membrane protein complex transfers electrons from NADH to ubiquinone, providing the energy to drive proton pumping at distant sites in the membrane arm. The critical steps of energy conversion are associated with the redox chemistry of ubiquinone. We report the cryo-EM structure of complete mitochondrial complex I from the aerobic yeast Yarrowia lipolytica both in the deactive form and after capturing the enzyme during steady-state activity. The site of ubiquinone binding observed during turnover supports a two-state stabilization change mechanism for complex I.
Crystallization and X-ray diffraction studies of a complete bacterial fatty-acid synthase type I
(2015)
While a deep understanding of the fungal and mammalian multi-enzyme type I fatty-acid synthases (FAS I) has been achieved in recent years, the bacterial FAS I family, which is narrowly distributed within the Actinomycetales genera Mycobacterium, Corynebacterium and Nocardia, is still poorly understood. This is of particular relevance for two reasons: (i) although homologous to fungal FAS I, cryo-electron microscopic studies have shown that bacterial FAS I has unique structural and functional properties, and (ii) M. tuberculosis FAS I is a drug target for the therapeutic treatment of tuberculosis (TB) and therefore is of extraordinary importance as a drug target. Crystals of FAS I from C. efficiens, a homologue of M. tuberculosis FAS I, were produced and diffracted X-rays to about 4.5 Å resolution.
Biogenesis of mitochondrial cytochrome c oxidase (COX) is a complex process involving the coordinate expression and assembly of numerous subunits (SU) of dual genetic origin. Moreover, several auxiliary factors are required to recruit and insert the redox-active metal compounds, which in most cases are buried in their protein scaffold deep inside the membrane. Here we used a combination of gel electrophoresis and pull-down assay techniques in conjunction with immunostaining as well as complexome profiling to identify and analyze the composition of assembly intermediates in solubilized membranes of the bacterium Paracoccus denitrificans. Our results show that the central SUI passes through at least three intermediate complexes with distinct subunit and cofactor composition before formation of the holoenzyme and its subsequent integration into supercomplexes. We propose a model for COX biogenesis in which maturation of newly translated COX SUI is initially assisted by CtaG, a chaperone implicated in CuB site metallation, followed by the interaction with the heme chaperone Surf1c to populate the redox-active metal-heme centers in SUI. Only then the remaining smaller subunits are recruited to form the mature enzyme which ultimately associates with respiratory complexes I and III into supercomplexes.
Mutations causing aberrant splicing are frequently implicated in human diseases including cancer. Here, we establish a high-throughput screen of randomly mutated minigenes to decode the cis-regulatory landscape that determines alternative splicing of exon 11 in the proto-oncogene MST1R (RON). Mathematical modelling of splicing kinetics enables us to identify more than 1000 mutations affecting RON exon 11 skipping, which corresponds to the pathological isoform RON∆165. Importantly, the effects correlate with RON alternative splicing in cancer patients bearing the same mutations. Moreover, we highlight heterogeneous nuclear ribonucleoprotein H (HNRNPH) as a key regulator of RON splicing in healthy tissues and cancer. Using iCLIP and synergy analysis, we pinpoint the functionally most relevant HNRNPH binding sites and demonstrate how cooperative HNRNPH binding facilitates a splicing switch of RON exon 11. Our results thereby offer insights into splicing regulation and the impact of mutations on alternative splicing in cancer.
Recently, the conserved intracellular digestion mechanism ‘autophagy’ has been considered to be involved in early tumorigenesis and its blockade proposed as an alternative treatment approach. However, there is an ongoing debate about whether blocking autophagy has positive or negative effects in tumor cells. Since there is only poor data about the clinico-pathological relevance of autophagy in gliomas in vivo, we first established a cell culture based platform for the in vivo detection of the autophago-lysosomal components. We then investigated key autophagosomal (LC3B, p62, BAG3, Beclin1) and lysosomal (CTSB, LAMP2) molecules in 350 gliomas using immunohistochemistry, immunofluorescence, immunoblotting and qPCR. Autophagy was induced pharmacologically or by altering oxygen and nutrient levels. Our results show that autophagy is enhanced in astrocytomas as compared to normal CNS tissue, but largely independent from the WHO grade and patient survival. A strong upregulation of LC3B, p62, LAMP2 and CTSB was detected in perinecrotic areas in glioblastomas suggesting micro-environmental changes as a driver of autophagy induction in gliomas. Furthermore, glucose restriction induced autophagy in a concentration-dependent manner while hypoxia or amino acid starvation had considerably lesser effects. Apoptosis and autophagy were separately induced in glioma cells both in vitro and in vivo. In conclusion, our findings indicate that autophagy in gliomas is rather driven by micro-environmental changes than by primary glioma-intrinsic features thus challenging the concept of exploitation of the autophago-lysosomal network (ALN) as a treatment approach in gliomas.
A dozen mRNAs are edited by multiple insertions and/or deletions of uridine residues in the mitochondrion of Trypanosoma brucei. Several protein complexes have been implicated in performing this type of RNA editing, including the mitochondrial RNA-binding complex 1 (MRB1). Two paralogous novel RNA-binding proteins, MRB8170 and MRB4160, are loosely associated with the core MRB1 complex. Their roles in RNA editing and effects on target mRNAs are so far not well understood. In this study, individual-nucleotide-resolution UV-cross-linking and affinity purification (iCLAP) revealed a preferential binding of both proteins to mitochondrial mRNAs, which was positively correlated with their extent of editing. Integrating additional in vivo and in vitro data, we propose that binding of MRB8170 and/or MRB4160 onto pre-mRNA marks it for the initiation of editing and that initial binding of both proteins may facilitate the recruitment of other components of the RNA editing/processing machinery to ensure efficient editing. Surprisingly, MRB8170 also binds never-edited mRNAs, suggesting that at least this paralog has an additional role outside RNA editing to shape the mitochondrial transcriptome.
We previously proposed that the dimeric cytochrome bc(1) complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc(1) monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289-22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc(1) complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10-16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c(1) by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wild-type dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.
In prokaryotes, RNA thermometers regulate a number of heat shock and virulence genes. These temperature sensitive RNA elements are usually located in the 5'-untranslated regions of the regulated genes. They repress translation initiation by base pairing to the Shine–Dalgarno sequence at low temperatures. We investigated the thermodynamic stability of the temperature labile hairpin 2 of the Salmonella fourU RNA thermometer over a broad temperature range and determined free energy, enthalpy and entropy values for the base-pair opening of individual nucleobases by measuring the temperature dependence of the imino proton exchange rates via NMR spectroscopy. Exchange rates were analyzed for the wild-type (wt) RNA and the A8C mutant. The wt RNA was found to be stabilized by the extraordinarily stable G14–C25 base pair. The mismatch base pair in the wt RNA thermometer (A8–G31) is responsible for the smaller cooperativity of the unfolding transition in the wt RNA. Enthalpy and entropy values for the base-pair opening events exhibit linear correlation for both RNAs. The slopes of these correlations coincide with the melting points of the RNAs determined by CD spectroscopy. RNA unfolding occurs at a temperature where all nucleobases have equal thermodynamic stabilities. Our results are in agreement with a consecutive zipper-type unfolding mechanism in which the stacking interaction is responsible for the observed cooperativity. Furthermore, remote effects of the A8C mutation affecting the stability of nucleobase G14 could be identified. According to our analysis we deduce that this effect is most probably transduced via the hydration shell of the RNA.
In mammalian species, including humans, the hippocampal dentate gyrus (DG) is a primary region of adult neurogenesis. Aberrant adult hippocampal neurogenesis is associated with neurological pathologies. Understanding the cellular mechanisms controlling adult hippocampal neurogenesis is expected to open new therapeutic strategies for mental disorders. Microglia is intimately associated with neural progenitor cells in the hippocampal DG and has been implicated, under varying experimental conditions, in the control of the proliferation, differentiation and survival of neural precursor cells. But the underlying mechanisms remain poorly defined. Using fluorescent in situ hybridization we show that microglia in brain express the ADP-activated P2Y13 receptor under basal conditions and that P2ry13 mRNA is absent from neurons, astrocytes, and neural progenitor cells. Disrupting P2ry13 decreases structural complexity of microglia in the hippocampal subgranular zone (SGZ). But it increases progenitor cell proliferation and new neuron formation. Our data suggest that P2Y13 receptor-activated microglia constitutively attenuate hippocampal neurogenesis. This identifies a signaling pathway whereby microglia, via a nucleotide-mediated mechanism, contribute to the homeostatic control of adult hippocampal neurogenesis. Selective P2Y13R antagonists could boost neurogenesis in pathological conditions associated with impaired hippocampal neurogenesis.
DNA damage in oocytes induces a switch of the quality control factor TAp63α from dimer to tetramer
(2011)
TAp63a, a homolog of the p53 tumor suppressor, is a quality control factor in the female germline. Remarkably, already undamaged oocytes express high levels of the protein, suggesting that TAp63a’s activity is under tight control of an inhibitory mechanism. Biochemical studies have proposed that inhibition requires the C-terminal transactivation inhibitory domain. However, the structural mechanism of TAp63a inhibition remains unknown. Here, we show that TAp63a is kept in an inactive dimeric state. We reveal that relief of inhibition leads to tetramer formation with ~20-fold higher DNA affinity. In vivo, phosphorylation-triggered tetramerization of TAp63a is not reversible by dephosphorylation. Furthermore, we show that a helix in the oligomerization domain of p63 is crucial for tetramer stabilization and competes with the transactivation domain for the same binding site. Our results demonstrate how TAp63a is inhibited by complex domain-domain interactions that provide the basis for regulating quality control in oocytes.
Spheroids resemble features of tissues and serve as model systems to study cell–cell and cell–ECM interactions in non-adhesive three-dimensional environments. Although it is generally accepted that mature spheroids resemble tissue properties very well, no studies relate different phases in the spheroid formation processes that contribute to tissue integrity. Tissue integrity involves the cellular processes adhesion formation, adhesion reinforcement, rearrangement as well as proliferation. They maintain the structure and function of tissues and, upon dysregulation, contribute to malignancy. We investigated spheroid formation dynamics in cell lines of different metastatic potential. We dissected spheroid formation into phases of aggregation, compaction and growth to identify the respective contributions of E-cadherin, actin, microtubules and FAK. E-cadherin, actin and microtubules drive the first two phases. Microtubules and FAK are involved in the proliferation phase. FAK activity correlates with the metastatic potential of the cells. A robust computational model based on a very large number of experiments reveals the temporal resolution of cell adhesion. Our results provide novel hypotheses to unveil the general mechanisms that contribute to tissue integrity.
The discovery of antibiotics represented a key milestone in the history of medicine. However, with the rise of these life-saving drugs came the awareness that bacteria deploy defense mechanisms to resist these antibiotics, and they are good at it. Today, we appear at a crossroads between discovery of new potent drugs and omni-resistant superbugs. Moreover, the misuse of antibiotics in different industries has increased the rate of resistance development by providing permanent selective pressure and, subsequently, enrichment of multidrug resistant pathogens. As a result, antimicrobial resistance has now become an urgent threat to public health worldwide (http:// www.who.int/drugresistance/documents/surveillancereport/en/). The development of multidrug resistance (MDR) in an increasing number of pathogens, including Pseudomonas, Acinetobacter, Klebsiella, Salmonella, Burkholderia, and other Gram-negative bacteria is a serious issue. Membrane efflux pump complexes of the Resistance-Nodulation-Division (RND) superfamily play a key role in the development of MDR in these bacteria. These pumps, together with other transporters, contribute to intrinsic and acquired resistance of bacteria toward most, if not all, of the compounds available in our antimicrobial arsenal. Given the enormous drug polyspecificity of MDR efflux pumps, studies on their mechanism of action are extremely challenging, and this has negatively impacted both on the development of new antibiotics that are able to evade these efflux pumps and on the design of pump inhibitors. The collection of articles in this eBook, published as a Research Topic in Frontiers in Microbiology, section of Antimicrobials, Resistance, and Chemotherapy, aims to update the reader about the latest advances on the structure and function of RND efflux transporters, their roles in the overall multidrug resistance phenotype of Gram-negative pathogens, and on the strategies to inhibit their activities. ...
Ein Zell-Atlas des kranken Herzens : Einzelzelltechniken ermöglichen neue Einsichten auf Zellebene
(2019)
Herz und Gefäße bilden ein hochkomplexes Organsystem, in dem unterschiedlichste Zellen korrekt zusammenarbeiten müssen, um alle Organe mit Blut zu versorgen. In den vergangenen Jahrzehnten hat die Herzbiologie ganze Gewebe oder Zellisolate in den Blick genommen. Doch jetzt erlauben neue Technologien, die Vielfalt der Zelltypen und ihre individuelle Antwort auf Signale bis auf die Ebene von Proteinen und Genen zu verfolgen. Forscher hoffen, kranken Herzen dadurch besser bei der Regeneration helfen zu können.
Endogenous AJAP1 associates with the cytoskeleton and attenuates angiogenesis in endothelial cells
(2017)
The adherens junction associated protein 1 (AJAP1, aka shrew-1) is presumably a type-I transmembrane protein localizing and interacting with the E-cadherin-catenin complex. In various tumors, AJAP1 expression is reduced or lost, including hepatocellular and esophageal squamous cell carcinoma, and glial-derived tumors. The aberrant expression of AJAP1 is associated with alterations in cell migration, invasion, increased tumor growth, and tumor vascularization, suggesting AJAP1 as a putative tumor suppressor. We show that AJAP1 attenuates sprouting angiogenesis by reducing endothelial migration and invasion capacities. Further, we show for the first time that endogenous AJAP1 is associated with the microtubule cytoskeleton. This linkage is independent from cell confluency and stable during angiogenic sprouting in vitro. Our work suggests that AJAP1 is a putative negative regulator of angiogenesis, reducing cell migration and invasion by interfering with the microtubule network. Based on our results and those of other authors, we suggest AJAP1 as a novel tumor suppressor and diagnostic marker.
Endothelial to mesenchymal transition in cardiovascular disease : JACC state-of-the-art review
(2019)
Endothelial to mesenchymal transition (EndMT) is a process whereby an endothelial cell undergoes a series of molecular events that lead to a change in phenotype toward a mesenchymal cell (e.g., myofibroblast, smooth muscle cell). EndMT plays a fundamental role during development, and mounting evidence indicates that EndMT is involved in adult cardiovascular diseases (CVDs), including atherosclerosis, pulmonary hypertension, valvular disease, and fibroelastosis. Therefore, the targeting of EndMT may hold therapeutic promise for treating CVD. However, the field faces a number of challenges, including the lack of a precise functional and molecular definition, a lack of understanding of the causative pathological role of EndMT in CVDs (versus being a “bystander-phenomenon”), and a lack of robust human data corroborating the extent and causality of EndMT in adult CVDs. Here, we review this emerging but exciting field, and propose a framework for its systematic advancement at the molecular and translational levels.
Fatty acids (FAs) are considered strategically important platform compounds that can be accessed by sustainable microbial approaches. Here we report the reprogramming of chain-length control of Saccharomyces cerevisiae fatty acid synthase (FAS). Aiming for short-chain FAs (SCFAs) producing baker’s yeast, we perform a highly rational and minimally invasive protein engineering approach that leaves the molecular mechanisms of FASs unchanged. Finally, we identify five mutations that can turn baker’s yeast into a SCFA producing system. Without any further pathway engineering, we achieve yields in extracellular concentrations of SCFAs, mainly hexanoic acid (C6-FA) and octanoic acid (C8-FA), of 464 mg l−1 in total. Furthermore, we succeed in the specific production of C6- or C8-FA in extracellular concentrations of 72 and 245 mg l−1, respectively. The presented technology is applicable far beyond baker’s yeast, and can be plugged into essentially all currently available FA overproducing microorganisms.
Diffuse invasion of the surrounding brain parenchyma is a major obstacle in the treatment of gliomas with various therapeutics, including anti-angiogenic agents. Here we identify the epi-/genetic and microenvironmental downregulation of ephrinB2 as a crucial step that promotes tumour invasion by abrogation of repulsive signals. We demonstrate that ephrinB2 is downregulated in human gliomas as a consequence of promoter hypermethylation and gene deletion. Consistently, genetic deletion of ephrinB2 in a murine high-grade glioma model increases invasion. Importantly, ephrinB2 gene silencing is complemented by a hypoxia-induced transcriptional repression. Mechanistically, hypoxia-inducible factor (HIF)-1α induces the EMT repressor ZEB2, which directly downregulates ephrinB2 through promoter binding to enhance tumour invasiveness. This mechanism is activated following anti-angiogenic treatment of gliomas and is efficiently blocked by disrupting ZEB2 activity. Taken together, our results identify ZEB2 as an attractive therapeutic target to inhibit tumour invasion and counteract tumour resistance mechanisms induced by anti-angiogenic treatment strategies.
Despite advances in myocardial reperfusion therapies, acute myocardial ischaemia/reperfusion injury and consequent ischaemic heart failure represent the number one cause of morbidity and mortality in industrialized societies. Although different therapeutic interventions have been shown beneficial in preclinical settings, an effective cardioprotective or regenerative therapy has yet to be successfully introduced in the clinical arena. Given the complex pathophysiology of the ischaemic heart, large scale, unbiased, global approaches capable of identifying multiple branches of the signalling networks activated in the ischaemic/reperfused heart might be more successful in the search for novel diagnostic or therapeutic targets. High-throughput techniques allow high-resolution, genome-wide investigation of genetic variants, epigenetic modifications, and associated gene expression profiles. Platforms such as proteomics and metabolomics (not described here in detail) also offer simultaneous readouts of hundreds of proteins and metabolites. Isolated omics analyses usually provide Big Data requiring large data storage, advanced computational resources and complex bioinformatics tools. The possibility of integrating different omics approaches gives new hope to better understand the molecular circuitry activated by myocardial ischaemia, putting it in the context of the human ‘diseasome’. Since modifications of cardiac gene expression have been consistently linked to pathophysiology of the ischaemic heart, the integration of epigenomic and transcriptomic data seems a promising approach to identify crucial disease networks. Thus, the scope of this Position Paper will be to highlight potentials and limitations of these approaches, and to provide recommendations to optimize the search for novel diagnostic or therapeutic targets for acute ischaemia/reperfusion injury and ischaemic heart failure in the post-genomic era.
We have analyzed the distribution of mitochondrial contact site and cristae organizing system (MICOS) complex proteins and mitochondrial intermembrane space bridging complex (MIB) proteins over (sub)complexes and over species. The MICOS proteins are associated with the formation and maintenance of mitochondrial cristae. Indeed, the presence of MICOS genes in genomes correlates well with the presence of cristae: all cristae containing species have at least one MICOS gene and cristae-less species have none. Mic10 is the most widespread MICOS gene, while Mic60 appears be the oldest one, as it originates in the ancestors of mitochondria, the proteobacteria. In proteobacteria the gene occurs in clusters with genes involved in heme synthesis while the protein has been observed in intracellular membranes of the alphaproteobacterium Rhodobacter sphaeroides. In contrast, Mic23 and Mic27 appear to be the youngest MICOS proteins, as they only occur in opisthokonts. The remaining MICOS proteins, Mic10, Mic19, Mic25 and Mic12, the latter we show to be orthologous to human C19orf70/QIL1, trace back to the root of the eukaryotes. Of the remaining MIB proteins, also DNAJC11 shows a high correlation with the presence of cristae. In mitochondrial protein complexome profiles, the MIB complex occurs as a defined complex and as separate subcomplexes, potentially reflecting various assembly stages. We find three main forms of the complex: A) The MICOS complex, containing all the MICOS proteins, B) a membrane bridging subcomplex, containing in addition SAMM50, MTX2 and the previously uncharacterized MTX3, and C) the complete MIB complex containing in addition DNAJC11 and MTX1.
The direct study of transcription or DNA–protein-binding events, requires imaging of individual genes at molecular resolution. Electron microscopy (EM) can show local detail of the genome. However, direct visualization and analysis of specific individual genes is currently not feasible as they cannot be unambiguously localized in the crowded, landmark-free environment of the nucleus. Here, we present a method for the genomic insertion of gene clusters that can be localized and imaged together with their associated protein complexes in the EM. The method uses CRISPR/Cas9 technology to incorporate several genes of interest near the 35S rRNA gene, which is a frequently occurring, easy-to-identify genomic locus within the nucleolus that can be used as a landmark in micrographs. As a proof of principle, we demonstrate the incorporation of the locus-native gene RDN5 and the locus-foreign gene HSX1. This led to a greater than 7-fold enrichment of RNA polymerase III (Pol III) complexes associated with the genes within the field of view, allowing for a significant increase in the analysis yield. This method thereby allows for the insertion and direct visualization of gene clusters for a range of analyses, such as changes in gene activity upon alteration of cellular or external factors.
Proton pumping respiratory complex I is a major player in mitochondrial energy conversion. Yet little is known about the molecular mechanism of this large membrane protein complex. Understanding the details of ubiquinone reduction will be prerequisite for elucidating this mechanism. Based on a recently published partial structure of the bacterial enzyme, we scanned the proposed ubiquinone binding cavity of complex I by site-directed mutagenesis in the strictly aerobic yeast Yarrowia lipolytica. The observed changes in catalytic activity and inhibitor sensitivity followed a consistent pattern and allowed us to define three functionally important regions near the ubiquinone-reducing iron-sulfur cluster N2. We identified a likely entry path for the substrate ubiquinone and defined a region involved in inhibitor binding within the cavity. Finally, we were able to highlight a functionally critical structural motif in the active site that consisted of Tyr-144 in the 49-kDa subunit, surrounded by three conserved hydrophobic residues.
The turnover of endoplasmic reticulum (ER) ensures the correct biological activity of its distinct domains. In mammalian cells, the ER is degraded via a selective autophagy pathway (ER-phagy), mediated by two specific receptors: FAM134B, responsible for the turnover of ER sheets and SEC62 that regulates ER recovery following stress. Here, we identified reticulon 3 (RTN3) as a specific receptor for the degradation of ER tubules. Oligomerization of the long isoform of RTN3 is sufficient to trigger fragmentation of ER tubules. The long N-terminal region of RTN3 contains several newly identified LC3-interacting regions (LIR). Binding to LC3s/GABARAPs is essential for the fragmentation of ER tubules and their delivery to lysosomes. RTN3-mediated ER-phagy requires conventional autophagy components, but is independent of FAM134B. None of the other reticulon family members have the ability to induce fragmentation of ER tubules during starvation. Therefore, we assign a unique function to RTN3 during autophagy.
Mitochondrial complex I, the largest and most complicated proton pump of the respiratory chain, links the electron transfer from NADH to ubiquinone to the pumping of four protons from the matrix into the intermembrane space. In humans, defects in complex I are involved in a wide range of degenerative disorders. Recent progress in the X-ray structural analysis of prokaryotic and eukaryotic complex I confirmed that the redox reactions are confined entirely to the hydrophilic peripheral arm of the L-shaped molecule and take place at a remarkable distance from the membrane domain. While this clearly implies that the proton pumping within the membrane arm of complex I is driven indirectly via long-range conformational coupling, the molecular mechanism and the number, identity, and localization of the pump-sites remains unclear. Here, we report that upon deletion of the gene for a small accessory subunit of the Yarrowia complex I, a stable subcomplex (nb8m delta) is formed that lacks the distal part of the membrane domain as revealed by single particle analysis. The analysis of the subunit composition of holo and subcomplex by three complementary proteomic approaches revealed that two (ND4 and ND5) of the three subunits with homology to bacterial Mrp-type Na+/H+ antiporters that have been discussed as prime candidates for harbouring the proton pumps were missing in nb8m delta. Nevertheless, nb8m delta still pumps protons at half the stoichiometry of the complete enzyme. Our results provide evidence that the membrane arm of complex I harbours two functionally distinct pump modules that are connected in series by the long helical transmission element recently identified by X-ray structural analysis.
We investigate complexes of two paramagnetic metal ions Gd3+ and Mn2+ to serve as polarizing agents for solid-state dynamic nuclear polarization (DNP) of 1H, 13C, and 15N at magnetic fields of 5, 9.4, and 14.1 T. Both ions are half-integer high-spin systems with a zero-field splitting and therefore exhibit a broadening of the mS = −1/2 ↔ +1/2 central transition which scales inversely with the external field strength. We investigate experimentally the influence of the chelator molecule, strong hyperfine coupling to the metal nucleus, and deuteration of the bulk matrix on DNP properties. At small Gd-DOTA concentrations the narrow central transition allows us to polarize nuclei with small gyromagnetic ratio such as 13C and even 15N via the solid effect. We demonstrate that enhancements observed are limited by the available microwave power and that large enhancement factors of >100 (for 1H) and on the order of 1000 (for 13C) can be achieved in the saturation limit even at 80 K. At larger Gd(III) concentrations (≥10 mM) where dipolar couplings between two neighboring Gd3+ complexes become substantial a transition towards cross effect as dominating DNP mechanism is observed. Furthermore, the slow spin-diffusion between 13C and 15N, respectively, allows for temporally resolved observation of enhanced polarization spreading from nuclei close to the paramagnetic ion towards nuclei further removed. Subsequently, we present preliminary DNP experiments on ubiquitin by site-directed spin-labeling with Gd3+ chelator tags. The results hold promise towards applications of such paramagnetically labeled proteins for DNP applications in biophysical chemistry and/or structural biology.
Crescentic rapidly progressive glomerulonephritis (RPGN) represents the most aggressive form of acquired glomerular disease. While most therapeutic approaches involve potentially toxic immunosuppressive strategies, the pathophysiology remains incompletely understood. Podocytes are glomerular epithelial cells that are normally growth-arrested because of the expression of cyclin-dependent kinase (CDK) inhibitors. An exception is in RPGN where podocytes undergo a deregulation of their differentiated phenotype and proliferate. Here we demonstrate that microRNA-92a (miR-92a) is enriched in podocytes of patients and mice with RPGN. The CDK inhibitor p57Kip2 is a major target of miR-92a that constitutively safeguards podocyte cell cycle quiescence. Podocyte-specific deletion of miR-92a in mice de-repressed the expression of p57Kip2 and prevented glomerular injury in RPGN. Administration of an anti-miR-92a after disease initiation prevented albuminuria and kidney failure, indicating miR-92a inhibition as a potential therapeutic strategy for RPGN. We demonstrate that miRNA induction in epithelial cells can break glomerular tolerance to immune injury.
Global response of diacylglycerol kinase towards substrate binding observed by 2D and 3D MAS NMR
(2019)
Escherichia coli diacylglycerol kinase (DGK) is an integral membrane protein, which catalyses the ATP-dependent phosphorylation of diacylglycerol (DAG) to phosphatic acid (PA). It is a unique trimeric enzyme, which does not share sequence homology with typical kinases. It exhibits a notable complexity in structure and function despite of its small size. Here, chemical shift assignment of wild-type DGK within lipid bilayers was carried out based on 3D MAS NMR, utilizing manual and automatic analysis protocols. Upon nucleotide binding, extensive chemical shift perturbations could be observed. These data provide evidence for a symmetric DGK trimer with all of its three active sites concurrently occupied. Additionally, we could detect that the nucleotide substrate induces a substantial conformational change, most likely directing DGK into its catalytic active form. Furthermore, functionally relevant interprotomer interactions are identified by DNP-enhanced MAS NMR in combination with site-directed mutagenesis and functional assays.
Regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking in response to neuronal activity is critical for synaptic function and plasticity. Here, we show that neuronal activity induces the binding of ephrinB2 and ApoER2 receptors at the postsynapse to regulate de novo insertion of AMPA receptors. Mechanistically, the multi-PDZ adaptor glutamate-receptor-interacting protein 1 (GRIP1) binds ApoER2 and bridges a complex including ApoER2, ephrinB2, and AMPA receptors. Phosphorylation of ephrinB2 in a serine residue (Ser-9) is essential for the stability of such a complex. In vivo, a mutation on ephrinB2 Ser-9 in mice results in a complete disruption of the complex, absence of ApoER2 downstream signaling, and impaired activity-induced and ApoER2-mediated AMPA receptor insertion. Using compound genetics, we show the requirement of this complex for long-term potentiation (LTP). Together, our findings uncover a cooperative ephrinB2 and ApoER2 signaling at the synapse, which serves to modulate activity-dependent AMPA receptor dynamic changes during synaptic plasticity.
Heat stress transcription factors (HSFs) regulate transcriptional response to a large number of environmental influences, such as temperature fluctuations and chemical compound applications. Plant HSFs represent a large and diverse gene family. The HSF members vary substantially both in gene expression patterns and molecular functions. HEATSTER is a web resource for mining, annotating, and analyzing members of the different classes of HSFs in plants. A web-interface allows the identification and class assignment of HSFs, intuitive searches in the database and visualization of conserved motifs, and domains to classify novel HSFs.
Hepatitis Delta virus (HDV) is a satellite of Hepatitis B virus with a single-stranded circular RNA genome. HDV RNA genome synthesis is carried out in infected cells by cellular RNA polymerases with the assistance of the small hepatitis delta antigen (S-HDAg). Here we show that S-HDAg binds the bromodomain (BRD) adjacent to zinc finger domain 2B (BAZ2B) protein, a regulatory subunit of BAZ2B-associated remodeling factor (BRF) ISWI chromatin remodeling complexes. shRNA-mediated silencing of BAZ2B or its inactivation with the BAZ2B BRD inhibitor GSK2801 impairs HDV replication in HDV-infected human hepatocytes. S-HDAg contains a short linear interacting motif (SLiM) KacXXR, similar to the one recognized by BAZ2B BRD in histone H3. We found that the integrity of the S-HDAg SLiM sequence is required for S-HDAg interaction with BAZ2B BRD and for HDV RNA replication. Our results suggest that S-HDAg uses a histone mimicry strategy to co-activate the RNA polymerase II-dependent synthesis of HDV RNA and sustain HDV replication.
Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I–V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.
High-resolution NMR structure of an RNA model system : the 14-mer cUUCGg tetraloop hairpin RNA
(2009)
We present a high-resolution nuclear magnetic resonance (NMR) solution structure of a 14-mer RNA hairpin capped by cUUCGg tetraloop. This short and very stable RNA presents an important model system for the study of RNA structure and dynamics using NMR spectroscopy, molecular dynamics (MD) simulations and RNA force-field development. The extraordinary high precision of the structure (root mean square deviation of 0.3 Å) could be achieved by measuring and incorporating all currently accessible NMR parameters, including distances derived from nuclear Overhauser effect (NOE) intensities, torsion-angle dependent homonuclear and heteronuclear scalar coupling constants, projection-angle-dependent cross-correlated relaxation rates and residual dipolar couplings. The structure calculations were performed with the program CNS using the ARIA setup and protocols. The structure quality was further improved by a final refinement in explicit water using OPLS force field parameters for non-bonded interactions and charges. In addition, the 2'-hydroxyl groups have been assigned and their conformation has been analyzed based on NOE contacts. The structure currently defines a benchmark for the precision and accuracy amenable to RNA structure determination by NMR spectroscopy. Here, we discuss the impact of various NMR restraints on structure quality and discuss in detail the dynamics of this system as previously determined.
Spectroscopic responses of the potentiometric probe 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) were investigated in living cells by means of a time- and space-correlated single photon counting technique. Spatially resolved fluorescence decays from single mitochondria or only a very few organelles of XTH2 cells exhibited three-exponential decay kinetics. Based on DASPMI photophysics in a variety of solvents, these lifetimes were attributed to the fluorescence from the locally excited state, intramolecular charge transfer state, and twisted intramolecular charge transfer state. A considerable variation in lifetimes among mitochondria of different morphologies and within single cells was evident, corresponding to high physiological variations within single cells. Considerable shortening of the short lifetime component ({tau}1) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents. Under these conditions {tau}2 and {tau}3 increased with decreasing contribution. Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence. Increased DASPMI fluorescence under conditions that elevate the mitochondrial membrane potential has been attributed to uptake according to Nernst distributions, delocalization of {pi}-electrons, quenching processes of the methyl pyridinium moiety, and restricted torsional dynamics at the mitochondrial inner membrane. Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential. The direct influence of the local electric field on the transition dipole moment of the probe and its torsional dynamics monitor changes in mitochondrial energy status within living cells.
HUWE1 E3 ligase promotes PINK1/PARKIN-independent mitophagy by regulating AMBRA1 activation via IKKα
(2018)
The selective removal of undesired or damaged mitochondria by autophagy, known as mitophagy, is crucial for cellular homoeostasis, and prevents tumour diffusion, neurodegeneration and ageing. The pro-autophagic molecule AMBRA1 (autophagy/beclin-1 regulator-1) has been defined as a novel regulator of mitophagy in both PINK1/PARKIN-dependent and -independent systems. Here, we identified the E3 ubiquitin ligase HUWE1 as a key inducing factor in AMBRA1-mediated mitophagy, a process that takes place independently of the main mitophagy receptors. Furthermore, we show that mitophagy function of AMBRA1 is post-translationally controlled, upon HUWE1 activity, by a positive phosphorylation on its serine 1014. This modification is mediated by the IKKα kinase and induces structural changes in AMBRA1, thus promoting its interaction with LC3/GABARAP (mATG8) proteins and its mitophagic activity. Altogether, these results demonstrate that AMBRA1 regulates mitophagy through a novel pathway, in which HUWE1 and IKKα are key factors, shedding new lights on the regulation of mitochondrial quality control and homoeostasis in mammalian cells.
Precise knowledge on the binding sites of an RNA-binding protein (RBP) is key to understanding the complex post-transcriptional regulation of gene expression. This information can be obtained from individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) experiments. Here, we present a complete data analysis workflow to reliably detect RBP binding sites from iCLIP data. The workflow covers all steps from the initial quality control of the sequencing reads up to peak calling and quantification of RBP binding. For each tool, we explain the specific requirements for iCLIP data analysis and suggest optimised parameter settings.
The identification of heat stress (HS)-resilient germplasm is important to ensure food security under less favorable environmental conditions. For that, germplasm with an altered activity of factors regulating the HS response is an important genetic tool for crop improvement. Heat shock binding protein (HSBP) is one of the main negative regulators of HS response, acting as a repressor of the activity of HS transcription factors. We identified a TILLING allele of Solanum lycopersicum (tomato) HSBP1. We examined the effects of the mutation on the functionality of the protein in tomato protoplasts, and compared the thermotolerance capacity of lines carrying the wild-type and mutant alleles of HSBP1. The methionine-to-isoleucine mutation in the central heptad repeats of HSBP1 leads to a partial loss of protein function, thereby reducing the inhibitory effect on Hsf activity. Mutant seedlings show enhanced basal thermotolerance, while mature plants exhibit increased resilience in repeated HS treatments, as shown by several physiological parameters. Importantly, plants that are homozygous for the wild-type or mutant HSBP1 alleles showed no significant differences under non-stressed conditions. Altogether, these results indicate that the identified mutant HSBP1 allele can be used as a genetic tool in breeding, aiming to improve the thermotolerance of tomato varieties.
Advanced colorectal carcinoma is currently incurable, and new therapies are urgently needed. We report that phosphotyrosine-dependent Eph receptor signaling sustains colorectal carcinoma cell survival, thereby uncovering a survival pathway active in colorectal carcinoma cells. We find that genetic and biochemical inhibition of Eph tyrosine kinase activity or depletion of the Eph ligand EphrinB2 reproducibly induces colorectal carcinoma cell death by autophagy. Spautin and 3-methyladenine, inhibitors of early steps in the autophagic pathway, significantly reduce autophagy-mediated cell death that follows inhibition of phosphotyrosine-dependent Eph signaling in colorectal cancer cells. A small-molecule inhibitor of the Eph kinase, NVP-BHG712 or its regioisomer NVP-Iso, reduces human colorectal cancer cell growth in vitro and tumor growth in mice. Colorectal cancers express the EphrinB ligand and its Eph receptors at significantly higher levels than numerous other cancer types, supporting Eph signaling inhibition as a potential new strategy for the broad treatment of colorectal carcinoma.
Maintenance of mitochondria is achieved by several mechanisms, including the regulation of mitochondrial proteostasis. The matrix protease CLPXP, involved in protein quality control, has been implicated in ageing and disease. However, particularly due to the lack of knowledge of CLPXP's substrate spectrum, only little is known about the pathways and mechanisms controlled by this protease. Here we report the first comprehensive identification of potential mitochondrial CLPXP in vivo interaction partners and substrates using a combination of tandem affinity purification and differential proteomics. This analysis reveals that CLPXP in the fungal ageing model Podospora anserina is mainly associated with metabolic pathways in mitochondria, e.g. components of the pyruvate dehydrogenase complex and the tricarboxylic acid cycle as well as subunits of electron transport chain complex I. These data suggest a possible function of mitochondrial CLPXP in the control and/or maintenance of energy metabolism. Since bioenergetic alterations are a common feature of neurodegenerative diseases, cancer, and ageing, our data comprise an important resource for specific studies addressing the role of CLPXP in these adverse processes.
NMR and chromatography methods combined with mass spectrometry are the most important analytical techniques employed for plant metabolomics screening. Metabolomic analysis integrated to transcriptome screening add an important extra dimension to the information flow from DNA to RNA to protein. The most useful NMR experiment in metabolomics analysis is the proton spectra due the high receptivity of 1H and important structural information, through proton–proton scalar coupling. Routinely, databases have been used in identification of primary metabolites, however, there is currently no comparable data for identification of secondary metabolites, mainly, due to signal overlap in normal 1H NMR spectra and natural variation of plant. Related to spectra overlap, alternatively, better resolution can be find using 1H pure shift and 2D NMR pulse sequence in complex samples due to spreading the resonances in a second dimension. Thus, in data brief we provide a catalogue of metabolites and expression levels of genes identified in soy leaves and roots under flooding stress.
Mitochondrial complex I (NADH:ubiquinone oxidoreductase) undergoes reversible deactivation upon incubation at 30–37 °C. The active/deactive transition could play an important role in the regulation of complex I activity. It has been suggested recently that complex I may become modified by S-nitrosation under pathological conditions during hypoxia or when the nitric oxide:oxygen ratio increases. Apparently, a specific cysteine becomes accessible to chemical modification only in the deactive form of the enzyme. By selective fluorescence labeling and proteomic analysis, we have identified this residue as cysteine-39 of the mitochondrially encoded ND3 subunit of bovine heart mitochondria. Cysteine-39 is located in a loop connecting the first and second transmembrane helix of this highly hydrophobic subunit. We propose that this loop connects the ND3 subunit of the membrane arm with the PSST subunit of the peripheral arm of complex I, placing it in a region that is known to be critical for the catalytic mechanism of complex I. In fact, mutations in three positions of the loop were previously reported to cause Leigh syndrome with and without dystonia or progressive mitochondrial disease.
Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF1 inhibitor protein in substoichiometric amounts. Alternatively ATP synthase can be isolated in dimeric and higher oligomeric states using digitonin for membrane solubilization and blue native or clear native electrophoresis for separation of the native mitochondrial complexes. Using blue native electrophoresis we could identify two ATP synthase-associated membrane proteins with masses smaller than 7 kDa and isoelectric points close to 10 that previously had been removed during purification. We show that in the mitochondrial membrane both proteins are almost quantitatively bound to ATP synthase. Both proteins had been identified earlier in a different context, but their association with ATP synthase was unknown. The first one had been named 6.8-kDa mitochondrial proteolipid because it can be isolated by chloroform/methanol extraction from mitochondrial membranes. The second one had been denoted as diabetes-associated protein in insulin-sensitive tissue (DAPIT), which may provide a clue for further functional and clinical investigations.
Improved risk stratification in prevention by use of a panel of selected circulating microRNAs
(2017)
Risk stratification is crucial in prevention. Circulating microRNAs have been proposed as biomarkers in cardiovascular disease. Here a miR panel consisting of miRs related to different cardiovascular pathophysiologies, was evaluated to predict outcome in the context of prevention. MiR-34a, miR-223, miR-378, miR-499 and miR-133 were determined from peripheral blood by qPCR and combined to a risk panel. As derivation cohort, 178 individuals of the DETECT study, and as validation cohort, 129 individuals of the SHIP study were used in a case-control approach. Overall mortality and cardiovascular events were outcome measures. The Framingham Risk Score(FRS) and the SCORE system were applied as risk classification systems. The identified miR panel was significantly associated with mortality given by a hazard ratio(HR) of 3.0 (95% (CI): 1.09–8.43; p = 0.034) and of 2.9 (95% CI: 1.32–6.33; p = 0.008) after adjusting for the FRS in the derivation cohort. In a validation cohort the miR-panel had a HR of 1.31 (95% CI: 1.03–1.66; p = 0.03) and of 1.29 (95% CI: 1.02–1.64; p = 0.03) in a FRS/SCORE adjusted-model. A FRS/SCORE risk model was significantly improved to predict mortality by the miR panel with continuous net reclassification index of 0.42/0.49 (p = 0.014/0.005). The present miR panel of 5 circulating miRs is able to improve risk stratification in prevention with respect to mortality beyond the FRS or SCORE.
Coronavirus disease 2019 (COVID-19) spawned a global health crisis in late 2019 and is caused by the novel coronavirus SARS-CoV-2. SARS-CoV-2 infection can lead to elevated markers of endothelial dysfunction associated with higher risk of mortality. It is unclear whether endothelial dysfunction is caused by direct infection of endothelial cells or is mainly secondary to inflammation. Here, we investigate whether different types of endothelial cells are susceptible to SARS-CoV-2. Human endothelial cells from different vascular beds including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells were inoculated in vitro with SARS-CoV-2. Viral spike protein was only detected in HCAECs after SARS-CoV-2 infection but not in the other endothelial cells tested. Consistently, only HCAEC expressed the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2), required for virus infection. Infection with the SARS-CoV-2 variants B.1.1.7, B.1.351, and P.2 resulted in significantly higher levels of viral spike protein. Despite this, no intracellular double-stranded viral RNA was detected and the supernatant did not contain infectious virus. Analysis of the cellular distribution of the spike protein revealed that it co-localized with endosomal calnexin. SARS-CoV-2 infection did induce the ER stress gene EDEM1, which is responsible for clearance of misfolded proteins from the ER. Whereas the wild type of SARS-CoV-2 did not induce cytotoxic or pro-inflammatory effects, the variant B.1.1.7 reduced the HCAEC cell number. Of the different tested endothelial cells, HCAECs showed highest viral uptake but did not promote virus replication. Effects on cell number were only observed after infection with the variant B.1.1.7, suggesting that endothelial protection may be particularly important in patients infected with this variant.
The 3′ untranslated regions (3′ UTRs) of transcripts serve as important hubs for posttranscriptional gene expression regulation. Here, we find that the exonisation of intergenic Alu elements introduced new terminal exons and polyadenylation sites during human genome evolution. While Alu exonisation from introns has been described previously, we shed light on a novel mechanism to create alternative 3′ UTRs, thereby opening opportunities for differential posttranscriptional regulation. On the mechanistic level, we show that intergenic Alu exonisation can compete both with alternative splicing and polyadenylation in the upstream gene. Notably, the Alu-derived isoforms are often expressed in a tissue-specific manner, and the Alu-derived 3′ UTRs can alter mRNA stability. In summary, we demonstrate that intergenic elements can affect processing of preceding genes, and elucidate how intergenic Alu exonisation can contribute to tissue-specific posttranscriptional regulation by expanding the repertoire of 3′ UTRs.
The RNA-chaperone Hfq catalyses the annealing of bacterial small RNAs (sRNAs) with target mRNAs to regulate gene expression in response to environmental stimuli. Hfq acts on a diverse set of sRNA-mRNA pairs using a variety of different molecular mechanisms. Here, we present an unusual crystal structure showing two Hfq-RNA complexes interacting via their bound RNA molecules. The structure contains two Hfq6:A18 RNA assemblies positioned face-to-face, with the RNA molecules turned towards each other and connected via interdigitating base stacking interactions at the center. Biochemical data further confirm the observed interaction, and indicate that RNA-mediated contacts occur between Hfq-RNA complexes with various (ARN)X motif containing RNA sequences in vitro, including the stress response regulator OxyS and its target, fhlA. A systematic computational survey also shows that phylogenetically conserved (ARN)X motifs are present in a subset of sRNAs, some of which share similar modular architectures. We hypothesise that Hfq can co-opt RNA-RNA base stacking, an unanticipated structural trick, to promote the interaction of (ARN)X motif containing sRNAs with target mRNAs on a "speed-dating" fashion, thereby supporting their regulatory function.
Intracarotid administration of human bone marrow mononuclear cells in rat photothrombotic ischemia
(2010)
Background: Increasing evidence suggests that cell therapy improves functional recovery in experimental models of stroke and myocardial infarction. So far only small pilot trials tested the effects of cell therapy in stroke patients, whereas large clinical trials were conducted in patients with ischemic heart disease. To investigate the therapeutic benefit of cell therapy to improve the recovery after stroke, we determined the efficacy of bone marrow derived mononuclear cells, which were shown to improve the recovery in experimental and clinical acute myocardial infarction studies, in a rat stroke model. Methods: Adult male Wistar rats were randomly assigned to receive either five million human bone marrow mononuclear cells (hBMC) or placebo intraarterially 3 days after photothrombotic ischemia. For immunosuppression the animals received daily injections of cyclosporine throughout the experiment, commencing 24 hours before the cell transplantation. A battery of behavioural tests was performed before and up to 4 weeks after ischemia. Results: Body temperature and body weight revealed no difference between groups. Neurological deficits measured by the Rotarod test, the adhesive-removal test and the cylinder test were not improved by hBMC transplantation compared to placebo. Conclusions: This study demonstrates that hBMC do not improve functional recovery when transplanted intraaterially 3 days after the onset of focal cerebral ischemia. A possible reason for the failed neurological improvement after cell therapy might be the delayed treatment initiation compared to other experimental stroke studies that showed efficacy of bone marrow mononuclear cells.
The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.
Ribosomal proteins are assumed to stabilize specific RNA structures and promote compact folding of the large rRNA. The conformational dynamics of the protein between the bound and unbound state play an important role in the binding process. We have studied those dynamical changes in detail for the highly conserved complex between the ribosomal protein L11 and the GTPase region of 23S rRNA. The RNA domain is compactly folded into a well defined tertiary structure, which is further stabilized by the association with the C-terminal domain of the L11 protein (L11ctd). In addition, the N-terminal domain of L11 (L11ntd) is implicated in the binding of the natural thiazole antibiotic thiostrepton, which disrupts the elongation factor function. We have studied the conformation of the ribosomal protein and its dynamics by NMR in the unbound state, the RNA bound state and in the ternary complex with the RNA and thiostrepton. Our data reveal a rearrangement of the L11ntd, placing it closer to the RNA after binding of thiostrepton, which may prevent binding of elongation factors. We propose a model for the ternary L11–RNA–thiostrepton complex that is additionally based on interaction data and conformational information of the L11 protein. The model is consistent with earlier findings and provides an explanation for the role of L11ntd in elongation factor binding.
Ribosome biogenesis is essential for cellular function and involves rRNA synthesis, rRNA processing and modification, and ribosomal protein assembly. Ribosome biogenesis factors and small nucleolar RNA assist these events. Ribosomal maturation takes place in the nucleolus, the nucleoplasm, and the cytosol in a coordinated and controlled manner. For example, some ribosomal proteins are thought to be assembled in the cytoplasm based on the observations in Saccharomyces cerevisiae. Here, we used cellular fractionation to demonstrate that cleavage of the 20S intermediate, the precursor to mature 18S rRNA, does not occur in the nucleoplasm of Arabidopsis thaliana. It most likely occurs in the cytoplasm. Further, we verified the proposed localization of RPS10e, RPS26e, and RPL24a/b in the nucleus and RPP1 in the nucleolus of A. thaliana by ribosome profiling, immunofluorescence, and analysis of the localization of GFP fusion proteins. Our results suggest that the order of events during ribosomal protein assembly in the ribosome biogenesis pathway differs between plants and yeast.
Lieblingsbild
(2017)
Dieses Bild ist wichtig, weil wir daran verstanden haben, wie in der Zelle fehlerhaftes Spleißen verhindert wird. Dazu muss man wissen, dass unsere Gene sich aus Exons und dazwischenliegenden Introns zusammensetzen. Während des Spleißens werden die Introns entfernt und die Exons in ein reifes Transkript zusammengefügt, das dann für ein Protein kodiert. Allerdings gibt es innerhalb der Introns viele Bereiche, die einem Exon sehr ähnlich sehen. Werden diese sogenannten "PseudoExons" fälschlicherweise während des Spleißprozesses erkannt und in das reife Transkript eingebaut, kann das fatale Folgen für das kodierte Protein und oft die gesamte Zelle haben. ...
Lieblingsbild
(2017)
Das rechte Bild stellt die Elektronendichte eines menschlichen Proteins dar, gewonnen durch die Röntgenstrahl-Beugung an Kristallen dieses Proteins. Die Struktur hat Sagar Bhogaraju 2016 aufgeklärt. Das linke Bild stellt ein erstes Strukturmodell auf der Basis der gemessenen Elektronendichte dar. ...
The full-length translation-regulating add adenine riboswitch (Asw) from Vibrio vulnificus has a more complex conformational space than its isolated aptamer domain. In addition to the predicted apo (apoA) and holo conformation that feature the conserved three-way junctional purine riboswitch aptamer, it adopts a second apo (apoB) conformation with a fundamentally different secondary structure. Here, we characterized the ligand-dependent conformational dynamics of the full-length add Asw by NMR and by single-molecule FRET (smFRET) spectroscopy. Both methods revealed an adenine-induced secondary structure switch from the apoB-form to the apoA-form that involves no tertiary structural interactions between aptamer and expression platform. This strongly suggests that the add Asw triggers translation by capturing the apoA-form secondary structure in the holo state. Intriguingly, NMR indicated a homogenous, docked aptamer kissing loop fold for apoA and holo, while smFRET showed persistent aptamer kissing loop docking dynamics between comparably stable, undocked and docked substates of the apoA and the holo conformation. Unraveling the folding of large junctional riboswitches thus requires the integration of complementary solution structural techniques such as NMR and smFRET.