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A plethora of modified nucleotides extends the chemical and conformational space for natural occurring RNAs. tRNAs constitute the class of RNAs with the highest modification rate. The extensive modification modulates their overall stability, the fidelity and efficiency of translation. However, the impact of nucleotide modifications on the local structural dynamics is not well characterized. Here we show that the incorporation of the modified nucleotides in tRNAfMet from Escherichia coli leads to an increase in the local conformational dynamics, ultimately resulting in the stabilization of the overall tertiary structure. Through analysis of the local dynamics by NMR spectroscopic methods we find that, although the overall thermal stability of the tRNA is higher for the modified molecule, the conformational fluctuations on the local level are increased in comparison to an unmodified tRNA. In consequence, the melting of individual base pairs in the unmodified tRNA is determined by high entropic penalties compared to the modified. Further, we find that the modifications lead to a stabilization of long-range interactions harmonizing the stability of the tRNA’s secondary and tertiary structure. Our results demonstrate that the increase in chemical space through introduction of modifications enables the population of otherwise inaccessible conformational substates.
The ligand-sensing transcription factor Nurr1 emerges as a promising therapeutic target for neurodegenerative pathologies but Nurr1 ligands for functional studies and therapeutic validation are lacking. Here pronounced Nurr1 modulation by statins for which clinically relevant neuroprotective effects are demonstrated, is reported. Several statins directly affect Nurr1 activity in cellular and cell-free settings with low micromolar to sub-micromolar potencies. Simvastatin as example exhibits anti-inflammatory effects in astrocytes, which are abrogated by Nurr1 knockdown. Differential gene expression analysis in native and Nurr1-silenced cells reveals strong proinflammatory effects of Nurr1 knockdown while simvastatin treatment induces several neuroprotective mechanisms via Nurr1 involving changes in inflammatory, metabolic and cell cycle gene expression. Further in vitro evaluation confirms reduced inflammatory response, improved glucose metabolism, and cell cycle inhibition of simvastatin-treated neuronal cells. These findings suggest Nurr1 involvement in the well-documented but mechanistically elusive neuroprotection by statins.
Thermally stable and highly conductive SAMs on Ag substrate — the impact of the anchoring group
(2021)
Self-assembled monolayers (SAMs) on metal substrates are an important part of modern interfacial chemistry and nanotechnology. The robustness of SAMs strongly depends on their thermal stability, which, together with electric conductivity, crucial for their applications in molecular/organic electronics. In this context, using a multidisciplinary approach, the structure, stability, and conductivity properties of conjugated aromatic SAMs featuring the naphthalene backbone and S, Se, or COO group, mediating bonding to the Ag substrate are addressed. Whereas thermal stability of these SAMs exhibits a strong dependence on anchoring group, their conductivity is similar, which is rationalized by tentative model considering redistribution of charge density along the molecular framework. The thermal stability of model naphthalenethiol SAM, emphasized by desorption energy of ≈1.69 eV, is better than that of typical N-heterocyclic carbene (NHC) monolayers considered currently as the most stable SAMs on metal substrates. However, in contrast to NHC SAMs, which are highly insulating, the naphtalene-based SAM, with S, Se or COO anchoring groups, are highly conductive, even in comparison with analogous oligophenyl SAMs (by a factor of 10). A unique combination of the ultimate thermal stability and superior conductivity for the naphthalenethiol SAM on Ag makes it highly attractive for applications.
Alzheimer’s disease (AD) is characterized by the deposition of aggregated species of amyloid beta (Aβ) in the brain, which leads to progressive cognitive deficits and dementia. Aβ is generated by the successive cleavage of the amyloid precursor protein (APP), first by β-site APP cleaving enzyme 1 (BACE1) and subsequently by the γ-secretase complex. Those conditions which enhace or reduce its clearance predispose to Aβ aggregation and the development of AD. In vitro studies have demonstrated that Aβ assemblies spark a feed-forward loop heightening Aβ production. However, the underlying mechanism remains unknown. Here, we show that oligomers and fibrils of Aβ enhance colocalization and physical interaction of APP and BACE1 in recycling endosomes of human neurons derived from induced pluripotent stem cells and other cell types, which leads to exacerbated amyloidogenic processing of APP and intracellular accumulation of Aβ42. In cells that are overexpressing the mutant forms of APP which are unable to bind Aβ or to activate Go protein, we have found that treatment with aggregated Aβ fails to increase colocalization of APP with BACE1 indicating that Aβ-APP/Go signaling is involved in this process. Moreover, inhibition of Gβγ subunit signaling with βARKct or gallein prevents Aβ-dependent interaction of APP and BACE1 in endosomes, β-processing of APP, and intracellular accumulation of Aβ42. Collectively, our findings uncover a signaling mechanism leading to a feed-forward loop of amyloidogenesis that might contribute to Aβ pathology in the early stages of AD and suggest that gallein could have therapeutic potential.
Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate specificity. Similarly to many other RND efflux pump systems, AcrAD-TolC confers resistance toward SDS, novobiocin and deoxycholate. In contrast to the other pumps, however, it in addition confers resistance against aminoglycosides and dianionic β-lactams, such as sulbenicillin, aztreonam and carbenicillin. Here, we could show that AcrD from Salmonella typhimurium confers resistance toward several hitherto unreported AcrD substrates such as temocillin, dicloxacillin, cefazolin and fusidic acid. In order to address the molecular determinants of the S. typhimurium AcrD substrate specificity, we conducted substitution analyses in the putative access and deep binding pockets and in the TM1/TM2 groove region. The variants were tested in E. coli ΔacrBΔacrD against β-lactams oxacillin, carbenicillin, aztreonam and temocillin. Deep binding pocket variants N136A, D276A and Y327A; access pocket variant R625A; and variants with substitutions in the groove region between TM1 and TM2 conferred a sensitive phenotype and might, therefore, be involved in anionic β-lactam export. In contrast, lower susceptibilities were observed for E. coli cells harbouring deep binding pocket variants T139A, D176A, S180A, F609A, T611A and F627A and the TM1/TM2 groove variant I337A. This study provides the first insights of side chains involved in drug binding and transport for AcrD from S. typhimurium.
Human serum albumin (HSA) nanoparticles represent a promising tool for targeted drug delivery to tumor cells. The coupling of the antibody trastuzumab to nanoparticles uses the capability of human epidermal growth factor receptor 2 (HER2)-positive cells to incorporate agents linked to HER2. In our present study, we developed targeted nanoparticles loaded with antisense oligonucleotides (ASOs) against polo-like kinase 1 (Plk1). We evaluated the receptor-mediated uptake into HER2-positive and -negative breast cancer and murine cell lines. We performed quantitative real-time PCR and Western blot analyses to monitor the impact on Plk1 expression in HER2-positive breast cancer cells. Antibody-conjugated nanoparticles showed a specific targeting to HER2-overexpressing cells with cellular uptake by receptor-mediated endocytosis and a release into HER2-positive BT-474 cells. We observed a significant reduction of Plk1 mRNA and protein expression and increased activation of Caspase 3/7. Thus, this is the first report about ASO-loaded HSA nanoparticles, where an impact on gene expression could be observed. The data provide the basis for the further development of carrier systems for Plk1-specific ASOs to reduce off-target effects evoked by systemically administered ASOs and to achieve a better penetration into primary and metastatic target cells. Treatment of tumors using trastuzumab-conjugated ASO-loaded HSA nanoparticles could be a promising approach to reach this goal.
To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an alpha-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15N and 2H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is approximately 15 degrees (+/- 7 degrees ). The alignment of both structured domains exhibits a mosaic spread of approximately 10 degrees . A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.
Through its role in intron cleavage, tRNA splicing endonuclease (TSEN) plays a critical function in the maturation of intron-containing pre-tRNAs. The catalytic mechanism and core requirement for this process is conserved between archaea and eukaryotes, but for decades, it has been known that eukaryotic TSENs have evolved additional modes of RNA recognition, which have remained poorly understood. Recent research identified new roles for eukaryotic TSEN, including processing or degradation of additional RNA substrates, and determined the first structures of pre-tRNA-bound human TSEN complexes. These recent discoveries have changed our understanding of how the eukaryotic TSEN targets and recognizes substrates. Here, we review these recent discoveries, their implications, and the new questions raised by these findings.
Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin 1beta (IL-1beta). We tested whether ligands of the peroxisome proliferator-activated receptor (PPARalpha) could influence the cytokine-induced expression of MMP-9. Different PPARalpha agonists dose-dependently inhibited the IL-1beta-triggered increase in gelatinolytic activity mainly by decreasing the MMP-9 steady-state mRNA levels. PPARalpha agonists on their own had no effects on MMP-9 mRNA levels and gelatinolytic activity. Surprisingly, the reduction of MMP-9 mRNA levels by PPARalpha activators contrasted with an amplification of cytokine-mediated MMP-9 gene promoter activity and mRNA expression. The potentiation of MMP-9 promoter activity functionally depends on an upstream peroxisome proliferator-responsive element-like binding site, which displayed an increased DNA binding of a PPARalpha immunopositive complex. In contrast, the IL-1beta-induced DNA-binding of nuclear factor kappaB was significantly impaired by PPARalpha agonists. Most interestingly, in the presence of an inducible nitric-oxide synthase (iNOS) inhibitor, the PPARalpha-mediated suppression switched to a strong amplification of IL-1beta-triggered MMP-9 mRNA expression. Concomitantly, activators of PPARalpha potentiated the cytokine-induced iNOS expression. Using actinomycin D, we found that NO, but not PPARalpha activators, strongly reduced the stability of MMP-9 mRNA. In contrast, the stability of MMP-9 protein was not affected by PPARalpha activators. In summary, our data suggest that the inhibitory effects of PPARalpha agonists on cytokine-induced MMP-9 expression are indirect and primarily due to a superinduction of iNOS with high levels of NO reducing the half-life of MMP-9 mRNA.
An efficient route for delivering specific proteins and peptides into neurons could greatly accelerate the development of therapies for various diseases, especially those involving intracellular defects such as Parkinson disease. Here we report the novel use of polybutylcyanoacrylate nanoparticles for delivery of intact, functional proteins into neurons and neuronal cell lines. Uptake of these particles is primarily dependent on endocytosis via the low density lipoprotein receptor. The nanoparticles are rapidly turned over and display minimal toxicity to cultured neurons. Delivery of three different functional cargo proteins is demonstrated. When primary neuronal cultures are treated with recombinant Escherichia coli beta-galactosidase as nanoparticle cargo, persistent enzyme activity is measured beyond the period of nanoparticle degradation. Delivery of the small GTPase rhoG induces neurite outgrowth and differentiation in PC12 cells. Finally, a monoclonal antibody directed against synuclein is capable of interacting with endogenous alpha-synuclein in cultured neurons following delivery via nanoparticles. Polybutylcyanoacrylate nanoparticles are thus useful for intracellular protein delivery in vitro and have potential as carriers of therapeutic proteins for treatment of neuronal disorders in vivo.
Nicotinamide adenine dinucleotide (NAD) serves as a cap-like structure on cellular RNAs (NAD-RNAs) in all domains of life including the bacterium Escherichia coli. NAD also acts as a key molecule in phage-host interactions, where bacterial immune systems deplete NAD to abort phage infection. Nevertheless, NAD-RNAs have not yet been identified during phage infections of bacteria and the mechanisms of their synthesis and degradation are unknown in this context. The T4 phage that specifically infects E. coli presents an important model to study phage infections, but a systematic analysis of the presence and dynamics of NAD-RNAs during T4 phage infection is lacking. Here, we investigate the presence of NAD-RNAs during T4 phage infection in a dual manner. By applying time-resolved NAD captureSeq, we identify NAD-capped host and phage transcripts and their dynamic regulation during phage infection. We provide evidence that NAD-RNAs are – as reported earlier – generated by the host RNA polymerase by initiating transcription with NAD at canonical transcription start sites. In addition, we characterize NudE.1 – a T4 phage-encoded Nudix hydrolase – as the first phage-encoded NAD-RNA decapping enzyme. T4 phages carrying inactive NudE.1 display a delayed lysis phenotype. This study investigates for the first time the dual epitranscriptome of a phage and its host, thereby introducing epitranscriptomics as an important field of phage research.
Targeted protein degradation (TPD) has recently emerged as an exciting new drug modality. However, the strategy of developing small molecule-based protein degraders has evolved over the past two decades and has now established molecular tags that are already in clinical use, as well as chimeric molecules, PROteolysis TArgeting Chimeras (PROTACs), based mainly on ligand systems developed for the two E3 ligases CRBN and VHL. The large size of the human E3 ligase family suggests that PROTACs can be developed by targeting a large diversity of E3 ligases, some of which have restricted expression patterns with the potential to design disease- or tissue-specific degraders. Indeed, many new E3 ligands have been published recently, confirming the druggability of E3 ligases. This review summarises recent data on E3 ligases and highlights the challenges in developing these molecules into efficient PROTACs rivalling the established degrader systems.
The enzyme acetyl-CoA carboxylase (ACC) plays a crucial role in fatty acid metabolism. In recent years, ACC has been recognized as a promising drug target for treating different diseases. However, the role of ACC in vascular endothelial cells (ECs) has been neglected so far. To characterize the role of ACC, we used the ACC inhibitor, soraphen A, as a chemical tool, and also a gene silencing approach. We found that ACC1 was the predominant isoform in human umbilical vein ECs as well as in human microvascular ECs and that soraphen A reduced the levels of malonyl-CoA. We revealed that ACC inhibition shifted the lipid composition of EC membranes. Accordingly, membrane fluidity, filopodia formation, and migratory capacity were reduced. The antimigratory action of soraphen A depended on an increase in the cellular proportion of PUFAs and, most importantly, on a decreased level of phosphatidylglycerol. Our study provides a causal link between ACC, membrane lipid composition, and cell migration in ECs. Soraphen A represents a useful chemical tool to investigate the role of fatty acid metabolism in ECs and ACC inhibition offers a new and valuable therapeutic perspective for the treatment of EC migration-related diseases.
5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2+ ions and phospholipids that both act via the N-terminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca2+ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 x g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca2+, OAG caused no stimulation of 5-LO. Also, phospholipids or cellular membranes abolished the effects of OAG. As found previously for Ca2+, OAG renders 5-LO activity resistant against inhibition by glutathione peroxidase activity, and this effect of OAG is reversed by phospholipids. Intriguingly, a 5-LO mutant lacking tryptophan residues (Trp-13, -75, and -102) important for the binding of the 5-LO C2-like domain to phospholipids was not stimulated by OAG. We conclude that OAG directly stimulates 5-LO by acting at a phospholipid binding site located within the C2-like domain.
Recently, we reported that in crude enzyme preparations, a monocyte-derived soluble protein (M-DSP) renders 5-lipoxygenase (5-LO) activity Ca2+-dependent. Here we provide evidence that this M-DSP is glutathione peroxidase (GPx)-1. Thus, the inhibitory effect of the M-DSP on 5-LO could be overcome by the GPx-1 inhibitor mercaptosuccinate and by the broad spectrum GPx inhibitor iodoacetate, as well as by addition of 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPODE). Also, the chromatographic characteristics and the estimated molecular mass (80-100 kDa) of the M-DSP fit to GPx-1 (87 kDa), and GPx-1, isolated from bovine erythrocytes, mimicked the effects of the M-DSP. Intriguingly, only a trace amount of thiol (10 micro M GSH) was required for reduction of 5-LO activity by GPx-1 or the M-DSP. Moreover, the requirement of Ca2+ allowing 5-LO product synthesis in various leukocytes correlated with the respective GPx-1 activities. Mutation of the Ca2+ binding sites within the C2-like domain of 5-LO resulted in strong reduction of 5-LO activity by M-DSP and GPx-1, also in the presence of Ca2+. In summary, our data suggest that interaction of Ca2+ at the C2-like domain of 5-LO protects the enzyme against the effect of GPx-1. Apparently, in the presence of Ca2+, a low lipid hydroperoxide level is sufficient for 5-LO activation.
5-lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis, is expressed in a tissue- and cell differentiation-specific manner. The 5-LO core promoter required for basal promoter activity has a unique (G+C)-rich sequence that contains five tandem Sp1 consensus sequences. The mechanisms involved in the regulation of cell type-specific 5-LO expression are unknown. Here we show that 5-LO expression is regulated by DNA methylation. Treatment of the 5-LO-negative cell lines U937 and HL-60TB with the demethylating agent 5-aza-2'-deoxycytidine (AdC) up-regulated expression of 5-LO primary transcripts and mature mRNA in a similar fashion, indicating that AdC stimulates 5-LO gene transcription. Analysis of the methylation status of the 5-LO promoter revealed that the core promoter region was methylated in U937 and HL-60TB cells, whereas it was unmethylated in the 5-LO-positive parent HL-60 cell line. Reporter gene assays with 5-LO promoter constructs gave up to 68- and 655-fold repression of 5-LO promoter activity in HeLa and Mono Mac 6 cells by methylation. 1,25-dihydroxyvitamin D(3) and transforming growth factor-beta (TGFbeta), potent inducers of the 5-LO pathway in myeloid cell lines, increased 5-LO RNA expression in HL-60TB and U937 cells, but co-treatment with AdC was required to achieve 5-LO expression levels in HL-60TB cells that were comparable with wild-type HL-60 cells. In reporter gene assays, 1,25-dihydroxyvitamin D(3) and TGFbeta were unable to induce promoter activity when the 5-LO promoter constructs were methylated, which suggests that 5-LO promoter demethylation is a prerequisite for the high level induction of 5-LO gene expression by 1,25-dihydroxyvitamin D(3) and TGFbeta and that the effects of both agents on 5-LO mRNA expression are not related to DNA methylation.
Nuclear magnetic resonance (NMR) spectroscopy is a powerful and popular technique for probing the molecular structures, dynamics and chemical properties. However the conventional NMR spectroscopy is bottlenecked by its low sensitivity. Dynamic nuclear polarization (DNP) boosts NMR sensitivity by orders of magnitude and resolves this limitation. In liquid-state this revolutionizing technique has been restricted to a few specific non-biological model molecules in organic solvents. Here we show that the carbon polarization in small biological molecules, including carbohydrates and amino acids, can be enhanced sizably by in situ Overhauser DNP (ODNP) in water at room temperature and at high magnetic field. An observed connection between ODNP 13C enhancement factor and paramagnetic 13C NMR shift has led to the exploration of biologically relevant heterocyclic compound indole. The QM/MM MD simulation underscores the dynamics of intermolecular hydrogen bonds as the driving force for the scalar ODNP in a long-living radical-substrate complex. Our work reconciles results obtained by DNP spectroscopy, paramagnetic NMR and computational chemistry and provides new mechanistic insights into the high-field scalar ODNP.
Upon antibiotic stress Gram-negative pathogens deploy resistance-nodulation-cell division-type tripartite efflux pumps. These include a H+/drug antiporter module that recognizes structurally diverse substances, including antibiotics. Here, we show the 3.5 Å structure of subunit AdeB from the Acinetobacter baumannii AdeABC efflux pump solved by single-particle cryo-electron microscopy. The AdeB trimer adopts mainly a resting state with all protomers in a conformation devoid of transport channels or antibiotic binding sites. However, 10% of the protomers adopt a state where three transport channels lead to the closed substrate (deep) binding pocket. A comparison between drug binding of AdeB and Escherichia coli AcrB is made via activity analysis of 20 AdeB variants, selected on basis of side chain interactions with antibiotics observed in the AcrB periplasmic domain X-ray co-structures with fusidic acid (2.3 Å), doxycycline (2.1 Å) and levofloxacin (2.7 Å). AdeABC, compared to AcrAB-TolC, confers higher resistance to E. coli towards polyaromatic compounds and lower resistance towards antibiotic compounds.
Coronavirus disease 2019 (COVID-19) is a global pandemic posing significant health risks. The diagnostic test sensitivity of COVID-19 is limited due to irregularities in specimen handling. We propose a deep learning framework that identifies COVID-19 from medical images as an auxiliary testing method to improve diagnostic sensitivity. We use pseudo-coloring methods and a platform for annotating X-ray and computed tomography images to train the convolutional neural network, which achieves a performance similar to that of experts and provides high scores for multiple statistical indices (F1 scores > 96.72% (0.9307, 0.9890) and specificity >99.33% (0.9792, 1.0000)). Heatmaps are used to visualize the salient features extracted by the neural network. The neural network-based regression provides strong correlations between the lesion areas in the images and five clinical indicators, resulting in high accuracy of the classification framework. The proposed method represents a potential computer-aided diagnosis method for COVID-19 in clinical practice.
Single-particle tracking enables the analysis of the dynamics of biomolecules in living cells with nanometer spatial and millisecond temporal resolution. This technique reports on the mobility of membrane proteins and is sensitive to the molecular state of a biomolecule and to interactions with other biomolecules. Trajectories describe the mobility of single particles over time and provide information such as the diffusion coefficient and diffusion state. Changes in particle dynamics within single trajectories lead to segmentation, which allows to extract information on transitions of functional states of a biomolecule. Here, mean-squared displacement analysis is developed to classify trajectory segments into immobile, confined diffusing, and freely diffusing states, and to extract the occurrence of transitions between these modes. We applied this analysis to single-particle tracking data of the membrane receptor MET in live cells and analyzed state transitions in single trajectories of the un-activated receptor and the receptor bound to the ligand internalin B. We found that internalin B-bound MET shows an enhancement of transitions from freely and confined diffusing states into the immobile state as compared to un-activated MET. Confined diffusion acts as an intermediate state between immobile and free, as this state is most likely to change the diffusion state in the following segment. This analysis can be readily applied to single-particle tracking data of other membrane receptors and intracellular proteins under various conditions and contribute to the understanding of molecular states and signaling pathways.
The covalent conjugation of ubiquitin-fold modifier 1 (UFM1) to proteins generates a signal that regulates transcription, response to cell stress, and differentiation. Ufmylation is initiated by ubiquitin-like modifier activating enzyme 5 (UBA5), which activates and transfers UFM1 to ubiquitin-fold modifier-conjugating enzyme 1 (UFC1). The details of the interaction between UFM1 and UBA5 required for UFM1 activation and its downstream transfer are however unclear. In this study, we described and characterized a combined linear LC3-interacting region/UFM1-interacting motif (LIR/UFIM) within the C terminus of UBA5. This single motif ensures that UBA5 binds both UFM1 and light chain 3/γ-aminobutyric acid receptor-associated proteins (LC3/GABARAP), two ubiquitin (Ub)-like proteins. We demonstrated that LIR/UFIM is required for the full biological activity of UBA5 and for the effective transfer of UFM1 onto UFC1 and a downstream protein substrate both in vitro and in cells. Taken together, our study provides important structural and functional insights into the interaction between UBA5 and Ub-like modifiers, improving the understanding of the biology of the ufmylation pathway.
RcsF, a proposed auxiliary regulator of the regulation of capsule synthesis (rcs) phosphorelay system, is a key element for understanding the RcsC-D-A/B signaling cascade, which is responsible for the regulation of more than 100 genes and is involved in cell division, motility, biofilm formation, and virulence. The RcsC-D-A/B system is one of the most complex bacterial signal transduction pathways, consisting of several membrane-bound and soluble proteins. RcsF is a lipoprotein attached to the outer membrane and plays an important role in activating the RcsC-d-A/B pathway. The exact mechanism of activation of the rcs phosphorelay by RcsF, however, remains unknown. We have analyzed the sequence of RcsF and identified three structural elements: 1) an N-terminal membrane-anchored helix (residues 3-13), 2) a loop (residues 14-48), and 3) a C-terminal folded domain (residues 49-134). We have determined the structure of this C-terminal domain and started to investigate its interaction with potential partners. Important features of its structure are two disulfide bridges between Cys-74 and Cys-118 and between Cys-109 and Cys-124. To evaluate the importance of this RcsF disulfide bridge network in vivo, we have examined the ability of the full-length protein and of specific Cys mutants to initiate the rcs signaling cascade. The results indicate that the Cys-74/Cys-118 and the Cys-109/Cys-124 residues correlate pairwise with the activity of RcsF. Interaction studies showed a weak interaction with an RNA hairpin. However, no interaction could be detected with reagents that are believed to activate the rcs phosphorelay, such as lysozyme, glucose, or Zn(2+) ions.
Hypoxia potentiates palmitate-induced pro-inflammatory activation of primary human macrophages
(2015)
Pro-inflammatory cytokines secreted by adipose tissue macrophages (ATMs) contribute to chronic low-grade inflammation and obesity-induced insulin resistance. Recent studies have shown that adipose tissue hypoxia promotes an inflammatory phenotype in ATMs. However, our understanding of how hypoxia modulates the response of ATMs to free fatty acids within obese adipose tissue is limited. We examined the effects of hypoxia (1% O2) on the pro-inflammatory responses of human monocyte-derived macrophages to the saturated fatty acid palmitate. Compared with normoxia, hypoxia significantly increased palmitate-induced mRNA expression and protein secretion of IL-6 and IL-1β. Although palmitate-induced endoplasmic reticulum stress and nuclear factor κB pathway activation were not enhanced by hypoxia, hypoxia increased the activation of JNK and p38 mitogen-activated protein kinase signaling in palmitate-treated cells. Inhibition of JNK blocked the hypoxic induction of pro-inflammatory cytokine expression, whereas knockdown of hypoxia-induced transcription factors HIF-1α and HIF-2α alone or in combination failed to reduce IL-6 and only modestly reduced IL-1β gene expression in palmitate-treated hypoxic macrophages. Enhanced pro-inflammatory cytokine production and JNK activity under hypoxia were prevented by inhibiting reactive oxygen species generation. In addition, silencing of dual-specificity phosphatase 16 increased normoxic levels of IL-6 and IL-1β and reduced the hypoxic potentiation in palmitate-treated macrophages. The secretome of hypoxic palmitate-treated macrophages promoted IL-6 and macrophage chemoattractant protein 1 expression in primary human adipocytes, which was sensitive to macrophage JNK inhibition. Our results reveal that the coexistence of hypoxia along with free fatty acids exacerbates macrophage-mediated inflammation.
Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic beta-cells, and small molecule activators of GK (GKAs) are under evaluation for the treatment of type 2 diabetes. In liver, GK activity is controlled by the GK regulatory protein (GKRP), which forms an inhibitory complex with the enzyme. Here, we performed isothermal titration calorimetry and surface plasmon resonance experiments to characterize GK-GKRP binding and to study the influence that physiological and pharmacological effectors of GK have on the protein-protein interaction. In the presence of fructose-6-phosphate, GK-GKRP complex formation displayed a strong entropic driving force opposed by a large positive enthalpy; a negative change in heat capacity was observed (Kd = 45 nm, DeltaH = 15.6 kcal/mol, TDeltaS = 25.7 kcal/mol, DeltaCp = -354 cal mol(-1) K(-1)). With k(off) = 1.3 x 10(-2) s(-1), the complex dissociated quickly. The thermodynamic profile suggested a largely hydrophobic interaction. In addition, effects of pH and buffer demonstrated the coupled uptake of one proton and indicated an ionic contribution to binding. Glucose decreased the binding affinity between GK and GKRP. This decrease was potentiated by an ATP analogue. Prototypical GKAs of the amino-heteroaryl-amide type bound to GK in a glucose-dependent manner and impaired the association of GK with GKRP. This mechanism might contribute to the antidiabetic effects of GKAs.
The β-subunits of Na,K-ATPase and H,K-ATPase have important functions in maturation and plasma membrane targeting of the catalytic α-subunit but also modulate the transport activity of the holoenzymes. In this study, we show that tryptophan replacement of two highly conserved tyrosines in the transmembrane domain of both Na,K- and gastric H,K-ATPase β-subunits resulted in considerable shifts of the voltage-dependent E1P/E2P distributions toward the E1P state as inferred from presteady-state current and voltage clamp fluorometric measurements of tetramethylrhodamine-6-maleimide-labeled ATPases. The shifts in conformational equilibria were accompanied by significant decreases in the apparent affinities for extracellular K+ that were moderate for the Na,K-ATPase β-(Y39W,Y43W) mutation but much more pronounced for the corresponding H,K-ATPase β-(Y44W,Y48W) variant. Moreover in the Na,K-ATPase β-(Y39W,Y43W) mutant, the apparent rate constant for reverse binding of extracellular Na+ and the subsequent E2P-E1P conversion, as determined from transient current kinetics, was significantly accelerated, resulting in enhanced Na+ competition for extracellular K+ binding especially at extremely negative potentials. Analogously the reverse binding of extracellular protons and subsequent E2P-E1P conversion was accelerated by the H,K-ATPase β-(Y44W,Y48W) mutation, and H+ secretion was strongly impaired. Remarkably tryptophan replacements of residues in the M7 segment of Na,K- and H,K-ATPase α-subunits, which are at interacting distance to the β-tyrosines, resulted in similar E1 shifts, indicating their participation in stabilization of E2. Thus, interactions between selected residues within the transmembrane regions of α- and β-subunits of P2C-type ATPases exert an E2-stabilizing effect, which is of particular importance for efficient H+ pumping by H,K-ATPase under in vivo conditions.
The Na+/K+-ATPase maintains the physiological Na+ and K+ gradients across the plasma membrane in most animal cells. The functional unit of the ion pump is comprised of two mandatory subunits including the α-subunit, which mediates ATP hydrolysis and ion translocation, as well as the β-subunit, which acts as a chaperone to promote proper membrane insertion and trafficking in the plasma membrane. To examine the conformational dynamics between the α- and β-subunits of the Na+/K+-ATPase during ion transport, we have used fluorescence resonance energy transfer, under voltage clamp conditions on Xenopus laevis oocytes, to differentiate between two models that have been proposed for the relative orientation of the α- and β-subunits. These experiments were performed by measuring the time constant of irreversible donor fluorophore destruction with fluorescein-5-maleimide as the donor fluorophore and in the presence or absence of tetramethylrhodamine-6-maleimide as the acceptor fluorophore following labeling on the M3-M4 or M5-M6 loop of the α-subunit and the β-subunit. We have also used fluorescence resonance energy transfer to investigate the relative movement between the two subunits as the ion pump shuttles between the two main conformational states (E1 and E2) as described by the Albers-Post scheme. The results from this study have identified a model for the orientation of the β-subunit in relation to the α-subunit and suggest that the α- and β-subunits move toward each other during the E2 to E1 conformational transition.
Macrophages respond to the Th2 cytokine IL-4 with elevated expression of arachidonate 15-lipoxygenase (ALOX15). Although IL-4 signaling elicits anti-inflammatory responses, 15-lipoxygenase may either support or inhibit inflammatory processes in a context-dependent manner. AMP-activated protein kinase (AMPK) is a metabolic sensor/regulator that supports an anti-inflammatory macrophage phenotype. How AMPK activation is linked to IL-4-elicited gene signatures remains unexplored. Using primary human macrophages stimulated with IL-4, we observed elevated ALOX15 mRNA and protein expression, which was attenuated by AMPK activation. AMPK activators, e.g. phenformin and aminoimidazole-4-carboxamide 1-β-d-ribofuranoside inhibited IL-4-evoked activation of STAT3 while leaving activation of STAT6 and induction of typical IL-4-responsive genes intact. In addition, phenformin prevented IL-4-induced association of STAT6 and Lys-9 acetylation of histone H3 at the ALOX15 promoter. Activating AMPK abolished cellular production of 15-lipoxygenase arachidonic acid metabolites in IL-4-stimulated macrophages, which was mimicked by ALOX15 knockdown. Finally, pretreatment of macrophages with IL-4 for 48 h increased the mRNA expression of the proinflammatory cytokines IL-6, IL-12, CXCL9, and CXCL10 induced by subsequent stimulation with lipopolysaccharide. This response was attenuated by inhibition of ALOX15 or activation of AMPK during incubation with IL-4. In conclusion, limiting ALOX15 expression by AMPK may promote an anti-inflammatory phenotype of IL-4-stimulated human macrophages.
By translocating proteasomal degradation products into the endoplasmic reticulum for loading of major histocompatibility complex I molecules, the ABC transporter TAP plays a focal role in the adaptive immunity against infected or malignantly transformed cells. A key question regarding the transport mechanism is how the quality of the incoming peptide is detected and how this information is transmitted to the ATPase domains. To identify residues involved in this process, we evolved a Trojan horse strategy in which a small artificial protease is inserted into antigenic epitopes. After binding, the TAP backbone in contact is cleaved, allowing the peptide sensor site to be mapped by mass spectrometry. Within this sensor site, we identified residues that are essential for tight coupling of peptide binding and transport. This sensor and transmission interface is restructured during the ATP hydrolysis cycle, emphasizing its important function in the cross-talk between the transmembrane and the nucleotide-binding domains. This allocrite sensor may be similarly positioned in other members of the ABC exporter family.
Biogenesis of mitochondrial cytochrome c oxidase (COX) relies on a large number of assembly factors, among them the transmembrane protein Surf1. The loss of human Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder caused by severe COX deficiency. In the bacterium Paracoccus denitrificans, two homologous proteins, Surf1c and Surf1q, were identified, which we characterize in the present study. When coexpressed in Escherichia coli together with enzymes for heme a synthesis, the bacterial Surf1 proteins bind heme a in vivo. Using redox difference spectroscopy and isothermal titration calorimetry, the binding of the heme cofactor to purified apo-Surf1c and apo-Surf1q is quantified: Each of the Paracoccus proteins binds heme a in a 1:1 stoichiometry and with Kd values in the submicromolar range. In addition, we identify a conserved histidine as a residue crucial for heme binding. Contrary to most earlier concepts, these data support a direct role of Surf1 in heme a cofactor insertion into COX subunit I by providing a protein-bound heme a pool.
Biological membranes are complex and dynamic assemblies of lipids and proteins. Poikilothermic organisms including bacteria, fungi, reptiles, and fish do not control their body temperature and must adapt their membrane lipid composition in order to maintain membrane fluidity in the cold. This adaptive response was termed homeoviscous adaptation and has been frequently studied with a specific focus on the acyl chain composition of membrane lipids. Mass spectrometry-based lipidomics can nowadays provide more comprehensive insights into the complexity of lipid remodeling during adaptive responses. Eukaryotic cells compartmentalize biochemical processes in organelles with characteristic surface properties, and the lipid composition of organelle membranes must be tightly controlled in order to maintain organelle function and identity during adaptive responses. Some highly differentiated cells such as neurons maintain unique lipid compositions with specific physicochemical properties. To date little is known about the sensory mechanisms regulating the acyl chain profile in such specialized cells or during adaptive responses. Here we summarize our current understanding of lipid metabolic networks with a specific focus on the role of physicochemical membrane properties for the regulation of the acyl chain profile during homeoviscous adaptation. By comparing the mechanisms of the bacterial membrane sensors with the prototypical eukaryotic lipid packing sensor Mga2 from Saccharomyces cerevisiae, we identify common operational principles that might guide our search for novel membrane sensors in different organelles, organisms, and highly specialized cells.
We demonstrated previously that 5-lipoxygenase (5-LO), a key enzyme in leukotriene biosynthesis, can be phosphorylated by p38 MAPK-regulated MAPKAP kinases (MKs). Here we show that mutation of Ser-271 to Ala in 5-LO abolished MK2 catalyzed phosphorylation and clearly reduced phosphorylation by kinases prepared from stimulated polymorphonuclear leukocytes and Mono Mac 6 cells. Compared with heat shock protein 27 (Hsp-27), 5-LO was a weak substrate for MK2. However, the addition of unsaturated fatty acids (i.e. arachidonate 1-50 microm) up-regulated phosphorylation of 5-LO, but not of Hsp-27, by active MK2 in vitro, resulting in a similar phosphorylation as for Hsp-27. 5-LO was phosphorylated also by other serine/threonine kinases recognizing the motif Arg-Xaa-Xaa-Ser (protein kinase A, Ca(2+)/calmodulin-dependent kinase II), but these activities were not increased by fatty acids. HeLa cells expressing wild type 5-LO or S271A-5-LO, showed prominent 5-LO activity when incubated with Ca(2+)-ionophore plus arachidonate. However, when stimulated with only exogenous arachidonic acid, activity for the S271A mutant was significantly lower as compared with wild type 5-LO. It appears that phosphorylation at Ser-271 is more important for 5-LO activity induced by a stimulus that does not prominently increase intracellular Ca(2+) and that arachidonic acid stimulates leukotriene biosynthesis also by promoting this MK2-catalyzed phosphorylation.
Malfunction of the actin cytoskeleton is linked to numerous human diseases including neurological disorders and cancer. LIMK1 (LIM domain kinase 1) and its paralogue LIMK2 are two closely related kinases that control actin cytoskeleton dynamics. Consequently, they are potential therapeutic targets for the treatment of such diseases. In the present review, we describe the LIMK conformational space and its dependence on ligand binding. Furthermore, we explain the unique catalytic mechanism of the kinase, shedding light on substrate recognition and how LIMK activity is regulated. The structural features are evaluated for implications on the drug discovery process. Finally, potential future directions for targeting LIMKs pharmacologically, also beyond just inhibiting the kinase domain, are discussed.
Epigenetic control of microsomal prostaglandin E synthase-1 by HDAC-mediated recruitment of p300
(2017)
Nonsteroidal anti-inflammatory drugs are the most widely used medicine to treat pain and inflammation, and to inhibit platelet function. Understanding the expression regulation of enzymes of the prostanoid pathway is of great medical relevance. Histone acetylation crucially controls gene expression. We set out to identify the impact of histone deacetylases (HDACs) on the generation of prostanoids and examine the consequences on vascular function. HDAC inhibition (HDACi) with the pan-HDAC inhibitor, vorinostat, attenuated prostaglandin (PG)E2 generation in the murine vasculature and in human vascular smooth muscle cells. In line with this, the expression of the key enzyme for PGE2 synthesis, microsomal PGE synthase-1 (PTGES1), was reduced by HDACi. Accordingly, the relaxation to arachidonic acid was decreased after ex vivo incubation of murine vessels with HDACi. To identify the underlying mechanism, chromatin immunoprecipitation (ChIP) and ChIP-sequencing analysis were performed. These results suggest that HDACs are involved in the recruitment of the transcriptional activator p300 to the PTGES1 gene and that HDACi prevented this effect. In line with the acetyltransferase activity of p300, H3K27 acetylation was reduced after HDACi and resulted in the formation of heterochromatin in the PTGES1 gene. In conclusion, HDAC activity maintains PTGES1 expression by recruiting p300 to its gene.
The accumulation and distribution of characteristic secondary products in the different organs of an Aloe plant (A. succotrina Lam.) were studied by high performance liquid chromatography for the first time. In the leaves of the Aloe plant, only anthrone-C-glycosyls of the 7-hydroxyaloin type and, for the first time in plant material, the free anthraquinone 7-hydroxyaloeemodin were found. In contrast to previous reports on the distribution of secondary products in Aloe plants, anthrone-C-glycosyls were also detected in flowers, bracts and the inflorescence axis of the species examined. Aloesaponol I, a tetrahydroanthracene aglycone, was only present in the underground organs and in the stem. The 2-alkylchromone-C-glucosyl aloeresin B showed no specific occurrence as it was found in every type of organ. Based on these results and the findings of recent studies on Aloe roots and flowers, a distribution scheme of polyketide types in the Aloe plant was established. It suggests a separate and independent anthranoid metabolism for underground Aloe organs and stem on the one hand, and for leaves and inflorescence organs on the other hand. In the latter structures anthranoid metabolism seems to be additionally compartmentalized as the anthranoid pro files of inflorescence organs and leaves differ in two points relevant to anthranoid biosynthe sis: firstly, the occurrence of anthrone aglycones and secondly, the individual content of corresponding anthrone-C-glucosyl diastereomers.
From the leaf exudate of Aloe lateritia ENGLER the C-glucosyl com pounds homonataloin, aloeresin A and aloesin (synon. aloeresin B) were isolated together with the anthraquinone nataloeem odin-8-methylether and spectroscopically identified. Hom onataloin, widely distributed in Aloe species, was separated into homonataloin A and B by combined TLC and DCCC. In their 1 D and 2D 1H NMR spectra only the shifts of the 2′-hydroxyl protons of both glucosyl residues differ significantly, indicative of 10 S (A) resp. 10 S (B) configurations. In both com pounds the anthrone is in β-position of the D-glucopyranosyl, as determined by the large coupling constants of the anomeric protons. The 13C NMR signals are unambiguously assigned by the use of DEPT, APT and gated-decoupling methods. Only the chemical shifts of C -11 and C -14 show significant differences between both diastereomers due to the adjacent 2′-sugar hydroxyls. The two homonataloins differ mostly in optical rotation and circulardichroism due to different configurations at C - 10 of the anthrone part. The absolute configurations of the diastereomers are determined by correlation of their CD spectra with the CD spectra of the structural analogues 7-hydroxyaloins A and B, which shows that hom onataloin A is the 10 S, 1′S-compound and that homonataloin B has 10 R, 1′S-configuration.
To better understand the role of sphingolipids in the multifactorial process of inflammatory bowel disease (IBD), we elucidated the role of CerS4 in colitis and colitis-associated cancer (CAC). For this, we utilized the azoxymethane/dextran sodium sulphate (AOM/DSS)-induced colitis model in global CerS4 knockout (CerS4 KO), intestinal epithelial (CerS4 Vil/Cre), or T-cell restricted knockout (CerS4 LCK/Cre) mice. CerS4 KO mice were highly sensitive to the toxic effect of AOM/DSS, leading to a high mortality rate. CerS4 Vil/Cre mice had smaller tumors than WT mice. In contrast, CerS4 LCK/Cre mice frequently suffered from pancolitis and developed more colon tumors. In vitro, CerS4-depleted CD8+ T-cells isolated from the thymi of CerS4 LCK/Cre mice showed impaired proliferation and prolonged cytokine production after stimulation in comparison with T-cells from WT mice. Depletion of CerS4 in human Jurkat T-cells led to a constitutively activated T-cell receptor and NF-κB signaling pathway. In conclusion, the deficiency of CerS4 in T-cells led to an enduring active status of these cells and prevents the resolution of inflammation, leading to a higher tumor burden in the CAC mouse model. In contrast, CerS4 deficiency in epithelial cells resulted in smaller colon tumors and seemed to be beneficial. The higher tumor incidence in CerS4 LCK/Cre mice and the toxic effect of AOM/DSS in CerS4 KO mice exhibited the importance of CerS4 in other tissues and revealed the complexity of general targeting CerS4.
Today, the term buchu refers to the two species in commerce, Agathosma betulina (P.J.Bergius) Pillans and Agathosma crenulata (L.) Pillans (Rutaceae). Its traditional use in urinary tract infections and related ailments made it a popular remedy, specifically in the US, in 19th century, but with the advent of antibiotics it became largely obsolete. Recent focus is on technological use and on the essential oil for use in the perfume and food-flavouring industry. A review of the scarce pharmacological research revealed moderate antimicrobial activity for a leaf extract but not the essential oil of both species in the MIC assay. In the 5-lipoxygenase (5-LO) assay the essential oil of both species revealed IC50 values of 50.37 ± 1.87 μg/ml and 59.15 ± 7.44 μg/ml, respectively. In another study 98% inhibitory activity was determined for 250 μg/ml of an ethanolic extract of A. betulina on cyclooxygenase (COX)-1 and a 25% inhibitory activity on COX-2. Analgesic activity of an ethanolic extract of A. betulina was shown in mice. Moderate antioxidant activity was determined for methanol:dichlormethane extracts of A. betulina and A. crenulata and an aqueous extract of A. betulina showed a Trolox equivalent antioxidant capacity (TEAC) of 11.8 µM Trolox. Recent in vitro studies with a commercial aqueous extract of buchu revealed increased uptake of glucose added to 3T3-L1 cell line, significant inhibition of the respiratory burst of neutrophils and monocytes, reduction in the expression of adhesion molecules and inhibition of the release of IL-6 and TNF-α. In diabetic rats the ingestion of aqueous buchu extract completely normalized the glucose level and in rats receiving a high fat diet the consumption of aqueous buchu extract resulted in less weight gain and less intraperitoneal fat gain as well as reduction of elevated blood pressure to normal associated with cardioprotective effects. Limitations in the hitherto conducted research lie in the undisclosed composition of the buchu extracts used and the difficulty in extrapolating data from animal studies to humans. Health claims for buchu products need to be substantiated by randomized, double-blind and placebo-controlled studies. Only then can they be promoted for their true therapeutic potential.
The absolute configurations of the diastereomeric 10-hydroxyaloins, which may be regarded as parent structures for other naturally occurring oxanthrone-C-glucosyls, have been established as 10R, 16 R (A) and 10 S, 16 R (B) by an X-ray structure analysis of the A-octaacetyl derivative (C 16 is the anomeric glucosyl carbon atom). The determination was confirmed by CD spectroscopic comparison with the structural analogues aloins A and B, which should prove useful for making future configurational assignments within this class of compounds. A conformational analysis by the use of a molecular modeling method based on force-field calculations reveals the presence of an extra- and an intra-form, the extra-form of which is energetically preferred.
In the course of systematic investigations on sila-substituted parasympatholytics the diphenyl(2-aminoethoxymethyl)silanols 3b and 4b (and its carbon analogue 4a) were synthesized and characterized by their physical and chemical properties. In the solid state 4a and 4b form strong O-H---N hydrogen bonds, which are intramolecular (4a) and intermolecular (4b), respectively. 4a and 4b were found to be weak antimuscarinic agents (4b >4a) and strong papaverine-like spasmolytics (4a ≈4b).
In bioengineering, scaffold proteins have been increasingly used to recruit molecules to parts of a cell, or to enhance the efficacy of biosynthetic or signalling pathways. For example, scaffolds can be used to make weak or non-immunogenic small molecules immunogenic by attaching them to the scaffold, in this role called carrier. Here, we present the dodecin from Mycobacterium tuberculosis (mtDod) as a new scaffold protein. MtDod is a homododecameric complex of spherical shape, high stability and robust assembly, which allows the attachment of cargo at its surface. We show that mtDod, either directly loaded with cargo or equipped with domains for non-covalent and covalent loading of cargo, can be produced recombinantly in high quantity and quality in Escherichia coli. Fusions of mtDod with proteins of up to four times the size of mtDod, e.g. with monomeric superfolder green fluorescent protein creating a 437 kDa large dodecamer, were successfully purified, showing mtDod’s ability to function as recruitment hub. Further, mtDod equipped with SYNZIP and SpyCatcher domains for post-translational recruitment of cargo was prepared of which the mtDod/SpyCatcher system proved to be particularly useful. In a case study, we finally show that mtDod-peptide fusions allow producing antibodies against human heat shock proteins and the C-terminus of heat shock cognate 70 interacting protein (CHIP).
Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.
The Corona pandemic has painfully taught us the threat of new pathogens in a globalized world and how vital modern vaccines are. Platform technologies play an important role in the discovery of new vaccines as reducing the time for the development dramatically — time that saves lives. Here, we present the protein Dodecin and how it may be utilized as a versatile platform technology to produce cheap and robust new vaccines for everyone in all parts of the world.
Approximately 80 % of persistent wound infections are affected by the presence of bacterial biofilms, resulting in a severe clinical challenge associated with prolonged healing periods, increased morbidity, and high healthcare costs. Unfortunately, in vitro models for wound infection research almost exclusively focus on early infection stages with planktonic bacteria. In this study, we present a new approach to emulate biofilm-infected human wounds by three-dimensional human in vitro systems. For this purpose, a matured biofilm consisting of the clinical key wound pathogen Pseudomonas aeruginosa was pre-cultivated on electrospun scaffolds allowing for non-destructive transfer of the matured biofilm to human in vitro wound models. We infected tissue-engineered human in vitro skin models as well as ex vivo human skin explants with the biofilm and analyzed structural tissue characteristics, biofilm growth behavior, and biofilm-tissue interactions. The structural development of biofilms in close proximity to the tissue, resulting in high bacterial burden and in vivo-like morphology, confirmed a manifest wound infection on all tested wound models, validating their applicability for general investigations of biofilm growth and structure. The extent of bacterial colonization of the wound bed, as well as the subsequent changes in molecular composition of skin tissue, were inherently linked to the characteristics of the underlying wound models including their viability and origin. Notably, the immune response observed in viable ex vivo and in vitro models was consistent with previous in vivo reports. While ex vivo models offered greater complexity and closer similarity to the in vivo conditions, in vitro models consistently demonstrated higher reproducibility. As a consequence, when focusing on direct biofilm-skin interactions, the viability of the wound models as well as their advantages and limitations should be aligned to the particular research question of future studies. Altogether, the novel model allows for a systematic investigation of host-pathogen interactions of bacterial biofilms and human wound tissue, also paving the way for development and predictive testing of novel therapeutics to combat biofilm-infected wounds.
Approximately 80 % of persistent wound infections are affected by the presence of bacterial biofilms, resulting in a severe clinical challenge associated with prolonged healing periods, increased morbidity, and high healthcare costs. Unfortunately, in vitro models for wound infection research almost exclusively focus on early infection stages with planktonic bacteria. In this study, we present a new approach to emulate biofilm-infected human wounds by three-dimensional human in vitro systems. For this purpose, a matured biofilm consisting of the clinical key wound pathogen Pseudomonas aeruginosa was pre-cultivated on electrospun scaffolds allowing for non-destructive transfer of the matured biofilm to human in vitro wound models. We infected tissue-engineered human in vitro skin models as well as ex vivo human skin explants with the biofilm and analyzed structural tissue characteristics, biofilm growth behavior, and biofilm-tissue interactions. The structural development of biofilms in close proximity to the tissue, resulting in high bacterial burden and in vivo-like morphology, confirmed a manifest wound infection on all tested wound models, validating their applicability for general investigations of biofilm growth and structure. The extent of bacterial colonization of the wound bed, as well as the subsequent changes in molecular composition of skin tissue, were inherently linked to the characteristics of the underlying wound models including their viability and origin. Notably, the immune response observed in viable ex vivo and in vitro models was consistent with previous in vivo reports. While ex vivo models offered greater complexity and closer similarity to the in vivo conditions, in vitro models consistently demonstrated higher reproducibility. As a consequence, when focusing on direct biofilm-skin interactions, the viability of the wound models as well as their advantages and limitations should be aligned to the particular research question of future studies. Altogether, the novel model allows for a systematic investigation of host-pathogen interactions of bacterial biofilms and human wound tissue, also paving the way for development and predictive testing of novel therapeutics to combat biofilm-infected wounds.
Chromosomal translocations (CTs) are a genetic hallmark of cancer. They could be identified as recurrent genetic aberrations in hemato-malignancies and solid tumors. More than 40% of all “cancer genes” were identified in recurrent CTs. Most of these CTs result in the production of oncofusion proteins of which many have been studied over the past decades. They influence signaling pathways and/or alter gene expression. However, a precise mechanism for how these CTs arise and occur in a nearly identical fashion in individuals remains to be elucidated. Here, we performed experiments that explain the onset of CTs: (1) proximity of genes able to produce prematurely terminated transcripts, which lead to the production of (2) trans-spliced fusion RNAs, and finally, the induction of (3) DNA double-strand breaks which are subsequently repaired via EJ repair pathways. Under these conditions, balanced chromosomal translocations could be specifically induced. The implications of these findings will be discussed.
Major histocompatibility complex class I (MHC I) molecules present antigenic peptides to cytotoxic T cells to eliminate infected or cancerous cells. The transporter associated with antigen processing (TAP) shuttles proteasomally generated peptides into the ER for MHC I loading. As central part of the peptide-loading complex (PLC), TAP is targeted by viral factors, which inhibit peptide supply and thereby impact MHC I-mediated immune responses. However, it is still poorly understood how antigen presentation via different MHC I allotypes is affected by TAP inhibition. Here, we show that conditional expression of herpes simplex viral ICP47 suppresses surface presentation of HLA-A and HLA-C, but not of HLA-B, while the human cytomegaloviral US6 reduces surface levels of all MHC I allotypes. This marked difference in HLA-B antigen presentation is echoed by an enrichment of HLA-B allomorphs at US6-arrested PLC in comparison to ICP47-PLC. Although both viral factors prevent TAP-mediated peptide supply, our data imply that MHC I allomorphs favor different conformationally arrested states of the PLC, leading to differential downregulation of MHC I surface presentation. These findings will help understand MHC I biology in general and will even advance the targeted treatment of infections depending on patients’ allotypes.