Duyệt theo Kiểu tài liệu "Journal Article"
- Ấn phẩm3-hydroxykynurenine is a ROS-inducing cytotoxic tryptophan metabolite that disrupts the TCA cycle(bioRxiv, 2023) Buchanan, Jane L.Tryptophan is an essential amino acid that is extensively characterized as a regulator of cellular function through its metabolism by indoleamine 2,3-deoxygenase (IDO) into the kynurenine pathway. However, despite decades of research on tryptophan metabolism, the metabolic regulatory roles of it and its metabolites are not well understood. To address this, we performed an activity metabolomics screen of tryptophan and most of its known metabolites in cell culture. We discovered that treatment of human colon cancer cells (HCT116) with 3- hydroxykynurenine (3-HK), a metabolite of kynurenine, potently disrupted TCA cycle function. Citrate and aconitate levels were increased, while isocitrate and all downstream TCA metabolites were decreased, suggesting decreased aconitase function. We hypothesized that 3HK or one of its metabolites increased reactive oxygen species (ROS) and inhibited aconitase activity. Accordingly, we observed almost complete depletion of reduced glutathione and a decrease in total glutathione levels. We observed a dose-dependent decrease in cell viability after 48 hours of 3HK treatment.
- Ấn phẩm3D Graph Contrastive Learning for Molecular Property Prediction(bioRxiv, 2023) Moon, Kisung
- Ấn phẩmA bespoke analytical workflow for the confident identification of sulfopeptides and their discrimination from phosphopeptides(bioRxiv, 2023) Daly, Leonard A.Protein tyrosine sulfation (sY) is a post-translational modification (PTM) catalysed by Golgi-resident Tyrosyl Protein SulfoTransferases (TPSTs). Information on protein tyrosine sulfation is currently limited to ~50 human proteins with only a handful having verified sites of sulfation. The contribution of this chemical moiety for the regulation of biological processes, both inside and outside the cell, remains poorly defined, in large part due to analytical limitations. Mass spectrometry-based proteomics is the method of choice for PTM analysis, but has yet to be applied for the systematic investigation and large-scale analysis of biomolecular sulfation (constituting the ‘sulfome’), primarily due to issues associated with discrimination of sY- from phosphotyrosine (pY)-containing peptides. In this study, we developed a mass spectrometry (MS)-based workflow centred on the characterization of sY-peptides, incorporating optimised Zr4+-IMAC and TiO2 enrichment strategies. Extensive characterization of a panel of sY- and pY-peptides using an array of MS fragmentation regimes (CID, HCD, EThcC, ETciD, UVPD) highlights differences in the ability to generate site-determining product ions, which can be exploited to differentiate sulfated peptides from nominally isobaric phosphopeptides based on precursor ion neutral loss at low collision energy. Application of our analytical workflow to a HEK-293 cell extracellular secretome facilitated identification of 21 new sulfotyrosine-containing proteins, several of which we validate enzymatically using in vitro sulfation assays. This study demonstrates the applicability of our strategy for confident, high-throughput, ‘sulfomics’ studies, and reveals new sY interplay between enzymes relevant to both protein and glycan sulfation.
- Ấn phẩmA cautionary note on the use of N-acetylcysteine as a reactive oxygen species antagonist to assess copper mediated cell death(2023) Graham, ebecca E.A new form of cell death has recently been proposed involving copper-induced cell death, termed cuproptosis. This new form of cell death has been widely studied in relation to a novel class of copper ionophores, including elesclomol and disulfiram. However, the exact mechanism leading to cell death remains contentious. The oldest and mostly widely accepted biological mechanism is that the accumulated intracellular copper leads to excessive build-up of reactive oxygen species and that this is what ultimately leads to cell death. Most of this evidence is largely based on studies using N-acetylcysteine (NAC), an antioxidant, to relieve the oxidative stress and prevent cell death. However, here we have demonstrated using inductively coupled mass-spectrometry, that NAC pretreatment significantly reduces intracellular copper uptake triggered by the ionophores, elesclomol and disulfiram, suggesting that reduction in copper uptake, rather than the antioxidant activity of NAC, is responsible for the diminished cell death. We present further data showing that key mediators of reactive oxygen species are not upregulated in response to elesclomol treatment, and further that sensitivity of cancer cell lines to reactive oxygen species does not correlate with sensitivity to these copper ionophores
- Ấn phẩmA global LC-MS2-based methodology to identify and quantify anionic phospholipids in plant samples(bioRxiv, 2023) Genva, ManonAnionic phospholipids (PS, PA, PI, PIPs) are low abundant phospholipids with impactful functions in cell signaling, membrane trafficking and cell differentiation processes. They can be quickly metabolized and can transiently accumulate at define spots within the cell or an organ to respond to physiological or environmental stimuli. As even a small change in their composition profile will produce a significant effect on biological processes, it is crucial to develop a sensitive and optimized analytical method to accurately detect and quantify them. While thin layer chromatography (TLC) separation coupled with gas chromatography (GC) detection methods already exist, they do not allow for precise, sensitive and accurate quantification of all anionic phospholipid species. Here we developed a method based on high performance liquid chromatography (HPLC) combined with two-dimensional mass spectrometry (MS2) by MRM mode to detect and quantify all molecular species and classes of anionic phospholipids in one-shot. This method is based on a derivatization step by methylation that greatly enhances the ionization, the separation of each peaks, the peak resolution as well as the limit of detection and quantification for each individual molecular species, and more particularly for PA and PS. Our method universally works in various plant samples. Remarkably, we identified that PS is enriched with very long chain fatty acids in the roots but not in aerial organs of Arabidopsis thaliana.
- Ấn phẩmA global, integrated view of the ubiquitylation site occupancy and dynamics(2023) Prus, GabrielaUbiquitylation regulates virtually all proteins and biological processes in a cell. However, the global site-specific occupancy (stoichiometry) and turnover rate of ubiquitylation have never been quantified. Here, we present the first integrated picture of ubiquitylation site occupancy and half-life. Ubiquitylation occupancy spans four orders of magnitude, but the median ubiquitylation site occupancy is three orders of magnitude lower than that of 20 phosphorylation. The occupancy, turnover rate, and the regulation of sites by proteasome inhibitors show strong interrelationships. These properties can discriminate signalingrelevant sites from the sites involved in proteasomal degradation. The sites strongly upregulated by proteasome inhibitors have a longer half-life, and the half-life increases with increasing protein length. Importantly, a previously unknown surveillance mechanism 25 rapidly deubiquitylates all ubiquitin-specific E1 and E2 enzymes and protects them against bystander ubiquitylation accumulation. This work reveals general principles of ubiquitylation-dependent governance and offers conceptual insights into the dynamic regulation of the cell.
- Ấn phẩmA guide to the in vitro reconstitution of Cdc42 GTPase activity and its regulation(bioRxiv, 2023) Tschirpke, SophieCdc42 is a small Rho-type GTPase and the main regulator of cell division in eukaryotes. It is surrounded by a large network of regulatory proteins. To understand the processes around cell division, in-depth understanding of Cdc42 and its regulation is required. In vitro reconstitutions are a suitable tool for such detailed mechanistic studies, as they allow a high level of control over the conditions and components used and. For these Cdc42 and its regulators need to be expressed, purified, and tested for their activity. There are many methods described for this, but their details, possible difficulties, and points of failure are rarely discussed. This makes in vitro studies on Cdc42 less accessible to scientists that have a background different from biochemistry. We here present our experience with working with Cdc42 in vitro. We describe the recombinant expression and purification behaviour of 12 Cdc42, six Cdc42-mNeonGreenSW and four Cdc42-sfGFPSW constructs in E. coli. We explore Cdc42 dimerisation in vitro and assess its activity using GTPase Glo assays and Flag-pulldown assays. GTPase Glo assays turn out to be a reliable tool to quantitatively asses GTPase activities, wheareas pulldown experiments are more error prone. We find that most Cdc42 constructs, with the exception of those with an N-terminal Twin-Step-tag, show a similar GTPase activity and interaction with the GDP/GTP exchange factor Cdc24. We close with using enterokinase and TEV protease to generate untagged Cdc42.
- Ấn phẩmA robust method for measuring aminoacylation through tRNA-Seq(University of Washington, 2023) Davidsen, KristianCurrent methods to quantify the fraction of aminoacylated tRNAs, also known as the 8 tRNA charge, are limited by issues with either low throughput, precision, and/or accuracy. Here, we 9 present an optimized charge tRNA-Seq method that combines previous developments with newly 10 described approaches to establish a protocol for precise and accurate tRNA charge measurements. 11 We verify that this protocol provides robust quantification of tRNA aminoacylation and we provide 12 an end-to-end method that scales to hundreds of samples including software for data processing. 13 Additionally, we show that this method supports measurements of relative tRNA expression levels 14 and can be used to infer tRNA modifications through reverse transcription misincorporations, 15 thereby supporting multipurpose applications in tRNA biology.
- Ấn phẩmA widespread proteinaceous sulfur storage compartment in bacteria(bioRxiv, 2023) Benisch, RobertIntracellular compartmentalization is essential for all cells and enables the regulation and optimization of metabolism1. One of the main functions of subcellular compartments is the storage of nutrients2-4. As bacteria do generally not possess membrane-bound organelles, they often have to rely on functionally analogous protein-based compartments2,5-7. Encapsulin nanocompartments are one of the most prevalent protein-based compartmentalization strategies found in prokaryotes5,8. Here we show that desulfurase encapsulins represent a novel sulfur storage compartment in bacteria able to sequester large amounts of crystalline elemental sulfur. We determined the 1.78 Å cryo-EM structure of a 24 nm desulfurase-loaded encapsulin highlighting the molecular details of the protein shell and desulfurase encapsulation. We found that elemental sulfur crystals can be formed inside encapsulin shells in a desulfurase-dependent manner with L-cysteine acting as the sulfur donor. Intracellular sulfur accumulation can be influenced by the concentration and type of sulfur source in growth media. The selectively permeable protein shell allows the long-term intracellular storage of redox-labile elemental sulfur by excluding cellular reducing agents from its interior.
- Ấn phẩmAcetic acid is a superior acidifier for sub-nanogram and single cell proteomic studies(2023) Orsburn, Benjamin CAmyloid β (Aβ) peptides accumulating in the brain are proposed to trigger Alzheimer's disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for Aβ42 toxicity that arises from its proven affinity for γ-secretases. We hypothesized that the reported increases in Aβ42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on γ-secretases, and thereby impair downstream signaling events. We show that human Aβ42 peptides, but neither murine Aβ42 nor human Aβ17-42 (p3), inhibit γ-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75 and pan-cadherin. Moreover, Aβ42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems.
- Ấn phẩmActivity-Guided Proteomic Profiling of Proteasomes Uncovers a Variety of Active (And Inactive) Proteasome Species(bioRxiv, 2023) Sahoo, Manisha PriyadarsiniProteasomes are multi-subunit, multi-catalytic protein complexes present in eukaryotic cells that degrade misfolded, damaged, or unstructured proteins. In this study, we used an activity-guided proteomic methodology based on a fluorogenic peptide substrate to characterize the composition of proteasome complexes in WT yeast, and the changes these complexes undergo upon the deletion of Pre9 (Δα3) or of Sem1 (ΔSem1). A comparison of whole-cell proteomic analysis to activity-guided proteasome profiling indicates that the amounts of proteasomal proteins and proteasome interacting proteins in the assembled active proteasomes differ significantly from their total amounts in the cell as a whole. Using this activityguided approach, we characterized the changes in the abundance of subunits of various active proteasome species in different strains, quantified the relative abundance of active proteasomes across these strains, and charted the overall distribution of different proteasome species within each strain. The distributions obtained by our mass spectrometry-based quantification were markedly higher for some proteasome species than those obtained by activity-based quantification alone, suggesting that the activity of some of these species is impaired. The impaired activity appeared mostly among 20SBlm10 proteasome species which account for 20% of the active proteasomes in WT.
- Ấn phẩmAlgal kainoid synthases exhibit substrate-dependent hydroxylation and cyclization activities(bioRxiv, 2023) Hopiavuori, Austin R.FeII/a-ketoglutarate-dependent dioxygenases (Fe/aKG) are a large enzyme family that functionalize C-H bonds on diverse organic substrates. Although Fe/aKG homologs catalyze an array of chemically useful reactions, hydroxylation typically predominates. Microalgal DabC uniquely forms a novel C-C bond to construct the bioactive pyrrolidine ring in domoic acid biosynthesis. However, this kainoid synthase exclusively performs a stereospecific hydroxylation reaction on its cis substrate regioisomer. Mechanistic and kinetic analyses with native and alternative substrates identified a 20-fold rate increase in DabC radical cyclization over β-hydroxylation, with no observable 1,5-hydrogen atom transfer. Moreover, this dual activity was conserved among macroalgal RadC1 and KabC homologs and provided insight into substrate recognition and reactivity trends. Investigation of this substratedependent chemistry improves our understanding of Fe/aKG enzymes and their biocatalytic application.
- Ấn phẩmAlzheimer's disease linked Aβ42 exerts product feedback inhibition on γsecretase impairing downstream cell signaling(2023) Orsburn, Benjamin C.A recent study demonstrated a substantial signal increase when employing a 0.5% acetic acid buffer additive instead of the traditional 0.1% formic acid used in shotgun proteomics. In this study I compare these two buffers for a dilution series of tryptic digests down to 20 picograms peptide on column on a TIMSTOF single cell proteome (SCP) system. I observe a comparable relative level of signal increase as previously reported, which translates to improvements in proteome coverage at every peptide load assessed. The relative increase in peptide identifications is more apparent at lower concentrations with a striking 1.8-fold more peptides identified at 20 pg peptide load, resulting in over 2,000 protein groups identified in 30 minutes on this system. These results translate well to isolated single human cancer cells allowing over 1,000 protein groups to be identified in single human cells processed using a simple one step method in standard 96-well plates. All vendor raw and processed data has been made publicly available at www.massive.ucsd.edu and can be accessed as MSV000092563.
- Ấn phẩmAn Atypical E3 Ligase Module in UBR4 Mediates Destabilization of N-degron Substrates(bioRxiv, 2023) Greer, Lucy BarnsbyUBR4 is an E3 ligase (E3) of the N-degron pathway and is involved in neurodevelopment, age-associated muscular atrophy and cancer progression. The location and mechanistic classification of the E3 module within the 600 kDa protein UBR4 remains unknown. Herein, we identify and characterize, at a biochemical and structural level, a distinct E3 module within human UBR4 consisting of a novel “hemiRING” zinc finger, a helical-rich UBR Zinc-finger Interacting (UZI) subdomain, and a predicted backside interacting N-terminal helix. A structure of an E2 conjugating enzyme (E2)-E3 complex provides atomic level insight into the exquisite specificity of the hemiRING towards the E2s UBE2A/B. The UZI subdomain can be considered a component of the E3 module as it has a modest activating effect on the ubiquitin loaded E2 (E2∼Ub), which is complemented by the intrinsically high lysine reactivity of UBE2A. These findings reveal the mechanistic underpinnings of a neuronal N-degron E3 ligase, its specific recruitment of UBE2A, and highlight the underappreciated architectural diversity of cross-brace domains associated with ubiquitin E3 activity.
- Ấn phẩmAn engineered biosensor enables dynamic aspartate measurements in living cells(bioRxiv, 2023) Davidsen, KristianIntracellular levels of the amino acid aspartate are responsive to changes in metabolism in mammalian cells and can correspondingly alter cell function, highlighting the need for robust tools to measure aspartate abundance. However, comprehensive understanding of aspartate metabolism has been limited by the throughput, cost, and static nature of the mass spectrometry based measurements that are typically employed to measure aspartate levels. To address these issues, we have developed a GFP-based sensor of aspartate (jAspSnFR3), where the fluorescence intensity corresponds to aspartate concentration. As a purified protein, the sensor has a 20-fold increase in fluorescence upon aspartate saturation, with dose dependent fluorescence changes covering a physiologically relevant aspartate concentration range and no significant off target binding. Expressed in mammalian cell lines, sensor intensity correlated with aspartate levels measured by mass spectrometry and could resolve temporal changes in intracellular aspartate from genetic, pharmacological, and nutritional manipulations. These data demonstrate the utility of jAspSnFR3 and highlight the opportunities it provides for temporally resolved and high throughput applications of variables that affect aspartate levels.
- Ấn phẩmAnionic phospholipids stimulate the proton pumping activity of the plant plasma membrane P-type H+-ATPase(bioRxiv, 2023) Paweletz, Laura C.The activity of membrane proteins depends strongly on the surrounding lipid environment. Here, we characterize the lipid stimulation of the plant plasma membrane H+-ATPase AHA2 upon purification and reconstitution into liposomes of defined lipid compositions. We show that the proton pumping activity of AHA2 is stimulated by anionic phospholipids, especially by phosphatidylserine. Molecular dynamics simulations revealed several preferential contact sites for anionic phospholipids in the transmembrane domain of AHA2. These contact sites are partly conserved across functionally different P-type ATPases from several organisms, suggesting a general regulation mechanism by the membrane lipid environment. Our findings highlight the fact that anionic lipids play an important role in the control of H+-ATPase activity
- Ấn phẩmAntimicrobial Evaluation of Salvadora persica Methanolic Stem Extract and In vivo Modelling of its Identified Constituting Compounds Revealed Antibacterial Effect of 2-[(5-Iodosalicylidene)hydrazino]-4-morpholino-6-(1-pyrrolidinyl)-1,3,5-triazine in the Treatment of Dental Plague(bioRxiv, 2023) Adigun, Temidayo OlamideChallenges of resistance to synthetic antimicrobials have opened new vistas in the search for natural products. This research rigorously reviews plant used in oral health. The antimicrobial activities of Salvadora persica commonly used as chewing stick was investigated against 3 clinical strains of Escherichia coli, Staphylococcus aureus, Aspergillus nigar. The antibacterial and antifungal activities of the extracts were determined using the agar well diffusion. Salvadora persica, was active against all the isolates especially on the bacteria with a MIC and MBC of 12.5mg/mL and 25mg/mL respectively. Phytochemical screening revealed the presence of alkaloids, tannins, flavonoids, saponins and traces of terpenoids. In silico modelling of the compounds identified in the chewing stick extract reveals that the aromatic nitrogen-rich compound with PubChem ID: 135580681 is likely responsible for the mechanistic inhibition associated with antibacterial effect of the plant material against dental plagues
- Ấn phẩmApplication of monolayer graphene to cryo-electron microscopy grids for high-resolution structure determination(2023) Grassetti, andrew VThe application of support layers, such as graphene, to cryo-electron microscopy grids can increase the density of particles imaged, limit particle interactions with the air-water interface, reduce the extent of beam-induced motion, and, in some instances, improve the distribution of particle orientations. This paper describes a robust protocol for coating cryo-EM grids with a monolayer of graphene for improved cryo-sample preparation. Single particle cryo-electron microscopy (cryo-EM) has evolved into a widely used method for visualizing biological macromolecules1. Fueled by advances in direct electron detection2-4, data acquisition5, and image processing algorithms6-10, cryo-EM is now capable of producing near-atomic resolution 3D structures of a fast growing number of macromolecules11. Moreover, by leveraging the single-molecule nature of the approach, users can now determine multiple structures from a single sample12-15, highlighting the promise of using these data to understand heterogeneous structural ensembles16,17. Despite this progress, bottlenecks in cryo-specimen grid preparation persist.
- Ấn phẩmBeyond the MEP Pathway: a novel kinase required for prenol utilization by malaria parasites(2023) Crispim, MarcellPlasmodium falciparum causes the most severe form of human malaria, a parasitic disease with a high global burden. In 2021, the World Health Organization reported an estimated 247 million cases of malaria and 619,000 malaria-related deaths, with the majority occurring among children and pregnant women in Sub-Saharan Africa. In 2021, 96% of all malaria-related deaths occurred in this region. Resistance to current antimalarial drugs is a significant challenge for malaria control, leading to increased morbidity and mortality (WHO, 2022). Therefore, the identification and development of novel antimalarial therapies are urgently needed.
- Ấn phẩmBifunctional small molecules that induce nuclear localization and targeted transcriptional regulation(bioRxiv, 2023) Gibson, William J.The aberrant localization of proteins in cells is a key factor in the development of various diseases, including cancer and neurodegenerative disease. To better understand and potentially manipulate protein localization for therapeutic purposes, we engineered bifunctional compounds that bind to proteins in separate cellular compartments. We show these compounds induce nuclear import of cytosolic cargoes, using nuclear-localized BRD4 as a “carrier” for co-import and nuclear trapping of cytosolic proteins. We use this system to calculate kinetic constants for passive diffusion across the nuclear pore and demonstrate single-cell heterogeneity in response to these bifunctional molecules, with cells requiring high carrier to cargo expression for complete import. We also observe incorporation of cargoes into BRD4-containing condensates. Proteins shown to be substrates for nuclear transport include oncogenic mutant nucleophosmin (NPM1c) and mutant PI3K catalytic subunit alpha (PIK3CAE545K), suggesting potential applications to cancer treatment. In addition, we demonstrate that chemical-induced localization of BRD4 to cytosolic-localized DNA-binding proteins, namely, IRF1 with a nuclear export signal, induces target gene expression. These results suggest that induced localization of proteins with bifunctional molecules enables the rewiring of cell circuitry with significant implications for disease therapy.
- Ấn phẩmBorrelia PeptideAtlas: A proteome resource of common Borrelia burgdorferi isolates for Lyme research(bioRxiv, 2023) Reddy, Panga Jaipal.Lyme disease , caused by an infection with the spirochete Borrelia burgdorferi, is the most common 36 vector-borne disease in North America. B. burgdorferi strains harbor extensive genomic and 37 proteomic variability and further comparison is key to understanding the spirochetes infectivity and 38 biological impacts of identified sequence variants. To achieve this goal, both transcript and mass 39 spectrometry (MS)-based proteomics was applied to assemble peptide datasets of laboratory 40 strains B31, MM1, B31-ML23, infective isolates B31-5A4, B31-A3, and 297, and other public 41 datasets, to provide a publicly available Borrelia PeptideAtlas 42 (http://www.peptideatlas.org/builds/borrelia/). Included is information on total proteome, 43 secretome, and membrane proteome of these B. burgdorferi strains. Proteomic data collected from 44 35 different experiment datasets, with a total of 855 mass spectrometry runs, identified 76,936 45 distinct peptides at a 0.1% peptide false-discovery-rate, which map to 1,221 canonical proteins (924 46 core canonical and 297 noncore canonical) and covers 86% of the total base B31 proteome. The 47 diverse proteomic information from multiple isolates with credible data presented by the Borrelia 48 PeptideAtlas can be useful to pinpoint potential protein targets which are common to infective 49 isolates and may be key in the infection process.
- Ấn phẩmCardiomyopathy-Associated Variants Alter the Structure and Function of the α-Actinin-2 Actin-Binding Domain(bioRxiv, 2023) E Atang, AlexandraHypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy (RCM) are characterized by thickening, thinning, or stiffening, respectively, of the ventricular myocardium, resulting in diastolic or systolic dysfunction that can lead to heart failure and sudden cardiac death. Recently, variants in the ACTN2 gene, encoding the protein α-actinin-2, have been reported in HCM, DCM, and RCM patients. However, functional data supporting the pathogenicity of these variants is limited, and potential mechanisms by which these variants cause disease are largely unexplored. Currently, NIH ClinVar lists 34 ACTN2 missense variants, identified in cardiomyopathy patients, which we predict are likely to disrupt actin binding, based on their localization to specific substructures in the α-actinin-2 actin binding domain (ABD). We investigated the molecular consequences of three ABD localized, HCM-associated variants: A119T, M228T and T247M. Using circular dichroism, we demonstrate that the mutant ABD proteins can attain a well-folded state.
- Ấn phẩmCatalytic bias and redox-driven inactivation of ancestral FeFe hydrogenases from group B2(bioRxiv, 2023) Fasano, AndreaThe biodiversity of hydrogenases, the enzymes that oxidize and produce H2, is only just beginning to be explored. Here we use direct electrochemistry to characterize two enzymes from a subgroup of ancestral FeFe hydrogenases, defined by the presence of three adjacent cysteine residues near the active site: the third FeFe hydrogenase from Clostridium pasteurianum (CpIII) and the second from Megasphaera elsdenii (MeII). To examine the functional role of the unusual TSCCCP motif, which defines the group B2 and is replaced with TSCCP in group A hydrogenases, we also produced a CpIII variant where the supernumerary cysteine is deleted. CpIII and MeII inactivate under oxidative conditions in a manner that is distinct from all other previously characterized hydrogenases from group A. Our results suggest that the supernumerary cysteine allows the previously observed sulfide-independent formation of the Hinact state in these enzymes. We also evidence a second reversible, oxidative inactivation process. Because of their inactivation under oxidative conditions, these enzymes are inefficient H2 oxidation catalysts, but their active site itself is not tuned to make them more active in one particular direction.
- Ấn phẩmCell-penetrating peptides stimulate protein transport on the Twin-arginine translocation pathway: evidence for a membrane thinning and toroidal pore mechanism(bioRxiv, 2023) McNeilage, RobertThe Tat pathway is essential for photosynthetic protein transport across plant thylakoid membranes and is also ubiquitous throughout prokaryotes and archaea. The Tat pathway is quite unique amongst protein translocation pathways as it specializes in transporting folded proteins driven by a proton motive force. Mechanistic details of the actual translocation step (s) of the pathway remain elusive. Here, we show that membrane thinning stimulates Tat transport and, conversely, membrane strengthening abolishes Tat transport. We draw parallels from the Tat machinery to cell penetrating peptides and propose that the Tat pore could be toroidal, as in most pores formed by cell penetrating peptides.