Proton-coupled Transporter Research Articles

Proton-coupled transport mechanism of the efflux pump NorA.

Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to how acid/base chemistry drives drug efflux. Here, we uncover the proton-coupling mechanism of the Staphylococcus aureus efflux pump NorA by elucidating structures in various protonation states of two essential acidic residues using cryo-EM. Protonation of Glu222 and Asp307 within the C-terminal domain stabilized the inward-occluded conformation by forming hydrogen bonds between the acidic residues and a single helix within the N-terminal domain responsible for occluding the substrate binding pocket. Remarkably, deprotonation of both Glu222 and Asp307 is needed to release interdomain tethering interactions, leading to opening of the pocket for antibiotic entry. Hence, the two acidic residues serve as a "belt and suspenders" protection mechanism to prevent simultaneous binding of protons and drug that enforce NorA coupling stoichiometry and confer antibiotic resistance.

Molecular Mechanisms Underlying Abnormal Activation of T Cells in Idiopathic Aplastic Anemia and Hypoplastic Myelodysplastic Neoplasms

Idiopathic aplastic anemia (AA) and hypoplastic myelodysplastic neoplasms (MDS-h) are severe hematopoietic disorders characterized by pancytopenia and hypoplastic bone marrow. There is compelling evidence that these distinct clinical entities share a common pathophysiology based on the damage of hematopoietic stem and progenitor cells (HSPCs) by cytotoxic T cells. Expanded T cells overproduce proinflammatory cytokines, resulting in decreased proliferation and increased apoptosis of HSPCs. To uncover the molecular mechanisms underlying this abnormal immune response, we used RNA-Seq for the transcriptome analysis of T cells from patients with idiopathic AA and MDS-h. CD3+ cells were isolated from peripheral blood of 15 patients with AA or MDS-h at diagnosis and 6 healthy blood donors. The RNA-Seq library was constructed by NEB Next Ultra II Directional RNA Library Prep Kit and the data were analysed by R software 4.0.2. Functional annotation of differently expressed genes was performed using DAVID database and Gene Set Enrichment Analysis (GSEA). Principal component analysis-based clustering of RNA-Seq data defined the three major components for AA and MDS-h patients, and healthy individuals (Fig. 1A). As expected, the control samples formed a distinct group, meanwhile patient samples partially clustered together. The same results were obtained using unsupervised hierarchical clustering. The differential expression analysis (DEA) identified 256 significantly upregulated and 96 downregulated genes (ІlogFCІ>1, FDR<0.05) in patients compared to controls. Notably, the DEA detected many long noncoding RNAs (lncRNAs) including upregulated lncRNAs associated with immunological disturbances (e.g. CDC42-IT1 and NEAT1), and oncogenesis (e.g. ACAP2-IT1, and FAM238A). Particularly, NEAT1 positively regulates differentiation of CD4+ T cells into Th17 cells through STAT3 protein. Autoreactive T cells may be further stimulated by increased expression of S100A8/A9 detected in the patient cells. Gene Ontology (GO) analysis annotated the upregulated genes in AA/MDS-h to biological processes associated with oxygen transport, B cell receptor signaling pathway, innate immune response, positive regulation of inflammatory response, and apoptotic process, etc. (adjusted p<0.05) (Fig. 1B). The downregulated genes were significantly enriched in processes related to adaptive immune response, cell surface receptor signaling pathway, mitochondrial ATP synthesis coupled proton transport, etc. (adjusted p<0.05) (Fig.1B). B cell receptor signaling pathway was enriched by upregulated genes such as BANK1, MEF2C, and SYK whose deregulation is associated with autoimmune disorders. Herein, the activation of apoptosis is likely driven via AP-1 positive modulators that showed increased expressions in the patients. These modulators are essential for T cell activation and regulatory T cell differentiation. Pathway enrichment analysis determined the following signaling pathways (adjusted p<0.05): B cell receptor signaling pathway, osteoclast differentiation, oxidative phosphorylation, chemical carcinogenesis - reactive oxygen species, and cytokine-cytokine receptor interaction. The GSEA using the Hallmark gene sets showed that DNA Repair and Oxidative Phosphorylation were significantly negatively correlated with patient phenotype (Fig. 1C). Both GSEA and pathway analysis revealed that oxidative phosphorylation was supressed in patient-derived T cells. This is consistent with previous metabolic findings demonstrating that a transition from oxidative phosphorylation to glycolysis is a sign of T cell activation and it is critical for support of rapid cell growth. In conclusion, our study suggests the possible mechanisms of aberrant T cellular-immunity in idiopathic AA and MDS-h. Consistent with abnormal biological features of T cells, a large number of deregulated genes in the patients was involved in immune and inflammatory responses. Furthermore, our analyses revealed novel pathways, such as oxidative phosphorylation, as well as candidates ( TLR2, and CDC42-IT1) for functional studies. The deregulation of multiple lncRNAs in the patients indicates their implication in the molecular pathogenesis and thus their roles in bone marrow failure need to be explored. Supported by AZV (NU21-03-00565), and MH CZ-DRO (UHKT, 00023736).

Intestinal monocarboxylate transporter 1 mediates lactate transport in the gut and regulates metabolic homeostasis of mouse in a sex-dimorphic pattern

Abstract The monocarboxylate transporter 1 (MCT1), encoded by gene Slc16a1, is a proton-coupled transporter for lactate and other monocarboxylates. MCT1-mediated lactate transport was recently found to regulate various biological functions. However, how MCT1 and lactate in the intestine modulate the physiology and pathophysiology of the body is unclear. In this study, we generated a mouse model with specific deletion of Slc16a1 in the intestinal epithelium (Slc16a1IKO mice) and investigated the functions of MCT1 in the gut. When fed a high-fat diet, Slc16a1IKO male mice had improvement in glucose tolerance and insulin sensitivity, while Slc16a1IKO female mice only had increased adiposity. Deficiency of intestinal MCT1 in male mice was associated with downregulation of pro-inflammatory pathways, together with decreased circulating levels of inflammatory cytokines including tumor necrosis factor alpha (TNFα) and C–C motif chemokine ligand 2 (CCL2). Lactate had a stimulatory effect on pro-inflammatory macrophages in vitro. The number of intestinal macrophages was reduced in Slc16a1IKO male mice in vivo. Intestinal deletion of Slc16a1 in male mice reduced interstitial lactate level in the intestine. In addition, treatment of male mice with estrogen lowered interstitial lactate level in the intestine and abolished the difference in glucose homeostasis between Slc16a1IKO and wild-type mice. Deficiency of intestinal MCT1 also blocked the transport of lactate and short-chain fatty acids from the intestine to the portal vein. The effect of Slc16a1 deletion on glucose homeostasis in male mice was partly mediated by alterations in gut microbiota. In conclusion, our work reveals that intestinal MCT1 regulates glucose homeostasis in a sex-dependent manner.

Mechanisms by which water-mediated hydrogen-bond networks govern pH-dependent reactions at membrane interfaces.

pH-dependent reactions at membrane interfaces are used by cells to maintain pH homeostasis and to ensure normal cell function. Membrane-bound proteins and peptide systems that sense pH typically bind protons using a few titratable sidechains that communicate with the bulk. The identity of these pH-sensing sidechains is central to hypotheses about protein reaction mechanisms and for the rational design of proton detectors. But, as protein groups hypothesized to sense pH in proteins and peptide systems are part of dynamic water-mediated networks, evaluating their pH-sensing functionality may prove difficult. We developed efficient graph-based algorithms and methodologies to evaluate dynamic hydrogen-bond networks of protein and peptide systems known to have a pH-sensing functionality, and to identify sites of hydrogen-bond networks where protons are likely to bind. We find that the assembly of dynamic water-mediated hydrogen-bond networks that include potential proton-sensing groups is a key event along the reaction coordinates of pH-dependent membrane proteins and membrane-bound peptides, and implement graph-based algorithms to characterize the dynamics of these water networks. We further find that polar and basic protein sidechains largely govern the dynamics of the water-mediated hydrogen-bond networks of proton-binding groups, and help maintain acidic sidechains within clusters that collectively can bind and store protons. Taken together, the ensemble of hydrogen-bond network computations we performed for pH-coupled proteins and peptide systems provide a framework to decipher the roles of dynamic water-mediated hydrogen-bond networks in proton binding at membrane interfaces. Research was supported in part by the European Union's Horizon 2020 Research and Innovation Program under the Marie Sklodowska-Curie grant agreement No 860592, Innovative Training Network ‘Proton and proton-coupled transport’, and by computing time from the Physics Department of the Freie Universität Berlin and from the Forschungszentrum Jülich (JURECA-DC, allocation PHDPORES).

Residues involved in proton-coupled transport of antimalarial drugs by PfCRT.

A major setback in the fight against malaria has been the emergence of P. falciparum parasites resistant to chloroquine (CQ), the primary antimalarial drug. Neutral CQ enters the parasite's digestive vacuole (DV) and obtains two protons preventing its passive efflux through the membrane. The trapped CQ2+ kills the parasite by disrupting the detoxification process of heme released from proteolyzed host hemoglobin. CQ-resistant parasites show mutations in a DV membrane transporter, P. falciparum CQ resistance transporter (PfCRT), that enables active, proton-coupled efflux of CQ2+. While biochemical measurements established the coupling of PfCRT's transport function to the DV membrane proton gradient, the proton-coupled transport mechanism has remained elusive. Particularly it is still unclear what the proton binding site(s) is(are). Recently, the cryo-EM structure of PfCRT from a CQ-resistant variant was determined with a bound Fab fragment occupying its binding pocket. Using us-long all-atom molecular dynamics (MD) simulations we examined the stability of the transporter in the DV-open conformation in the absence of bound Fab fragments, discovering surprising protein stability upon removal of Fab. Next, utilizing constant pH (cpHMD) simulations at 7 different pH values, and considering Galvani potential offset corrections, we determined the pKa of the four titratable residues (H97, D137, D326, and D329) lining the binding pocket. These simulations allowed unambiguous determination of the primary site responding to DV's acidic environment. Finally, with protonation states compatible with the acidic pH of the DV for the aforementioned residues, we studied binding of CQ2+ to the transporter via a series of driven MD simulations. We then repeated the cpHMD simulations and determined the proton binding site in the presence of the bound CQ2+. Our results shed light on PfCRT's transport mechanism and open new possibilities to design new effective drugs against malaria.

Investigating the regulation and function of bacterial quaternary ammonium compound exporters.

Quaternary ammonium cations were introduced as commonly used disinfectant compounds in the 1930s. Today, they are often used in disinfectant solutions and wipes. Bacterial resistance to these antiseptic compounds is mediated by transporters from the small multidrug resistance (SMR) family of proteins. SMR transporters are bacterial proton-coupled transporters that export cationic substrates including quaternary ammonium compounds. Here we examine the genetic regulation of SMR expression. In addition, we use solid supported membrane (SSM) electrophysiology to observe transport of quaternary ammonium compounds by purified, reconstituted QacE proteins. Our findings allow us to functionally characterize and obtain insight into the evolution of these quaternary ammonium antiseptic transporters so that we can further investigate how to mitigate the spread of antiseptic resistance among bacterial populations.

Contribution of the Collective Excitations to the Coupled Proton and Energy Transport along Mitochondrial Cristae Membrane in Oxidative Phosphorylation System.

The results of many experimental and theoretical works indicate that after transport of protons across the mitochondrial inner membrane (MIM) in the oxidative phosphorylation (OXPHOS) system, they are retained on the membrane-water interface in nonequilibrium state with free energy excess due to low proton surface-to-bulk release. This well-established phenomenon suggests that proton trapping on the membrane interface ensures vectorial lateral transport of protons from proton pumps to ATP synthases (proton acceptors). Despite the key role of the proton transport in bioenergetics, the molecular mechanism of proton transfer in the OXPHOS system is not yet completely established. Here, we developed a dynamics model of long-range transport of energized protons along the MIM accompanied by collective excitation of localized waves propagating on the membrane surface. Our model is based on the new data on the macromolecular organization of the OXPHOS system showing the well-ordered structure of respirasomes and ATP synthases on the cristae membrane folds. We developed a two-component dynamics model of the proton transport considering two coupled subsystems: the ordered hydrogen bond (HB) chain of water molecules and lipid headgroups of MIM. We analytically obtained a two-component soliton solution in this model, which describes the motion of the proton kink, corresponding to successive proton hops in the HB chain, and coherent motion of a compression soliton in the chain of lipid headgroups. The local deformation in a soliton range facilitates proton jumps due to water molecules approaching each other in the HB chain. We suggested that the proton-conducting structures formed along the cristae membrane surface promote direct lateral proton transfer in the OXPHOS system. Collective excitations at the water-membrane interface in a form of two-component soliton ensure the coupled non-dissipative transport of charge carriers and elastic energy of MIM deformation to ATP synthases that may be utilized in ATP synthesis providing maximal efficiency in mitochondrial bioenergetics.

Novel Evolutionary Engineering Approach to Alter Substrate Specificity of Disaccharide Transporter Mal11 in Saccharomyces cerevisiae

A major challenge in the research of transport proteins is to understand how single amino acid residues contribute to their structure and biological function. Amino acid substitutions that result in a selective advantage in adaptive laboratory evolution experiments can provide valuable hints at their role in transport proteins. In this study, we applied an evolutionary engineering strategy to alter the substrate specificity of the proton-coupled disaccharide transporter Mal11 in Saccharomyces cerevisiae, which has affinity for sucrose, maltose and glucose. The introduction of MAL11 in a strain devoid of all other sugar transporters and disaccharide hydrolases restored growth on glucose but rendered the strain highly sensitive to the presence of sucrose or maltose. Evolution in glucose-limited continuous cultures with pulse-wise addition of a concentrated sucrose solution at increasing frequency resulted in the enrichment of spontaneous mutant cells that were less sensitive to the presence of sucrose and maltose. Sequence analysis showed that in each of the two independent experiments, three mutations occurred in MAL11, which were found responsible for the disaccharide-insensitive phenotype via reverse engineering. Our work demonstrates how laboratory evolution with proton-motive force-driven uptake of a non-metabolizable substrate can be a powerful tool to provide novel insights into the role of specific amino acid residues in the transport function of Mal11.

Monocarboxylate Transporters Are Involved in Extracellular Matrix Remodelling in Pancreatic Ductal Adenocarcinoma.

Simple SummaryMonocarboxylate transporters (MCTs) carry a variety of substrates, with MCT1–4 being well-characterised and involved in the proton-coupled transport of monocarboxylates (such as lactate), which can be used as metabolic fuel for cancer cells. Increased acidity of the tumour microenvironment via MCTs favours remodelling of the extracellular matrix (ECM), leading to desmoplasia associated with tumour metastasis and poor patient outcomes. Although MCT1, MCT2, and MCT4 are upregulated in several cancers, their expression and role in pancreatic ductal adenocarcinoma desmoplasia is little understood. Here, we aimed to understand the role of MCTs in desmoplasia through their association with ECM components. Our analysis using hypernetworks showed the presence of bidirectional associations of MCTs and ECMs, suggesting the presence of a causal relationship and the need to further investigate their functional associations. It confirmed the role of MCTs in desmoplasia, highlighting their importance as therapeutic targets alone or in combination with key ECM components to potentially improve patient outcomes.Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a five-year survival rate of <8%. PDAC is characterised by desmoplasia with an abundant extracellular matrix (ECM) rendering current therapies ineffective. Monocarboxylate transporters (MCTs) are key regulators of cellular metabolism and are upregulated in different cancers; however, their role in PDAC desmoplasia is little understood. Here, we investigated MCT and ECM gene expression in primary PDAC patient biopsies using RNA-sequencing data obtained from Gene Expression Omnibus. We generated a hypernetwork model from these data to investigate whether a causal relationship exists between MCTs and ECMs. Our analysis of stromal and epithelial tissues (n = 189) revealed nine differentially expressed MCTs, including the upregulation of SLC16A2/6/10 and the non-coding SLC16A1-AS1, and 502 ECMs, including collagens, laminins, and ECM remodelling enzymes (false discovery rate < 0.05). A causal hypernetwork analysis demonstrated a bidirectional relationship between MCTs and ECMs; four MCT and 255 ECM-related transcripts correlated with 90% of the differentially expressed ECMs (n = 376) and MCTs (n = 7), respectively. The hypernetwork model was robust, established by iterated sampling, direct path analysis, validation by an independent dataset, and random forests. This transcriptomic analysis highlights the role of MCTs in PDAC desmoplasia via associations with ECMs, opening novel treatment pathways to improve patient survival.

Asymmetric protonation of glutamate residues drives a preferred transport pathway in EmrE

EmrE is an Escherichia coli multidrug efflux pump and member of the small multidrug resistance (SMR) family that transports drugs as a homodimer by harnessing energy from the proton motive force. SMR family transporters contain a conserved glutamate residue in transmembrane 1 (Glu14 in EmrE) that is required for binding protons and drugs. Yet the mechanism underlying proton-coupled transport by the two glutamate residues in the dimer remains unresolved. Here, we used NMR spectroscopy to determine acid dissociation constants (pKa ) for wild-type EmrE and heterodimers containing one or two Glu14 residues in the dimer. For wild-type EmrE, we measured chemical shifts of the carboxyl side chain of Glu14 using solid-state NMR in lipid bilayers and obtained unambiguous evidence on the existence of asymmetric protonation states. Subsequent measurements of pKa values for heterodimers with a single Glu14 residue showed no significant differences from heterodimers with two Glu14 residues, supporting a model where the two Glu14 residues have independent pKa values and are not electrostatically coupled. These insights support a transport pathway with well-defined protonation states in each monomer of the dimer, including a preferred cytoplasmic-facing state where Glu14 is deprotonated in monomer A and protonated in monomer B under pH conditions in the cytoplasm of E. coli Our findings also lead to a model, hop-free exchange, which proposes how exchangers with conformation-dependent pKa values reduce proton leakage. This model is relevant to the SMR family and transporters comprised of inverted repeat domains.

MtNPF6.5 mediates chloride uptake and nitrate preference in Medicago roots.

The preference for nitrate over chloride through regulation of transporters is a fundamental feature of plant ion homeostasis. We show that Medicago truncatula MtNPF6.5, an ortholog of Arabidopsis thaliana AtNPF6.3/NRT1.1, can mediate nitrate and chloride uptake in Xenopus oocytes but is chloride selective and that its close homologue, MtNPF6.7, can transport nitrate and chloride but is nitrate selective. The MtNPF6.5 mutant showed greatly reduced chloride content relative to wild type, and MtNPF6.5 expression was repressed by high chloride, indicating a primary role for MtNPF6.5 in root chloride uptake. MtNPF6.5 and MtNPF6.7 were repressed and induced by nitrate, respectively, and these responses required the transcription factor MtNLP1. Moreover, loss of MtNLP1 prevented the rapid switch from chloride to nitrate as the main anion in nitrate-starved plants after nitrate provision, providing insight into the underlying mechanism for nitrate preference. Sequence analysis revealed three sub-types of AtNPF6.3 orthologs based on their predicted substrate-binding residues: A (chloride selective), B (nitrate selective), and C (legume specific). The absence of B-type AtNPF6.3 homologues in early diverged plant lineages suggests that they evolved from a chloride-selective MtNPF6.5-like protein.

Prognostic significance and oncogene function of cathepsin A in hepatocellular carcinoma

Cathepsin A (CTSA) is a lysosomal protease that regulates galactoside metabolism. The previous study has shown CTSA is abnormally expressed in various types of cancer. However, rarely the previous study has addressed the role of CTSA in hepatocellular carcinoma (HCC) and its prognostic value. To study the clinical value and potential function of CTSA in HCC, datasets from the Cancer Genome Atlas (TCGA) database and a 136 HCC patient cohort were analyzed. CTSA expression was found to be significantly higher in HCC patients compared with normal liver tissues, which was supported by immunohistochemistry (IHC) validation. Both gene ontology (GO) and The Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses demonstrated that CTSA co-expressed genes were involved in ATP hydrolysis coupled proton transport, carbohydrate metabolic process, lysosome organization, oxidative phosphorylation, other glycan degradation, etc. Survival analysis showed a significant reduction both in overall survival (OS) and recurrence-free survival (RFS) of patients with high CTSA expression from both the TCGA HCC cohort and 136 patients with the HCC cohort. Furthermore, CTSA overexpression has diagnostic value in distinguishing between HCC and normal liver tissue [Area under curve (AUC) = 0.864]. Moreover, Gene set enrichment analysis (GSEA) showed that CTSA expression correlated with the oxidative phosphorylation, proteasome, and lysosome, etc. in HCC tissues. These findings demonstrate that CTSA may as a potential diagnostic and prognostic biomarker in HCC.

Key computational findings reveal proton transfer as driving the functional cycle in the phosphate transporter PiPT

Phosphate is an indispensable metabolite in a wide variety of cells and is involved in nucleotide and lipid synthesis, signaling, and chemical energy storage. Proton-coupled phosphate transporters within the major facilitator family are crucial for phosphate uptake in plants and fungi. Similar proton-coupled phosphate transporters have been found in different protozoan parasites that cause human diseases, in breast cancer cells with elevated phosphate demand, in osteoclast-like cells during bone reabsorption, and in human intestinal Caco2BBE cells for phosphate homeostasis. However, the mechanism of proton-driven phosphate transport remains unclear. Here, we demonstrate in a eukaryotic, high-affinity phosphate transporter from Piriformospora indica (PiPT) that deprotonation of aspartate 324 (D324) triggers phosphate release. Quantum mechanics/molecular mechanics molecular dynamics simulations combined with free energy sampling have been employed here to identify the proton transport pathways from D324 upon the transition from the occluded structure to the inward open structure and phosphate release. The computational insights so gained are then corroborated by studies of D45N and D45E amino acid substitutions via mutagenesis experiments. Our findings confirm the function of the structurally predicted cytosolic proton exit tunnel and suggest insights into the role of the titratable phosphate substrate.

Cysteine 159 delineates a hinge region of the alternating access monocarboxylate transporter1 and is targeted by cysteine-modifying inhibitors.

Monocarboxylate transporter isoforms 1-4, MCT, of the solute carrier SLC16A family facilitate proton-coupled transport of l-lactate. Growth of tumors that exhibit the Warburg effect, that is, high rates of anaerobic glycolysis despite availability of oxygen, relies on swift l-lactate export, whereas oxygenic cancer cells import circulating l-lactate as a fuel. Currently, MCTs are viewed as promising anticancer targets. Small-molecule inhibitors have been found, and, recently, high-resolution protein structures have been obtained. Key questions, however, regarding the exact binding sites of cysteine-modifying inhibitors and the substrate translocation cycle lack a conclusive experimental basis. Here, we report Cys159 of the ubiquitous human MCT1 to reside in a critical hinge region of the alternating access-type transporter. We identified Cys159 as the binding site of the organomercurial pCMBS. The inhibitory effect of pCMBS was proposed to be indirect via modification of the chaperone basigin. We provide evidence that pCMBS locks MCT1 in its outward open conformation in a wedge-like fashion. We corroborated this finding using smaller cysteine-modifying reagents that size-dependently inhibited l-lactate transport. The smallest modifiers targeted additional cysteines as shown by a C159S mutant. We found a Cys399/Cys400 pair to constitute the second hinge of the transporter that tolerated only individual replacement by serine. The hinge cysteines, in particular the selectively addressable Cys159, provide natural anchors for placing probes into MCTs to report, for instance, on the electrostatics or hydration upon binding of the transported l-lactate substrate and the proton cosubstrate.

Extracellular Vesicles Derived From Adult and Fetal Bone Marrow Mesenchymal Stromal Cells Differentially Promote ex vivo Expansion of Hematopoietic Stem and Progenitor Cells.

Recently, we and others have illustrated that extracellular vesicles (EVs) have the potential to support hematopoietic stem and progenitor cell (HSPC) expansion; however, the mechanism and processes responsible for the intercellular communication by EVs are still unknown. In the current study, we investigate whether primary human bone marrow derived mesenchymal stromal cells (BMSC) EVs isolated from two different origins, fetal (fEV) and adult (aEV) tissue, can increase the relative low number of HSPCs found in umbilical cord blood (UCB) and which EV-derived components are responsible for ex vivo HSPC expansion. Interestingly, aEVs and to a lesser extent fEVs, showed supportive ex vivo expansion capacity of UCB-HSPCs. Taking advantage of the two BMSC sources with different supportive effects, we analyzed the EV cargo and investigated how gene expression is modulated in HSPCs after incubation with aEVs and fEVs. Proteomics analyses of the protein cargo composition of the supportive aEV vs. the less-supportive fEV identified 90% of the Top100 exosome proteins present in the ExoCarta database. Gene Ontology (GO) analyses illustrated that the proteins overrepresented in aEVs were annotated to oxidation-reduction process, mitochondrial ATP synthesis coupled proton transport, or protein folding. In contrast, the proteins overrepresented in fEVs were annotated to extracellular matrix organization positive regulation of cell migration or transforming growth factor beta receptor (TGFBR) signaling pathway. Small RNA sequencing identified different molecular signatures between aEVs and fEVs. Interestingly, the microRNA cluster miR-99b/let-7e/miR-125a, previously identified to increase the number of HSPCs by targeting multiple pro-apoptotic genes, was highly and significantly enriched in aEVs. Although we identified significant differences in the supportive effects of aEVs and fEVs, RNAseq analyses of the 24 h treated HSPCs indicated that a limited set of genes was differentially regulated when compared to cells that were treated with cytokines only. Together, our study provides novel insights into the complex biological role of EVs and illustrates that aEVs and fEVs differentially support ex vivo expansion capacity of UCB-HSPCs. Together opening new means for the application of EVs in the discovery of therapeutics for more efficient ex vivo HSPC expansion.

Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

Proton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.

Unidirectional electron injection and accelerated proton transport in bacteriorhodopsin based Bio-p-n junctions

Hampered by the absence of evidence and theoretical model of biological semiconductors, the unidirectional electron transport via the p-n junction between functional proteins and abiotic materials remains a challenge for bioelectronics. Bacteriorhodopsin (bR), a representative transmembrane protein, has demonstrated exceptional optoelectronic effects in bR/semiconductor hybrid materials and offers a possible pathway for addressing this challenge. In the present work, for the first time, bR is proved to be an n-type semiconductor with an indirect electron transition. Through the photo-electrochemical method used for studying the p-n junction effect in the bR and p-type semiconductor combined electrodes, we reached several important conclusions: The self-corrosion of bR integrated Cu2O electrodes is delayed for about 36 times; The photocurrent of bR integrated CuSCN electrodes is enhanced by about 400%, which is attributed to the directional migration of electrons via the p-n junction. Furthermore, the ultrafast kinetics we have explored, shows that the injection of electrons shortens the lifetime of the intermediate state O640 from 37.3 μs to 20.1μs, what means that the protons transport rate accompanying the bR photocycle process is accelerated. Therefore, we believe that the concept of the bio-p-n junction and the mechanism of electron coupled proton transport, which are discussed herein, will promote useful research on bioelectronic applications for bR and its homologs.

Modelling the Proton-Conductive Membrane in Practical Polymer Electrolyte Membrane Fuel Cell (PEMFC) Simulation: A Review.

Theoretical models used to describe the proton-conductive membrane in polymer electrolyte membrane fuel cells (PEMFCs) are reviewed, within the specific context of practical, physicochemical simulations of PEMFC device-scale performance and macroscopically observable behaviour. Reported models and their parameterisation (especially for Nafion 1100 materials) are compiled into a single source with consistent notation. Detailed attention is given to the Springer–Zawodzinski–Gottesfeld, Weber–Newman, and “binary friction model” methods of coupling proton transport with water uptake and diffusive water transport; alongside, data are compiled for the corresponding parameterisation of proton conductivity, water sorption isotherm, water diffusion coefficient, and electroosmotic drag coefficient. Subsequent sections address the formulation and parameterisation of models incorporating interfacial transport resistances, hydraulic transport of water, swelling and mechanical properties, transient and non-isothermal phenomena, and transport of dilute gases and other contaminants. Lastly, a section is dedicated to the formulation of models predicting the rate of membrane degradation and its influence on PEMFC behaviour.

Metaproteomics reveals the structural and functional diversity of Dermatocarpon miniatum (L.) W. Mann. Microbiota

Metaproteomics is a strategy to understand the taxonomy, functionality and metabolic pathways of the microbial communities. The relationship among the symbiotic microbiota in the entire lichen thallus, Dermatocarpon miniatum, was evaluated using the metaproteomic approach. Proteomic profiling using one-dimensional SDS-PAGE followed by LC-MS/MS analysis resulted in a total of 138 identified proteins via Mascot search against UniRef100 and Swiss-Prot databases. In addition to the fungal and algal partners, D. miniatum proteome encompasses proteins from prokaryotes, which is a multifarious community mainly dominated by cyanobacteria and proteobacteria. While proteins assigned to fungus were the most abundant (55 %), followed by protists (16 %), bacterial (13 %), plant (11 %), and viral (1 %) origin, whereas 4 % remained undefined. Various proteins were assigned to the different lichen symbionts by using Gene Ontology (GO) terms, e.g. fungal proteins involved in the oxidation-reduction process, protein folding and glycolytic process, while protists and bacterial proteins were involved in photosynthetic electron transport in photosystem II (PS II), ATP synthesis coupled proton transport, and carbon fixation. The presence of bacterial communities extended the traditional concept of fungal-algal lichen symbiotic interaction.

Electrophysiology Measurements of Metal Transport by MntH2 from Enterococcus faecalis.

Transition metals are essential trace elements and their high-affinity uptake is required for many organisms. Metal transporters are often characterised using metal-sensitive fluorescent dyes, limiting the metals and experimental conditions that can be studied. Here, we have tested whether metal transport by Enterococcus faecalis MntH2 can be measured with an electrophysiology method that is based on the solid-supported membrane technology. E. faecalis MntH2 belongs to the Natural Resistance-Associated Macrophage Protein (Nramp) family of proton-coupled transporters, which transport divalent transition metals and do not transport the earth metals. Electrophysiology confirms transport of Mn(II), Co(II), Zn(II) and Cd(II) by MntH2. However, no uptake responses for Cu(II), Fe(II) and Ni(II) were observed, while the presence of these metals abolishes the uptake signals for Mn(II). Fluorescence assays confirm that Ni(II) is transported. The data are discussed with respect to properties and structures of Nramp-type family members and the ability of electrophysiology to measure charge transport and not directly substrate transport.