Important Membrane Proteins Research Articles

VRAC channel inhibition as a novel strategy for the treatment of ischemia-reperfusion injury

Ischemia-reperfusion injury is a serious clinical pathology involving multiple organs such as the heart and brain. The injury results from oxidative stress, inflammatory response and cell death triggered by restoring tissue blood flow after ischemia, leading to severe cell and tissue damage. In recent years, the volume-regulated anion channel (VRAC) has gained attention as an important membrane protein complex. VRAC plays a dual role in ischemia-reperfusion injury: on the one hand, activated VRAC promotes the release of intracellular chloride and glutamate, exacerbating cellular swelling and excitotoxicity, and on the other hand, the regulatory effect of VRAC may also provide protection to cardiomyocytes. This article reviews the pathophysiological mechanisms of ischemia-reperfusion injury, existing therapeutic strategies and their limitations, focuses on the molecular structure of VRAC, its activation mechanism, and its role in ischemia-reperfusion injury, and concludes with a discussion of the potential of targeted inhibition of VRAC as an emerging therapeutic strategy and the challenges it faces. A deeper understanding of the role of VRAC in ischemia-reperfusion injury is expected to provide new therapeutic ideas to improve patient prognosis.

PKD2: An Important Membrane Protein in Organ Development.

PKD2 was first identified as the pathogenic protein for autosomal dominant polycystic kidney disease (ADPKD) and is widely recognized as an ion channel. Subsequent studies have shown that PKD2 is widely expressed in various animal tissues and plays a crucial role in tissue and organ development. Additionally, PKD2 is conserved from single-celled organisms to vertebrates. Here, we provide an overview of recent advances in the function of PKD2 in key model animals, focusing on the establishment of left-right organ asymmetry, renal homeostasis, cardiovascular development, and signal transduction in reproduction and mating. We specifically focus on the roles of PKD2 in development and highlight future prospects for PKD2 research.

O-glycosylation of intrinsically disordered regions regulates homeostasis of membrane proteins in streptococci.

Proteins harboring intrinsically disordered regions (IDRs) lacking stable secondary or tertiary structures are abundant across the three domains of life. These regions have not been systematically studied in prokaryotes. Our genome-wide analysis identifies extracytoplasmic serine/threonine-rich IDRs in several biologically important membrane proteins in streptococci. We demonstrate that these IDRs are O-glycosylated with glucose by glycosyltransferases GtrB and PgtC2 in Streptococcus pyogenes and Streptococcus pneumoniae, and with N-acetylgalactosamine by a Pgf-dependent mechanism in Streptococcus mutans. Absence of glycosylation leads to a defect in biofilm formation under ethanol-stressed conditions in S. mutans. We link this phenotype to the C-terminal IDR of a post-translocation secretion chaperone PrsA. O-glycosylation of the IDR protects this region from proteolytic degradation. The IDR length attenuates the efficiency of glycosylation and, consequently, the expression level of PrsA. Taken together, our data reveal that O-glycosylation of IDRs functions as a dynamic switch of protein homeostasis in streptococci.

SRT1720 inhibits bladder cancer cell progression by impairing autophagic flux

Bladder cancer (BC) is the most common cancer of the urinary tract, with poor survival, high recurrence rates, and lacking of targeted drugs. In this study, we constructed a library to screen compounds inhibiting bladder cancer cells growth. Among them, SRT1720 was identified to inhibit bladder cancer cell proliferation in vitro and in vivo. SRT1720 treatment also suppressed bladder cancer cells migration, invasion and induced apoptosis. Mechanism studies shown that SRT1720 promoted autophagosomes accumulation by inducing early-stage autophagy but disturbed the late-stage of autophagy by blocking fusion of autophagosomes and lysosomes. SRT1720 appears to induce autophagy related proteins expression and alter autophagy-related proteins acetylation to impede the autophagy flux. LAMP2, an important lysosomal associated membrane protein, may mediate SRT1720-inhibited autophagy flux as SRT1720 treatment significantly deacetylated LAMP2 which may influence its activity. Taken together, our results demonstrated that SRT1720 mediated apoptosis and autophagy flux inhibition may be a novel therapeutic strategy for bladder cancer treatment.

Role of the nucleotide-binding oligomerization domain-containing protein 1 pathway in the development of periodontitis

ObjectivesDuring innate immune defense, host pattern recognition receptors, including toll-like receptors and nucleotide-binding oligomerization domain-like receptors (NLRs), can activate downstream pathways by recognizing pathogen-associated molecular patterns produced by microorganisms, triggering immune responses. NOD1, an important cell membrane protein in the NLR-like receptor protein family, exerts anti-infective effects through γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) recognition. Oral epithelial cells resist bacterial invasion through iE-DAP-induced interleukin (IL)-8 production, recruiting neutrophils to sites of inflammation in response to bacterial threats to periodontal tissues. To date, the regulatory mechanisms of iE-DAP in gingival epithelial cells (GECs) are poorly understood. This study was conducted to investigate the role of the NOD1 pathway in the development of periodontitis by examining the effect of iE-DAP on IL-8 production in Ca9-22 cells. MethodsIL-8 production by iE-DAP-stimulated-Ca9-22 cells was assessed using an enzyme-linked immunosorbent assay. Phosphorylation levels of intracellular signaling molecules were evaluated using western blot analyses.Results: iE-DAP induced NOD1 receptor expression in Ca9-22 cells. Additionally, iE-DAP induced expression of pro-IL-1β protein without extracellular secretion. Our results suggest that iE-DAP regulates IL-8 production by activating p38 mitogen-activated protein kinase (MAPK) and ERK1/2 signaling pathways. iE-DAP also promoted nuclear factor kappa-B p65 phosphorylation, facilitating its nuclear translocation. Notably, p38 MAPK and ERK1/2 inhibitors suppressed iE-DAP-stimulated IL-8 production, suggesting that JNK is not involved in this mechanism. ConclusionsOur results indicate that p38 MAPK and ERK1/2, but not JNK, are involved in innate immune responses in GECs.

Polyvalent passive vaccine candidates from egg yolk antibodies (IgY) of important outer membrane proteins (PF1380 and ExbB) of Pseudomonas fluorescens in fish

Polyvalent antibodies can resist multiple bacterial species, and immunoglobulin Y (IgY) antibody can be economically prepared in large quantities from egg yolk; further, IgY polyvalent antibodies have application value in aquaculture. The outer membrane proteins (OMPs) PF1380 and ExbB of Pseudomonas fluorescens were expressed and purified, and the corresponding IgY antibodies were prepared. PF1380, ExbB, and the corresponding IgY antibodies could activate the innate immune responses of chicken and Carassius auratus. The passive immunization to C. auratus showed that the IgY antibodies of PF1380 and ExbB had an immune protection rate, down-regulated the expression of antioxidant-related factors (MDA, SOD, GSH-Px, and CAT) to reduce the antioxidant reaction, down-regulated the expression of inflammation-related genes (IL-6, IL-8, TNF-α, and IL-1β) to reduce the inflammatory reaction, maintained the integrity of visceral tissue structure, and reduced apoptosis and damage of tissue cells in relation to P. fluorescens and Aeromonas hydrophila infections. Thus, the IgY antibodies of PF1380 and ExbB could be considered as passive polyvalent vaccine candidates in aquaculture.

Side-chain dynamics of the α1B -adrenergic receptor determined by NMR via methyl relaxation.

G protein-coupled receptors (GPCRs) are medically important membrane proteins that sample inactive, intermediate, and active conformational states characterized by relatively slow interconversions (~μs-ms). On a faster timescale (~ps-ns), the conformational landscape of GPCRs is governed by the rapid dynamics of amino acid side chains. Such dynamics are essential for protein functions such as ligand recognition and allostery. Unfortunately, technical challenges have almost entirely precluded the study of side-chain dynamics for GPCRs. Here, we investigate the rapid side-chain dynamics of a thermostabilized α1B -adrenergic receptor (α1B -AR) as probed by methyl relaxation. We determined order parameters for Ile, Leu, and Val methyl groups in the presence of inverse agonists that bind orthosterically (prazosin, tamsulosin) or allosterically (conopeptide ρ-TIA). Despite the differences in the ligands, the receptor's overall side-chain dynamics are very similar, including those of the apo form. However, ρ-TIA increases the flexibility of Ile1764×56 and possibly of Ile2145×49 , adjacent to Pro2155×50 of the highly conserved P5×50 I3×40 F6×44 motif crucial for receptor activation, suggesting differences in the mechanisms for orthosteric and allosteric receptor inactivation. Overall, increased Ile side-chain rigidity was found for residues closer to the center of the membrane bilayer, correlating with denser packing and lower protein surface exposure. In contrast to two microbial membrane proteins, in α1B -AR Leu exhibited higher flexibility than Ile side chains on average, correlating with the presence of Leu in less densely packed areas and with higher protein-surface exposure than Ile. Our findings demonstrate the feasibility of studying receptor-wide side-chain dynamics in GPCRs to gain functional insights.

Ubiquitin-like protein MNSFβ regulates glycolysis and promotes cell proliferation with HSC70 assistance

Monoclonal non-specific suppressor factor β (MNSFβ) is a universally expressed ubiquitin-like protein that has multiple biological functions. MNSFβ modifies its target molecules through covalent conjugation. Most recently, we identified a molecular chaperone, HSC70, that facilitates the stabilization of aggregable MNSFβ. In the current study, we determined the role of HSC70 in stabilizing unstable MNSFβ. HSC70 promoted the correct folding of MNSFβ both in vitro and in vivo. We also examined the regulatory function of MNSFβ in cell proliferation and glycolysis. MNSFβ siRNA and HSC70 siRNA treatment attenuated lactate release from Raw264.7 macrophage-like cells. MNSFβ siRNA inhibited glucose uptake in Raw264.7 cells. We found that glucose transporter 1 (GLUT1) is an important membrane protein involved in the regulatory function of MNSFβ during glycolysis. MNSFβ siRNA inhibited the increased GLUT1 expression in LPS-stimulated cells, suggesting that MNSFβ controls the inflammatory response through GLUT1 regulation. We identified several important molecules, including lactate dehydrogenase A, which are regulated by MNSFβ and involved in glucose metabolism. Here we firstly report that MNSFβ regulates glycolysis and promotes cell proliferation.

Crystal Structure of an Archaeal Tyrosyl-tRNA Synthetase Bound to Photocaged L-Tyrosine and Its Potential Application to Time-Resolved X-ray Crystallography.

Genetically encoded caged amino acids can be used to control the dynamics of protein activities and cellular localization in response to external cues. In the present study, we revealed the structural basis for the recognition of O-(2-nitrobenzyl)-L-tyrosine (oNBTyr) by its specific variant of Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (oNBTyrRS), and then demonstrated its potential availability for time-resolved X-ray crystallography. The substrate-bound crystal structure of oNBTyrRS at a 2.79 Å resolution indicated that the replacement of tyrosine and leucine at positions 32 and 65 by glycine (Tyr32Gly and Leu65Gly, respectively) and Asp158Ser created sufficient space for entry of the bulky substitute into the amino acid binding pocket, while Glu in place of Leu162 formed a hydrogen bond with the nitro moiety of oNBTyr. We also produced an oNBTyr-containing lysozyme through a cell-free protein synthesis system derived from the Escherichia coli B95. ΔA strain with the UAG codon reassigned to the nonnatural amino acid. Another crystallographic study of the caged protein showed that the site-specifically incorporated oNBTyr was degraded to tyrosine by light irradiation of the crystals. Thus, cell-free protein synthesis of caged proteins with oNBTyr could facilitate time-resolved structural analysis of proteins, including medically important membrane proteins.

Uncovering lipopolysaccharide regulation in bacteria via the critical lipid binding tunnel of YciS/YciM

SummaryGram-negative bacteria contain an asymmetric outer membrane, in which the outer leaflet is composed of lipopolysaccharide (LPS). LPS, a drug target of polymyxin, plays an essential role in drug resistance, biofilm formation, and pathogenesis. An important inner membrane protein, YciM, may be responsible for the regulation of LPS biosynthesis and transport. Here, we report the crystal structure of YciM from Salmonella typhimurium in a complex with a non-specifically bond molecule, an ethylene glycol, which identified a tunnel that could bind lipids. Our in vitro assays showed that YciM could bind lipid molecules with affinity in the micromolar range, while mutagenic and functional studies confirmed that lipid-binding residues are critical for the function of YciM. Additionally, our data also showed that YciM accurately regulates LPS biosynthesis and transport with YciS, which could help to better understand the regulation mechanism of LPS.

Fluorescently Labeled α-Conotoxin TxID, a New Probe for α3β4 Neuronal Nicotinic Acetylcholine Receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) are important ion channel membrane proteins that are widely distributed in the central nervous system (CNS) and peripheral nervous system (PNS). As an important member, α3β4 nAChRs are related to pain sensation in PNS and nicotine addiction in CNS. However, research related to the α3β4 nAChRs is greatly limited by the lack of subtype-selective pharmacological tools. The α-conotoxin (α-CTx) TxID from the marine cone snail, Conus textile, is a selective α3β4 nAChR antagonist with relatively high potency. In this study, a fluorescent dye (5-TAMRA SE) was used to label TxID on the N-terminus of α-CTx TxID, and pure TxID-F (fluorescent analogue of TxID) was obtained by HPLC. At the same time, the potency and selectivity of TxID-F were detected by high-performance liquid chromatography (HPLC). Additionally, the potency and selectivity of TxID-F were determined by using a two-electrode voltage-clamp technique on various nAChRs expressed in the Xenopus oocyte expression system. The results obtained by electrophysiology showed that TxID-F maintained the same order of potency (IC50 73 nM) as the native toxin (IC50 25 nM) for the α3β4 nAChR subtype. In addition, the results of fluorescent spectroscopy and circular dichroism showed TxID-F has the same fluorescence as 5-TAMRA SE, as well as similar profiles as TxID. The results of flow cytometry showed that the histogram shifted significantly to the right for the RAW264.7 cells expressing α3β4-containing nAChRs stained with TxID-F and confirmed by live cell imaging. The study of fluorescent-labeled α-CTx TxID provides a rich pharmacological tool to explore the structure–function relationship, distribution, and ligand-binding domain of α3β4 nAChR subtype in the future.

A rapid anti-Helicobacter pylori biofilm drug screening biosensor based on AlpB outer membrane protein and colloidal gold/nanoporous gold framework

Inhibition or disruption of biofilms has been recognized as an important means to eradicate Helicobacter pylori (H. pylori) infection. However, a fast and efficient drug screening method against H. pylori biofilms has not yet been established. Therefore, AlpB, an important outer membrane protein in H. pylori biofilm formation, was selected as a biological recognition element to screen anti-biofilm drugs in this study. A novel AlpB/colloidal gold (CG)/nanoporous gold (NPG)/Nafion-reduced graphene oxide (rGO)/glassy carbon electrode (GCE) biosensor was constructed based on the heterologous expression of AlpB. The prepared AlpB-based biosensor not only successfully identified six anti-biofilm drugs, but also evaluated the sensitivity and action intensity of different anti-biofilm drugs binding to AlpB by interaction kinetics analysis. The sensitivity order of AlpB to the six anti-biofilm drugs was: allicin > erythromycin > SCC > curcumin > rifampicin > NAC and the action intensity of the six anti-biofilm drugs on AlpB was: rifampicin > NAC > allicin > erythromycin > SCC > curcumin. In addition, molecular docking results showed that the six anti-biofilm drugs might exert their anti-biofilm effects by spontaneously binding to the conserved region of AlpB protein. This study provided a rapid screening platform and a unified data processing method for potential anti-biofilm drug development.

Natural Compounds or Their Derivatives against Breast Cancer: A Computational Study.

Background Breast cancer is one of the most common types of cancer diagnosed and the second leading cause of death among women. Breast cancer susceptibility proteins of type 1 and 2 are human tumor suppressor genes. Genetic variations/mutations in these two genes lead to overexpression of human breast tumor suppressor genes (e.g., BRCA1, BRCA2), which triggers uncontrolled duplication of cells in humans. In addition, multidrug resistance protein 1 (MDR1), an important cell membrane protein that pumps many foreign substances from cells, is also responsible for developing resistance to cancer chemotherapy. Aim of the Study. The aim of this study was to analyze some natural compounds or their derivatives as part of the development of strong inhibitors for breast cancer. Methodology. Molecular docking studies were performed using compounds known in the literature to be effective against BRCA1 and BRCA2 and MDR1, with positive control being 5-fluorouracil, an antineoplastic drug as a positive control. Results The binding affinity of the compounds was analyzed, and it was observed that they had a better binding affinity for the target proteins than the standard drug 5-fluorouracil. Among the compounds analyzed, α-hederin, andrographolide, apigenin, asiatic acid, auricular acid, sinularin, curcumin, citrinin, hispolon, nerol, phytol, retinol palmitate, and sclareol showed the best binding affinity energy to the BRCA1, BRCA2, and MDR1 proteins, respectively. Conclusions α-Hederin, andrographolide, apigenin, asiatic acid, auricular acid, hispolon, sclareol, curcumin, citrinin, and sinularin or their derivatives can be a good source of anticancer agents in breast cancer.

Roadmap for Optimizing and Broadening Antibody-Based PROTACs for Degradation of Cell Surface Proteins.

Targeted protein degradation is a promising therapeutic strategy capable of overcoming the limitations of traditional occupancy-based inhibitors. By ablating all of the associated functions of a protein at once, the event-driven pharmacology of degrader technologies has recently enabled the targeting of proteins that have been historically deemed "undruggable". Most degradation strategies utilize the ubiquitin-proteasome system to mediate intracellular target degradation and are thus limited to targeting proteins with cytoplasmic domains. While some of these strategies, such as PROTACs, have shown great promise, there is a need for new modalities that can be applied to specifically target cell surface proteins. We previously described the development of an antibody-based PROTAC (AbTAC) that utilizes genetically encoded IgG bispecific antibody scaffolds to bring the cell surface E3-ligase RNF43 into the proximity of a membrane protein of interest (POI) to mediate its degradation. Here, we employ rational protein engineering strategies to interrogate and optimize the properties necessary for efficient degradation of two therapeutically important membrane proteins, PD-L1 and EGFR. We develop multiple antibodies to RNF43 and show that the specific antibody binding epitopes on RNF43 and the POI are more important than the affinities of the AbTAC antibodies. We further expand the available repertoire of E3 ligases by co-opting the E3-ligase ZNRF3 to degrade both PD-L1 and EGFR and show similar importance of epitope for degradation efficiency. Importantly, we show that both RNF43 and ZNRF3 AbTACs do not potentiate unwanted WNT signaling. Lastly, we find that these AbTACs can be even further improved by exploring various dual-binding and IgG scaffolds that range in flexibility, valency, and orientation of the binding arms. These structure-activity and mechanistic studies provide a roadmap for optimizing the development of AbTACs, thereby greatly expanding their utility for targeted cell surface protein degradation.

Repeated Exposure of Macrophages to Synthetic Amorphous Silica Induces Adaptive Proteome Changes and a Moderate Cell Activation.

Synthetic amorphous silica (SAS) is a nanomaterial used in a wide variety of applications, including the use as a food additive. Two types of SAS are commonly employed as a powder additive, precipitated silica and fumed silica. Numerous studies have investigated the effects of synthetic amorphous silica on mammalian cells. However, most of them have used an exposure scheme based on a single dose of SAS. In this study, we have used instead a repeated 10-day exposure scheme in an effort to better simulate the occupational exposure encountered in daily life by consumers and workers. As a biological model, we have used the murine macrophage cell line J774A.1, as macrophages are very important innate immune cells in the response to particulate materials. In order to obtain a better appraisal of the macrophage responses to this repeated exposure to SAS, we have used proteomics as a wide-scale approach. Furthermore, some of the biological pathways detected as modulated by the exposure to SAS by the proteomic experiments have been validated through targeted experiments. Overall, proteomics showed that precipitated SAS induced a more important macrophage response than fumed SAS at equal dose. Nevertheless, validation experiments showed that most of the responses detected by proteomics are indeed adaptive, as the cellular homeostasis appeared to be maintained at the end of the exposure. For example, the intracellular glutathione levels or the mitochondrial transmembrane potential at the end of the 10 days exposure were similar for SAS-exposed cells and for unexposed cells. Similarly, no gross lysosomal damage was observed after repeated exposure to SAS. Nevertheless, important functions of macrophages such as phagocytosis, TNFα, and interleukin-6 secretion were up-modulated after exposure, as was the expression of important membrane proteins such as the scavenger receptors, MHC-II, or the MAC-1 receptor. These results suggest that repeated exposure to low doses of SAS slightly modulates the immune functions of macrophages, which may alter the homeostasis of the immune system.

Crystal structure of CmABCB1 multi-drug exporter in lipidic mesophase revealed by LCP-SFX.

CmABCB1 is a Cyanidioschyzon merolae homolog of human ABCB1, a well known ATP-binding cassette (ABC) transporter responsible for multi-drug resistance in various cancers. Three-dimensional structures of ABCB1 homologs have revealed the snapshots of inward- and outward-facing states of the transporters in action. However, sufficient information to establish the sequential movements of the open-close cycles of the alternating-access model is still lacking. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has proven its worth in determining novel structures and recording sequential conformational changes of proteins at room temperature, especially for medically important membrane proteins, but it has never been applied to ABC transporters. In this study, 7.7 mono-acyl-glycerol with cholesterol as the host lipid was used and obtained well diffracting microcrystals of the 130 kDa CmABCB1 dimer. Successful SFX experiments were performed by adjusting the viscosity of the crystal suspension of the sponge phase with hy-droxy-propyl methyl-cellulose and using the high-viscosity sample injector for data collection at the SACLA beamline. An outward-facing structure of CmABCB1 at a maximum resolution of 2.22 Å is reported, determined by SFX experiments with crystals formed in the lipidic cubic phase (LCP-SFX), which has never been applied to ABC transporters. In the type I crystal, CmABCB1 dimers interact with adjacent molecules via not only the nucleotide-binding domains but also the transmembrane domains (TMDs); such an interaction was not observed in the previous type II crystal. Although most parts of the structure are similar to those in the previous type II structure, the substrate-exit region of the TMD adopts a different configuration in the type I structure. This difference between the two types of structures reflects the flexibility of the substrate-exit region of CmABCB1, which might be essential for the smooth release of various substrates from the transporter.

A Genetically Encoded F-19 NMR Probe Reveals the Allosteric Modulation Mechanism of Cannabinoid Receptor 1.

Due to the lack of genetically encoded probes for fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR), its utility for probing eukaryotic membrane protein dynamics is limited. Here we report an efficient method for the genetic incorporation of an unnatural amino acid (UAA), 3'-trifluoromenthyl-phenylalanine (mtfF), into cannabinoid receptor 1 (CB1) in the Baculovirus Expression System. The probe can be inserted at any environmentally sensitive site, while causing minimal structural perturbation to the target protein. Using 19F NMR and X-ray crystallography methods, we discovered that the allosteric modulator Org27569 and agonists synergistically stabilize a previously unrecognized pre-active state. An allosteric modulation model is proposed to explain Org27569's distinct behavior. We demonstrate that our site-specific 19F NMR labeling method is a powerful tool in decoding the mechanism of GPCR allosteric modulation. This new method should be broadly applicable for uncovering conformational states for many important eukaryotic membrane proteins.

GSK1702934A and M085 directly activate TRPC6 via a mechanism of stimulating the extracellular cavity formed by the pore helix and transmembrane helix S6

Transient receptor potential canonical (TRPC) channels, as important membrane proteins regulating intracellular calcium (Ca2+i) signaling, are involved in a variety of physiological and pathological processes. Activation and regulation of TRPC are more dependent on membrane or intracellular signals. However, how extracellular signals regulate TRPC6 function remains to be further investigated. Here, we suggest that two distinct small molecules, M085 and GSK1702934A, directly activate TRPC6, both through a mechanism of stimulation of extracellular sites formed by the pore helix (PH) and transmembrane (TM) helix S6. In silico docking scanning of TRPC6 identified three extracellular sites that can bind small molecules, of which only mutations on residues of PH and S6 helix significantly reduced the apparent affinity of M085 and GSK1702934A and attenuated the maximal response of TRPC6 to these two chemicals by altering channel gating of TRPC6. Combing metadynamics, molecular dynamics simulations, and mutagenesis, we revealed that W679, E671, E672, and K675 in the PH and N701 and Y704 in the S6 helix constitute an orthosteric site for the recognition of these two agonists. The importance of this site was further confirmed by covalent modification of amino acid residing at the interface of the PH and S6 helix. Given that three structurally distinct agonists M085, GSK1702934A, and AM-0883, act at this site, as well as the occupancy of lipid molecules at this position found in other TRP subfamilies, it is suggested that the cavity formed by the PH and S6 has an important role in the regulation of TRP channel function by extracellular signals.

Tissue engineered bovine saphenous vein extracellular matrix scaffolds produced via antigen removal achieve high in vivo patency rates

Diseases of small diameter blood vessels encompass the largest portion of cardiovascular diseases, with over 4.2 million people undergoing autologous vascular grafting every year. However, approximately one third of patients are ineligible for autologous vascular grafting due to lack of suitable donor vasculature. Acellular extracellular matrix (ECM) scaffolds derived from xenogeneic vascular tissue have potential to serve as ideal biomaterials for production of off-the-shelf vascular grafts capable of eliminating the need for autologous vessel harvest. A modified antigen removal (AR) tissue process, employing aminosulfabetaine-16 (ASB-16) was used to create off-the-shelf small diameter (< 3 mm) vascular graft from bovine saphenous vein ECM scaffolds with significantly reduced antigenic content, while retaining native vascular ECM protein structure and function. Elimination of native tissue antigen content conferred graft-specific adaptive immune avoidance, while retention of native ECM protein macromolecular structure resulted in pro-regenerative cellular infiltration, ECM turnover and innate immune self-recognition in a rabbit subpannicular model. Finally, retention of the delicate vascular basement membrane protein integrity conferred endothelial cell repopulation and 100% patency rate in a rabbit jugular interposition model, comparable only to Autograft implants. Alternatively, the lack of these important basement membrane proteins in otherwise identical scaffolds yielded a patency rate of only 20%. We conclude that acellular antigen removed bovine saphenous vein ECM scaffolds have potential to serve as ideal off-the-shelf small diameter vascular scaffolds with high in vivo patency rates due to their low antigen content, retained native tissue basement membrane integrity and preserved native ECM structure, composition and functional properties. Statement of significanceThe use of autologous vessels for the treatment of small diameter vascular diseases is common practice. However, the use of autologous tissue poses significant complications due to tissue harvest and limited availability. Developing an alternative vessel for use for the treatment of small diameter vessel diseases can potentially increase the success rate of autologous vascular grafting by eliminating complications related to the use of autologous vessel and increased availability. This manuscript demonstrates the potential of non-antigenic extracellular matrix (ECM) scaffolds derived from xenogeneic vascular tissue as off-the-shelf vascular grafts for the treatment of small diameter vascular diseases.

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis.

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae ADΔΔ is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae ADΔΔ, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.