Important Membrane Proteins Research Articles

Isolation and Identification of a Highly Divergent Kaeng Khoi Virus from Bat Flies (Eucampsipoda sundaica) in China.

Kaeng Khoi virus (KKV), which belongs to the genus Orthobunyavirus, family Perbunyaviridae, was originally isolated from the brain tissue of bats and may cause infection in humans. In this study, the KKV strain WDBC1403 was isolated from bat flies (Eucampsipoda sundaica), ectoparasites of the bat Rousettus leschenaultia, collected from Yunnan Province of China at the Sino-Burmese border. The bat fly specimens were ground and inoculated in culture cells. The WDBC1403 strain was shown to induce cytopathic effects in Vero, baby hamster kidney (BHK-21), and Tb1Lu cells, but not in C6/36 cells; however, viral gene amplification was detected in the supernatants of C6/36 cells. Using electron microscopy, the virus was determined to be spherical, enveloped, and 80-90 nm in diameter; it was also shown to form plaques in BHK-21 cells and the titer reached 1 × 106.57 plaque-forming units/mL 24 h after infection. The phylogenetic analysis showed that WDBC1403 is a KKV strain, but is independent of the original KKV strain (PSC-19). Viral genome analysis revealed that the nucleotide and amino acid sequence identities of the S, M, and L segments of WDBC1403 with PSC-19 were 88.2% and 96.1%, 76.7% and 85.0%, and 78.3% and 88.9%, respectively. Two amino acids were removed at the end of the open reading frame of the M segment, and 47 nucleotides were removed in the 3'-untranslated region of the M segment of the WDBC1403 strain compared with the PSC-19 strain. The WDBC1403 strain is a highly divergent KKV strain, suggesting that there are a variety of KKV strains that exhibit molecular differences. Moreover, because of the large variations in nucleotide and amino acid sequence in the M segment, which encodes an important membrane protein, further research on antigenicity and pathogenicity in humans and animals is needed.

Melatonin chelates iron and binds directly with phenylhydrazine to provide protection against phenylhydrazine induced oxidative damage in red blood cells along with its antioxidant mechanisms: an in vitro study

Oxidative stress is an important causative factor for a number of diseases. Phenylhydrazine (PHZ) is a widely accepted model for studying hemolytic anemia by induction of oxidative stress. In the present study, goat red blood cells (RBCs) were incubated in vitro with PHZ (1mM) to generate oxidative stress. To test whether melatonin exhibits protective effects on PHZ induced RBC damage and to explore its potential molecular mechanisms, different concentrations of melatonin (5, 10, 20 and 40 nmoles/ml) were also included. PHZ caused altered profiles on biomarkers of oxidative stress and antioxidative as well as glucose metabolic enzymes in RBCs. These alterations indicated a development of oxidative stress. Melatonin at a concentration of 40 nmoles/ml provided optimal protection against all alterations induced by PHZ. The important cellular membrane proteins, including spectrin and actin, were also damaged by PHZ and this led to RBC deformation similar to that of observed in severe β-thalassaemia; the RBC deformation was also prevented by melatonin. Binding profiles of melatonin with PHZ and ferrous iron indicated favorable binding of melatonin with both of them, respectively. Thus, in addition to the direct antioxidant and free radical scavenging capability, melatonin also inhibited iron overloading by chelating iron and binding with the PHZ. This action of melatonin further reduces free radical generation. Based on the results, melatonin may provide therapeutic relevance to ß-thalassemia and other hemolytic RBC disorders involving oxidative stress.

The 5'-AMP-Activated Protein Kinase Regulates the Function and Expression of Human Organic Anion Transporting Polypeptide 1A2.

The organic anion transporting polypeptides (OATPs) are important membrane proteins that mediate the cellular uptake of drugs and endogenous substances. OATP1A2 is widely distributed in many human tissues that are targeted in drug therapy; defective OATP1A2 leads to altered drug disposition influencing therapeutic outcomes. 5'-AMP-activated protein kinase (AMPK) signaling plays an important role in the pathogenesis of the metabolic syndrome characterized by an increased incidence of type II diabetes and nonalcoholic fatty liver disease. This study investigated the regulatory role of AMPK in OATP1A2 transport function and expression. We found that the treatment of AMPK-specific inhibitor compound C (dorsomorphin dihydrochloride) decreased OATP1A2-mediated uptake of estrone-3-sulfate in a concentration- and time-dependent manner. The impaired OATP1A2 function was associated with a reduced Vmax [154.6 ± 17.9 pmol × (μg × 4 minutes)-1 in compound C-treated cells vs. 413.6 ± 52.5 pmol × (μg × 4 minutes)-1 in controls]; the Km was unchanged. The cell-surface expression of OATP1A2 was decreased by compound C treatment, but total cellular expression was unchanged. The impaired cell-surface expression of OATP1A2 was associated with accelerated internalization and impaired targeting/recycling. Silencing of the AMPK α1-subunit using specific small interfering RNA corroborated the findings with compound C and revealed a role for AMPK in regulating OATP1A2 protein stability. Overall, this study implicated AMPK in the regulation of the function and expression of OATP1A2, which potentially impacts on the disposition of OATP1A2 drug substrates that may be used to treat patients with the metabolic syndrome and other diseases.

Increased expression of DRAM1 confers myocardial protection against ischemia via restoring autophagy flux

BackgroundDRAM1 (Damage-regulated autophagy modulator 1) was reported as one of the most important lysosome membrane protein that mediates the interaction between autophagosome and lysosome. Our aim was to investigate whether DRAM1 contributes to cardiac remodeling after acute myocardial infarction (AMI) and the underlying mechanisms. Methods and resultsAdenovirus harboring DRAM1 was injected in the peri-infarct zone in a rat model of AMI experimentally produced by permanent ligation of left anterior descending (LAD) coronary artery. Increased DRAM1 expression protected the cardiomyocytes from ischemia stress-induced autophagy flux obstacle and improved cardiac prognosis after AMI. DRAM1 overexpression attenuated the accumulation of autophagy substrate protein, LC3IIand p62/SQSTM1 obviously both in vivo and in vitro. An adenovirus harboring mRFP-GFP-LC3 showed that DRAM1 overexpression restored the autophagic flux by enhancing autophagosome conversion to autophagolysosome. Although Atg12 mRNA was up-regulated with DRAM1 overexpression the free Atg12 protein was decreased accompanied by increased Atg12-Atg5 conjugate both in vitro and in vivo. Of interest, immunoprecipitation assay showed that DRAM1 interacted with Atg7, but without direct interaction with Atg5 or Atg12. Notably, the effect of DRAM1 on autophagy flux and cardiomyocyte protection could be mitigated by Atg7 siRNA. ConclusionsOur results indicated that DRAM1 protected cardiomyocytes from ischemia stress-induced autophagy flux obstacle and uncovered a novel DRAM1-Atg7-Atg12/Atg5 autophagy flux regulation pathway under conditions of myocardial ischemic stress.

The role of disordered regions in GPCR‐G protein signaling

G-protein-coupled receptors (GPCRs) are physiologically important membrane proteins that sense signaling molecules such as hormones and neurotransmitters and are the targets of several prescribed drugs. The ~800 human GPCRs allosterically activate 16 G proteins and trigger GDP release to regulate intracellular signaling. In this presentation, I will present our findings on the allosteric mechanism governing Gα activation and the determinants of selective coupling between receptors and G proteins. In the first part of the talk, I will present evidence, which suggests that different GPCRs interact with and activate Gα proteins through a highly conserved mechanism. Through comparison of Gα with the small G protein Ras, we found that the evolution of short segments that undergo disorder-to-order transitions can decouple regions important for allosteric activation from receptor binding specificity [1]. In the second part of the talk, I will present the existence of a selectivity barcode (i.e., patterns of amino acids) within the disordered tail of each of the 16 human G proteins that is recognized by distinct regions on the ~800 human receptors. Although universally conserved positions in the barcode allow the receptors to bind and activate G proteins in a similar manner, different receptors recognize the unique positions of the G-protein barcode through distinct residues, like multiple keys (receptors) opening the same lock (G protein) using non-identical cuts. Considering the evolutionary history of GPCRs, specifically, the disordered intracellular loops allows the identification of these selectivity-determining residues [2]. These observations might explain how the GPCR-Gα system diversified rapidly while conserving the allosteric activation mechanism and provides a framework for understanding the molecular basis of coupling selectivity within individual receptors and G proteins. Support or Funding Information Medical Research Council (MC_U105185859) GPCR-G protein interface This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cyclophilin A is the potential receptor of the Mycoplasma genitalium adhesion protein

The Mycoplasma genitalium adhesion protein (MgPa), the most important outer membrane protein of M. genitalium, plays a vital role in the adhesion to and invasion of host cells by M. genitalium. Identification of MgPa receptors will help elucidate the pathogenic mechanism of M. genitalium. However, the receptor protein of MgPa has not been reported to date. In this study, an MgPa-binding protein with a molecular weight of approximately 17 kDa was screened from SV-HUC-1 cell membrane proteins by a modified virus overlay protein binding assay (VOPBA). Liquid chromatography-mass spectrometry (LC–MS) was used to analyze the protein components of the 17-kDa protein. The results demonstrated that the MgPa-binding protein was most likely Cyclophilin A (CyPA). The binding activity and distribution of CyPA in SV-HUC-1 cells were detected using indirect ELISA, western blotting, far-western blotting and indirect immunofluorescence. We found that recombinant MgPa (rMgPa) could bind with CyPA from SV-HUC-1 cell membrane proteins and to recombinant CyPA, which indicated that CyPA was predominant component of the 17-kDa protein band and can interact with rMgPa. In addition, an indirect immunofluorescence assay showed that CyPA was partially distributed on the membrane surfaces of SV-HUC-1 cells and could partially inhibit the adhesion of rMgPa and M. genitalium to SV-HUC-1 cells. Co-localization assays further indicated that rMgPa and M. genitalium can interact with CyPA. These results suggested that the CyPA located on SV-HUC-1 cell membranes may be the potential receptor of MgPa, which could provide an experimental basis for elucidating the function of MgPa and the possible pathogenic mechanism of M. genitalium.

Exploring G Protein-Coupled Receptors (GPCRs) Ligand Space via Cheminformatics Approaches: Impact on Rational Drug Design.

The primary goal of rational drug discovery is the identification of selective ligands which act on single or multiple drug targets to achieve the desired clinical outcome through the exploration of total chemical space. To identify such desired compounds, computational approaches are necessary in predicting their drug-like properties. G Protein-Coupled Receptors (GPCRs) represent one of the largest and most important integral membrane protein families. These receptors serve as increasingly attractive drug targets due to their relevance in the treatment of various diseases, such as inflammatory disorders, metabolic imbalances, cardiac disorders, cancer, monogenic disorders, etc. In the last decade, multitudes of three-dimensional (3D) structures were solved for diverse GPCRs, thus referring to this period as the “golden age for GPCR structural biology.” Moreover, accumulation of data about the chemical properties of GPCR ligands has garnered much interest toward the exploration of GPCR chemical space. Due to the steady increase in the structural, ligand, and functional data of GPCRs, several cheminformatics approaches have been implemented in its drug discovery pipeline. In this review, we mainly focus on the cheminformatics-based paradigms in GPCR drug discovery. We provide a comprehensive view on the ligand– and structure-based cheminformatics approaches which are best illustrated via GPCR case studies. Furthermore, an appropriate combination of ligand-based knowledge with structure-based ones, i.e., integrated approach, which is emerging as a promising strategy for cheminformatics-based GPCR drug design is also discussed.

Dawning of a new era in TRP channel structural biology by cryo-electron microscopy.

Cryo-electron microscopy (cryo-EM) permits the determination of atomic protein structures by averaging large numbers of individual projection images recorded at cryogenic temperatures-a method termed single-particle analysis. The cryo-preservation traps proteins within a thin glass-like ice layer, making literally a freeze image of proteins in solution. Projections of randomly adopted orientations are merged to reconstruct a 3D density map. While atomic resolution for highly symmetric viruses was achieved already in 2009, the development of new sensitive and fast electron detectors has enabled cryo-EM for smaller and asymmetrical proteins including fragile membrane proteins. As one of the most important structural biology methods at present, cryo-EM was awarded in October 2017 with the NobelPrize in Chemistry. The molecular understanding of Transient-Receptor-Potential (TRP) channels has been boosted tremendously by cryo-EM single-particle analysis. Several near-atomic and atomic structures gave important mechanistic insights, e.g., into ion permeation and selectivity, gating, as well as into the activation of this enigmatic and medically important membrane protein family by various chemical and physical stimuli. Lastly, these structures have set the starting point for the rational design of TRP channel-targeted therapeutics to counteract life-threatening channelopathies. Here, we attempt a brief introduction to the method, review the latest advances in cryo-EM structure determination of TRP channels, and discuss molecular insights into the channel function based on the wealth of TRP channel cryo-EM structures.

Helicobacter pylori bab characterization in clinical isolates from Bhutan, Myanmar, Nepal and Bangladesh.

BackgroundHelicobacter pylori BabA is an important outer membrane protein that involves in the attachment to the gastric mucosa and enhances the virulence property of the bacterium. This study was aimed to characterize the bab genotypes, to evaluate its association with cagA, vacA and clinical diseases as well as degree of gastric inflammation.MethodsH. pylori isolates from four countries were subjected for the characterization of bab. The locus specific forward and bab specific reverse primers were used to get the specific products by PCR, which could distinguish the three locus (A, B and C). The histological activities were evaluated according to the Updated Sydney system.ResultIn patients from high risk countries (Bhutan and Myanmar) relatively higher frequencies of strains with babA-positivity (91.8% and 90.7%, respectively), babA at locus A (98% and 91.2%, respectively) and with single babA (96.8% and 91.2%, respectively) were found. Strains with two loci occupied were the most prevalent in Bhutan (84.6%), Myanmar (74.7%), Nepal (58.3%) and Bangladesh (56.9%). The genotype babA at locus A/babB at locus B/bab-negative at locus C (babA/babB/-) was the most common genotype isolated from Bhutan (82.7%), Myanmar (58.7%), Nepal (32%) and Bangladesh (31.4%) among all genotypes assessed. This genotype was also associated with the peptic ulcer disease (P = 0.013) when compared to gastritis. babA-positive characteristics and the genotype babA/babB/- exhibited the enhanced histological activities.ConclusionsThe higher prevalence of virulence associated babA-positive characteristics and enhanced histological activities in Bhutan than in Myanmar, Nepal and Bangladesh might partly explain why the peoples in Bhutan are at higher risk for developing severe gastric complications.

Identification and Characterization of the Major Porin of Desulfovibrio vulgaris Hildenborough.

ABSTRACTDue in large part to their ability to facilitate the diffusion of a diverse range of solutes across the outer membrane (OM) of Gram-negative bacteria, the porins represent one of the most prominent and important bacterial membrane protein superfamilies. Notably, for the Gram-negative bacterium Desulfovibrio vulgaris Hildenborough, a model organism for studies of sulfate-reducing bacteria, no genes for porins have been identified or proposed in its annotated genome. Results from initial biochemical studies suggested that the product of the DVU0799 gene, which is one of the most abundant proteins of the D. vulgaris Hildenborough OM and purified as a homotrimeric complex, was a strong porin candidate. To investigate this possibility, this protein was further characterized biochemically and biophysically. Structural analyses via electron microscopy of negatively stained protein identified trimeric particles with stain-filled depressions and structural modeling suggested a β-barrel structure for the monomer, motifs common among the known porins. Functional studies were performed in which crude OM preparations or purified DVU0799 was reconstituted into proteoliposomes and the proteoliposomes were examined for permeability against a series of test solutes. The results obtained establish DVU0799 to be a pore-forming protein with permeability properties similar to those observed for classical bacterial porins, such as those of Escherichia coli. Taken together, these findings identify this highly abundant OM protein to be the major porin of D. vulgaris Hildenborough. Classification of DVU0799 in this model organism expands the database of functionally characterized porins and may also extend the range over which sequence analysis strategies can be used to identify porins in other bacterial genomes.IMPORTANCE Porins are membrane proteins that form transmembrane pores for the passive transport of small molecules across the outer membranes of Gram-negative bacteria. The present study identified and characterized the major porin of the model sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, observing its preference for anionic sugars over neutral ones. Its predicted architecture appears to be novel for a classical porin, as its core β-barrel structure is of a type typically found in solute-specific channels. Broader use of the methods employed here, such as assays for channel permeability and electron microscopy of purified samples, is expected to help expand the database of confirmed porin sequences and improve the range over which sequence analysis-based strategies can be used to identify porins in other Gram-negative bacteria. Functional characterization of these critical gatekeeping proteins from divergent Desulfovibrio species should offer an improved understanding of the physiological features that determine their habitat range and supporting activities.

Recent Advances in Structure-Based Drug Design Targeting Class A G Protein-Coupled Receptors Utilizing Crystal Structures and Computational Simulations.

G protein-coupled receptors (GPCRs) represent the largest and most physiologically important integral membrane protein family, and these receptors respond to a wide variety of physiological and environmental stimuli. GPCRs are among the most critical therapeutic targets for numerous human diseases, and approximately one-third of the currently marketed drugs target this receptor family. The recent breakthroughs in GPCR structural biology have significantly contributed to our understanding of GPCR function, ligand binding, and pharmacological action as well as to the design of new drugs. This perspective highlights the latest advances in GPCR structures with a focus on the receptor-ligand interactions of each receptor family in class A nonrhodopsin GPCRs as well as the structural features for their activation, biased signaling, and allosteric mechanisms. The current state-of-the-art methodologies of structure-based drug design (SBDD) approaches in the GPCR research field are also discussed.

A Novel Regulatory Cascade Involving BluR, YcgZ, and Lon Controls the Expression of Escherichia coli OmpF Porin

In Escherichia coli, OmpF is an important outer membrane protein, which serves as a passive diffusion pore for small compounds including nutrients, antibiotics, and toxic compounds. OmpF expression responds to environmental changes such as temperature, osmolarity, nutrients availability, and toxic compounds via complex regulatory pathways involving transcriptional and post-transcriptional regulation. Our study identified a new regulatory cascade that controls the expression of OmpF porin. This pathway involves BluR, a transcriptional regulator repressing the expression of the ycgZ-ymgABC operon. We showed that BluR was responsible for the temperature-dependent regulation of the ycgZ-ymgABC operon. Furthermore, our results showed that independent expression of YcgZ led to a decreased activity of the ompF promoter, while YmgA, YmgB, and YmgC expression had no effect. We also determined that YcgZ accumulates in the absence of the Lon protease. Thus, mutation in bluR leads to de-repression of ycgZ-ymgABC transcription. With a second mutation in lon, YcgZ protein accumulates to reach levels that do not allow increased expression of OmpF under growth conditions that usually would, i.e., low temperature. With BluR responding to blue-light and temperature, this study sheds a new light on novel signals able to regulate OmpF porin.

Imaging of protein-specific glycosylation by glycan metabolic tagging and in situ proximity ligation

Glycosylation is an important posttranslational modification, which regulates a number of critical biological processes including cell-cell recognition, signal transduction and disease progression. Probing the glycosylation status on a specific protein of interest enables an in-depth understanding of the role of glycosylation on protein structure and function. However, methods for monitoring protein-specific glycosylation are largely lacking. Here we describe a highly sensitive fluorescence imaging strategy to visualize the protein-specific glycosylation by combining glycan metabolic tagging and in situ proximity ligation (termed GPLA). We demonstrate the visualization of sialylation, fucosylation and GalNAcylation on several important membrane proteins. Notably, the high spatial resolution of this method allows subcellular localization of the glycosylated fraction of the proteins. We further show that our strategy can be applied to image the dimerization of endogenous epidermal growth factor receptor. Thus, our study provides a unique tool to monitor the protein-specific glycosylation in a dynamic cellular context.

Auto-induction of Pichia pastoris AOX1 promoter for membrane protein expression

Pichia pastoris is a highly successful recombinant protein expression system due to its ability to quickly generate large quantities of recombinant proteins in simple media. P. pastoris has been used to successfully generate milligram quantities of many important human membrane proteins, including G-protein coupled receptors, ion channels, and transporters, which are becoming increasingly important therapeutic targets. Despite these successes, protein expression in P. pastoris is still cumbersome due to a need to change growth media from glycerol media to methanol induction media, which minimizes inhibition of the AOX1 promoter by residual glycerol. Taking advantage of this behavior of the AOX1 promoter, we developed Buffered extra-YNB Glycerol Methanol (BYGM) auto-induction media (100 mM potassium phosphate pH 6.0, 2.68% w/v YNB, 0.4% v/v glycerol, 0.5% v/v methanol, and 8 × 10−5% w/v biotin) which not only simplified the protein expression process, but also optimized protein expression levels in P. pastoris. We successfully used this auto-induction method to overexpress the target in both MutS and Mut+ strains. Moreover, we show that this method can facilitate screening high-expressing clones, as well as enable parallel protein production in P. pastoris.

Cleave but not leave: Astrotactin proteins in development and disease.

Over the years, animal studies have identified astrotactins as important membrane proteins for glial-guided neuronal migration during central nervous system development and hair follicle polarity control during skin development. Biochemical studies have revealed intramembrane proteolysis as an important feature of astrotactins. The two fragments of astrotactins remain linked together by a disulfide bond after the proteolytic cleavage. In humans, mutations in astrotactin genes have also been linked to a wide range of diseases, including several developmental brain disorders, neurodegenerative diseases and cancer. In this review, I will summarize the current knowledge of the biological function of astrotactins in development, highlight the linkage between mutations in astrotactin genes and human disease and discuss several outstanding questions that remain unanswered. © 2017 IUBMB Life, 69(8):572-577, 2017.

Expansion of bone marrow-derived human mesenchymal stem/stromal cells (hMSCs) using a two-phase liquid/liquid system.

BACKGROUNDHuman mesenchymal stem/stromal cells (hMSCs) are at the forefront of regenerative medicine applications due to their relatively easy isolation and availability in adults, potential to differentiate and to secrete a range of trophic factors that could determine specialised tissue regeneration. To date, hMSCs have been successfully cultured in vitro on substrates such as polystyrene dishes (TCPS) or microcarriers. However, hMSC sub‐cultivation and harvest typically employs proteolytic enzymes that act by cleaving important cell membrane proteins resulting in long‐term cell damage. In a process where the cells themselves are the product, a non‐enzymatic and non‐damaging harvesting approach is desirable.RESULTSAn alternative system for hMSC expansion and subsequent non‐enzymatic harvest was investigated here. A liquid/liquid two‐phase system was proposed, comprising a selected perfluorocarbon (FC40) and growth medium (DMEM). The cells exhibited similar cell morphologies compared with TCPS. Moreover, they retained their identity and differentiation potential post‐expansion and post‐harvest. Further, no significant difference was found when culturing hMSCs in the culture systems prepared with either fresh or recycled FC40 perfluorocarbon.CONCLUSIONSThese findings make the FC40/DMEM system an attractive alternative for traditional cell culture substrates due to their ease of cell recovery and recyclability, the latter impacting on overall process costs. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The inhibitory effects of five alkaloids on the substrate transport mediated through human organic anion and cation transporters

1. Human solute carrier transporters (SLCs) are important membrane proteins mediate the cellular transport of many endogenous and exogenous substances. Organic anion/cation transporters (OATs/OCTs) and organic anion transporting polypeptides (OATPs) are essential SLCs involved in drug influx. Drug–drug/herb interactions through competing for specific SLCs often lead to unsatisfied therapeutic outcomes and/or unwanted side effects. In this study, we comprehensively investigated the inhibitory effects of five clinically relevant alkaloids (dendrobine, matrine, oxymatrine, tryptanthrin and chelerythrine) on the substrate transport through several OATs/OCTs and OATPs.2. We performed transport functional assay and kinetic analysis on the HEK-293 cells over-expressing each SLC gene.3. Our data showed tryptanthrin significantly inhibited the transport activity of OAT3 (IC50 = 0.93 ± 0.22 μM, Ki = 0.43 μM); chelerythrine acted as a potent inhibitor to the substrate transport mediated through OATP1A2 (IC50 = 0.63 ± 0.43 μM, Ki = 0.60 μM), OCT1 (IC50 = 13.60 ± 2.81 μM) and OCT2 (IC50 =10.80 ± 1.16 μM).4. Our study suggested tryptanthrin and chelerythrine could potently impact on the drug transport via specific OATs/OCTs. Therefore, the co-administration of these alkaloids with drugs could have clinical consequences due to drug–drug/herb interactions. Precautions should be warranted in the multi-drug therapies involving these alkaloids.

Relevance of CARC and CRAC Cholesterol-Recognition Motifs in the Nicotinic Acetylcholine Receptor and Other Membrane-Bound Receptors.

Cholesterol is a ubiquitous neutral lipid, which finely tunes the activity of a wide range of membrane proteins, including neurotransmitter and hormone receptors and ion channels. Given the scarcity of available X-ray crystallographic structures and the even fewer in which cholesterol sites have been directly visualized, application of in silico computational methods remains a valid alternative for the detection and thermodynamic characterization of cholesterol-specific sites in functionally important membrane proteins. The membrane-embedded segments of the paradigm neurotransmitter receptor for acetylcholine display a series of cholesterol consensus domains (which we have coined "CARC"). The CARC motif exhibits a preference for the outer membrane leaflet and its mirror motif, CRAC, for the inner one. Some membrane proteins possess the double CARC-CRAC sequences within the same transmembrane domain. In addition to in silico molecular modeling, the affinity, concentration dependence, and specificity of the cholesterol-recognition motif-protein interaction have recently found experimental validation in other biophysical approaches like monolayer techniques and nuclear magnetic resonance spectroscopy. From the combined studies, it becomes apparent that the CARC motif is now more firmly established as a high-affinity cholesterol-binding domain for membrane-bound receptors and remarkably conserved along phylogenetic evolution.

Catechins from green tea modulate neurotransmitter transporter activity in Xenopus oocytes

The GABAergic and glutamatergic systems play key roles in controlling activity of the central nervous system. Important membrane proteins in the mammalian central nervous system transporting extrac...

Development of a Sandwich ELISA for EHEC O157:H7 Intimin γ1.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen of worldwide importance that causes foodborne infections in humans. Intimin gamma 1 (intimin γ1) is one of the most important outer membrane proteins required for EHEC’s intimate adhesion to epithelial cells. Here, we generated a polyclonal antibody (pAb) and a monoclonal antibody (mAb) against intimin γ1 to develop a double antibody sandwich ELISA (DAS-ELISA) with increased sensitivity and specificity for measuring EHEC O157:H7. To achieve this goal, a rabbit pAb was used as a capture antibody, and a mouse mAb was a detection antibody. No cross-reactivity was observed with the other genera of pathogenic bacteria tested with the DAS-ELISA, which included Salmonella enteritidis, Shigella flexneri type 2, Listeria monocytogenes, Streptococcus suis type 2, and other 18 serotype E. coli. Detection limits of the DAS-ELISA were 1 × 103 CFU/mL for EHEC O157:H7 cultures, 1 × 104 CFU/g before enrichment, and 1 × 102 CFU/g after enrichment of contaminated samples. Field samples (n = 498) were tested using a previously established duplex-PCR method and compared to our DAS-ELISA. The DAS-ELISA had a specificity of 94.4%, a sensitivity of 91.5% and accuracy of 94.0% compared with duplex-PCR. The DAS-ELISA developed here can be applied to EHEC O157:H7 quantification in food, animal, and environmental samples.