Extraction Of Membrane Proteins Research Articles

An amphipathic polypeptide derived from poly-γ-glutamic acid for the stabilization of membrane proteins.

Difficulties in the extraction of membrane proteins from cell membrane and their solubilization in native conformations have hindered their structural and biochemical analysis. To overcome these difficulties, an amphipathic polypeptide was synthesized by the conjugation of octyl and glucosyl groups to the carboxyl groups of poly-γ-glutamic acid (PGA). This polymer, called amphipathic PGA (APG), self-assembles as mono-disperse oligomers consisted of 4-5 monomers. APG shows significantly low value of critical micelle concentration and stabilization activity toward membrane proteins. Most of the sodium dodecyl sulfate (SDS)-solubilized membrane proteins from Escherichia coli remain soluble state in the presence of APG even after the removal of SDS. In addition, APG stabilizes purified 7 transmembrane proteins such as bacteriorhodopsin and human endothelin receptor Type A (ETA ) in their active conformations. Furthermore, ETA in complex with APG is readily inserted into liposomes without disrupting the integrity of liposomes. These properties of APG can be applied to overcome the difficulties in the stabilization and reconstitution of membrane proteins.

Potential risk of blood transfusion‐transmitted brucellosis in an endemic area of China

Brucellosis is a severe zoonotic disease that is increasingly prevalent in north China. A study evaluating Brucella infection in blood donors was conducted at Kashi central blood station, Xinjiang, China. Four serologic and two molecular methods of detection of Brucella infection were used in plasma samples from blood donations collected from Kashi in northwest China, considered a brucellosis-endemic area. Blood donor samples collected in Shenzhen, southern China, a brucellosis-nonendemic area, were tested as a negative control group. In 3896 plasma samples collected from Kashi central blood station, 135 (3.5%) plasma samples were reactive by the Rose Bengal plate test (RBPT), and 120 (3.1%) of the 135 RBPT-reactive sample were also reactive with the standard tube agglutination test (SAT), respectively. All samples of the control group of 399 blood samples from Shenzhen blood center tested negative with RBPT and SAT. Of 135 seroreactive plasma samples, 39 (1.0%) reacted with B. melitensis membrane protein extracts by enzyme-linked immunosorbent assay and 25 were reactive to either rBP26 or rOMP31 by Western blot. Thirteen plasma samples and two follow-up blood samples were identified as carrying Brucella DNA by quantitative polymerase chain reaction (PCR) and nested PCR. Overall 15 (1:300) Kashi blood donations were found positive by nucleic acid testing, confirmed specific by DNA sequencing. The data indicate a probable high rate of Brucella bacteremia, suggesting a potential risk of transfusion-transmitted brucellosis. Blood donation screening for Brucella infection may be considered in the high Brucella-endemic areas of China.

Polar interactions trump hydrophobicity in stabilizing the self-inserting membrane protein Mistic.

Canonical integral membrane proteins are attached to lipid bilayers through hydrophobic transmembrane helices, whose topogenesis requires sophisticated insertion machineries. By contrast, membrane proteins that, for evolutionary or functional reasons, cannot rely on these machineries need to resort to driving forces other than hydrophobicity. A striking example is the self-inserting Bacillus subtilis protein Mistic, which is involved in biofilm formation and has found application as a fusion tag supporting the recombinant production and bilayer insertion of other membrane proteins. Although this unusual protein contains numerous polar and charged residues and lacks characteristic membrane-interaction motifs, it is tightly bound to membranes in vivo and membrane-mimetic systems in vitro. Therefore, we set out to quantify the contributions from polar and nonpolar interactions to the coupled folding and insertion of Mistic. To this end, we defined conditions under which the protein can be unfolded completely and reversibly from various detergent micelles by urea in a two-state equilibrium and where the unfolded state is independent of the detergent used for solubilizing the folded state. This enabled equilibrium unfolding experiments previously used for soluble and β-barrel membrane proteins, revealing that polar interactions with ionic and zwitterionic headgroups and, presumably, the interfacial dipole potential stabilize the protein much more efficiently than nonpolar interactions with the micelle core. These findings unveil the forces that allow a protein to tightly interact with a membrane-mimetic environment without major hydrophobic contributions and rationalize the differential suitability of detergents for the extraction and solubilization of Mistic-tagged membrane proteins.

Erratum to: Molecular Characterization of a Lysophosphatidylcholine Acyltransferase Gene Belonging to the MBOAT Family in Ricinus communis L.

In the original publication of the article, figure 7 results were incorrectly published. Because of the discrepancies between our results published in Lipids 48(7):663–674, and others in the field with regards to Membrane Bound O-Acyltranferase (MBOAT) activity with 18:2n-6 and 18:3n-3 as substrates, we measured the activity of the castor lysophosphatidylcholine acyltransferase (RcLPCAT) using freshly prepared substrates. The 18:3-CoA and 18:2-CoA were prepared by chemical synthesis [1] and kindly provided by Dr. Stymne (Swedish University of Agricultural Sciences, Alnarp, Sweden). As indicated by our results using the new substrates, LPCAT activity was similar for 18:2 and 18:3 thioesters as it was for 18:1 and 16:1 thioesters, in agreement with the specificity recorded for Brassica LPCAT. These new results do not alter the major conclusion stated in our paper that MBOAT has limited activity with ricinoleoyl-CoA as a substrate, but does indicate it has similar activity with 18:2-CoA and 18:3-CoA as it does with 18:1-CoA and 16:1-CoA. The correct version of figure 7 is given below: RcLPCAT specificity using diverse acyl-CoA donors. LPCAT activity was assayed using 50 μM lysoPtdCho as the acceptor and different acyl-CoA as substrates at two concentrations, 10 μM (grey bars) or 40 μM (black bars) as indicated. The assay was performed as described in “Materials and Methods” of the published paper, in the presence of 30 μg of membrane protein extract prepared from yeast ale1 cells expressing RcLPCAT. Background LPLAT activities were determined from cells transformed with the pYES2 empty vector and the appropriate background rates were subtracted from the measured rate. The specific activity is presented and is expressed as nmol of released CoA min−1 μg−1 of membrane protein

Interacción de FABP4 con proteínas de membrana de células endoteliales

IntroductionFatty acid binding protein (FABP4) is an adipose tissue-secreted adipokine implicated in the regulation of the energetic metabolism and inflammation. High levels of circulating FABP4 have been described in people with obesity, atherogenic dyslipidemia, diabetes and metabolic syndrome. Recent studies have demonstrated that FABP4 could have a direct effect on peripheral tissues and, specifically, on vascular function. It is still unknown how the interaction between FABP4 and the endothelial cells is produced to prompt these effects on vascular function. The objective of this work is studying the interaction between FABP4 and the plasma membrane proteins of endothelial cells. MethodologyHUVEC cells were incubated with and without FABP4 (100ng/ml) for 5minutes. Immunolocalization of FABP4 was studied by confocal microscopy. The results showed that FABP4 colocalizates with CD31, a membrane protein marker.A strategy which combines 6XHistidine-tag FABP4 (FABP4-His), incubations with or without FABP4-His (100ng/ml), formaldehyde cross-linking, cellular membrane protein extraction and western blot, was designed to study the FABP4 interactions with membrane proteins of HUVECs. ResultsThe results showed different western blot profiles depending of the incubation with or without FABP4-His. The immunoblot revelead three covalent protein complexes of about 108, 77 and 33kDa containing FAPB4 and its putative receptor. DiscussionThe existence of a specific binding protein complex able to bind FABP4 to endothelial cells is supported by these results. The obtained results will permit us advance in the molecular knowledge of FABP4 effects as well as use this protein and its receptor as therapeutic target to prevent cardiovascular.

Site-specific characterization of cell membrane N-glycosylation with integrated hydrophilic interaction chromatography solid phase extraction and LC–MS/MS

Glycosylation of membrane proteins plays an important role in cellular behaviors such as cell-cell interaction, immunologic recognition and cell signaling. However, the effective extraction of membrane proteins, the selective isolation of glycopeptides and the mass spectrometric characterization of glycosylation are challenging with current analytical techniques. In this study, a systematic approach was developed which combined: an integrated hydrophilic interaction chromatography solid phase interaction (HILIC SPE) for simultaneous detergent removal and glycopeptide enrichment, and mass spectrometric identification of both protein N-glycosylation sites and site-specific glycan composition. The HILIC SPE conditions were optimized to enable the use of a high concentration of strong detergents, such as SDS and Triton X-100 and to dissolve highly hydrophobic membrane proteins, thus increasing the yield of membrane protein extraction. We illustrated the performance of this approach for the study of membrane protein glycosylation from human embryonic kidney cell lines (HEK 293T). 200μg total protein digest was processed using this approach, leading to the identification of 811 N-glycosylation sites from 567 proteins within two experimental replicates. Furthermore, 177 glycopeptides representing 82 N-glycosites with both glycan composition and peptide sequence were identified by high energy collision dissociation. A method for systematic characterizing of cell membrane glycosylation has been developed in this manuscript. It is comprised of an integrated hydrophilic interaction chromatography solid phase extraction for the simultaneous detergent removal and intact glycopeptide enrichment. This HILIC SPE significantly increased the efficiency and sensitivity for glycosylation analysis and was combined with high energy collision dissociation to characterize site-specific N-glycosylation from HEK293 cell membrane. Totally 811 N-glycosylation sites from 567 proteins were identified and 177 intact glycopeptides with both glycan composition and peptides sequence were characterized, which provided a solution for site-specific N-glycosylation characterization of membrane.

Mycobacterium tuberculosispellicles express unique proteins recognized by the host humoral response

Mycobacterium tuberculosis (MTB) causes both acute and chronic infections in humans characterized by tolerance to antibiotics and reactivation to cause secondary tuberculosis. These characteristics have led to renewed interest in the in vitro pellicle, or biofilm mode of growth, where bacteria grow to produce a thick aggregate at the air-liquid interface and exhibit increased phenotypic resistance to antibiotics. We infected guinea pigs with the virulent H37Rv strain of MTB for 60 days at which point we collected blood. To identify antigenic proteins, membrane protein extracts of MTB H37Ra pellicles and shaken cultures grown for 3, 5, or 7 weeks were probed with the infected animals' sera after the proteins were separated by two-dimensional gel electrophoresis (2DGE). Antigenic proteins were then identified using MALDI-TOF/TOF mass spectrometry peptide mass fingerprinting. Antigenic pellicle proteins were compared across the three timepoints to identify those that were produced consistently during pellicle growth. They were also compared to those membrane proteins identified from harvested shaken cultures to determine pellicle-specific vs. universally expressed proteins. Using this technique, we identified 44 distinct antigenic proteins, nine of which were pellicle-specific. The sequence of antigenic pellicle-specific proteins was checked for sequence conservation across 15 sequenced MTB clinical isolates, three other members of the MTB complex, as well as M. avium and M. smegmatis. The antigenic pellicle-specific protein Rv0097 was found to have very high sequence conservation within the MTB complex but not with related mycobacteria, while FabG4 was highly conserved in all mycobacteria analyzed. These conserved pellicle-specific proteins represent targets for the development of future diagnostic tests and vaccines.

Enzymatic synthesis of amino sugar fatty acid esters

N‐Acetyl‐glucosamine fatty acid esters were synthesized by a lipase‐catalyzed transesterification with methyl hexanoate and N‐acetyl‐glucosamine (GlcNAc), which resulted in the formation of 2‐(acetylamino)‐2‐deoxy‐6‐O‐hexanoate‐D‐glucose, a novel glycolipid. Additionally N‐butyryl‐glucosamine (GlcNBu) was used for a similar synthesis, leading to the formation of 2‐(butyrylamino)‐2‐deoxy‐6‐O‐hexanoate‐D‐glucose. The higher hydrophobicity of GlcNBu led to an increase in the overall yield and the initial reaction rate when compared to the reaction with GlcNAc. By pre‐dissolving GlcNAc and GlcNBu in dimethyl sulfoxide (DMSO), it was possible to completely dissolve both sugars in the organic solvent, thus further enhancing the initial reaction rate and yield respectively.Practical applications: Glycolipids are used in a wide range of applications, ranging from food, cosmetic, and pharmaceutical formulations, where they can be used as emulsifiers or foaming agents to classic cleaning products, utilizing their good detergent properties. Further applications may include fields like membrane protein extraction, bioremediation, or tertiary oil recovery. Novel glycolipids with tailor‐made properties might be useful to improve any of the named applications and widen the diversity of available environmentally friendly surfactants, often termed “green surfactants.” Glycolipids are the most prominent example therefrom.

Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device.

We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (∼20 fmol) of membrane proteins could be isolated and recovered with ∼89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs.

Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device

Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device

Detergent-Free Extraction of Membrane Proteins into Native Nanodiscs. Application to the Reaction center of Rhodobacter Sphaeroides

Recently, it has been discovered that styrene-maleic acid (SMA) copolymers are able to solubilize membrane proteins in the form of nanodiscs (SMALPs), also called ‘native nanodiscs’, directly from their native membrane [1,2]. To understand what physical properties of membranes and SMA modulate this unique property of SMA, we first studied the solubilization of vesicles of synthetic phospholipids by SMA using transmission experiments. We found that SMA is an excellent membrane solubilizer, able to solubilize different types of membranes below, at and above the gel to crystalline-liquid phase temperature. Based on the kinetics of solubilization under different experimental conditions we developed a model for the mode of action of SMA that will also explain why SMA is an efficient solubilizer, whereas membrane scaffold proteins (MSPs) and amphipols are not.Next we used the SMA technology to purify and characterize reaction centers (RCs) from the purple bacterium Rhodobacter sphaeroides in the form of native nanodiscs. Monitoring of heat stability and recombination kinetics of P+QB- in photo-excited RCs in native nanodiscs showed that (1) RCs are much more stable in native nanodiscs than in detergent, and (2) the charge recombination kinetics display native-membrane like behavior.Our study contributes fundamental knowledge about the mode of action of SMA that is essential for optimizing methods to extract membrane proteins from different organisms and it promotes the general applicability of SMALPs as host for membrane proteins in studies on interactions of proteins with native lipids and in protein structure determination studies.1. Knowles et al., 2009, JACS, 131, 7484-7485.2. Long et al., 2013, BMC Biotechnology, 13:41.

Cell Surface Protein-protein Binding on COS-7 Cells.

Examination of interactions between a transmembrane protein and a soluble protein by pull-down or immunoprecipitation assays can be tricky and complicated due to the detergent extraction of membrane proteins during the lysate preparation step. The choice and concentration of detergents must be determined empirically and the procedure can be burdensome. Here, we describe a simplified binding assay by expressing the membrane protein of interest in COS-7 cells and applying detergent-free solutions containing an extracellular protein to be tested. The binding is then examined by immunocytochemistry.

Insights into the TOR-S6K Signaling Pathway in Maize (Zea mays L.). Pathway Activation by Effector–Receptor Interaction

The primordial TOR pathway, known to control growth and cell proliferation, has still not been fully described for plants. Nevertheless, in maize, an insulin-like growth factor (ZmIGF) peptide has been reported to stimulate this pathway. This research provides further insight into the TOR pathway in maize, using a biochemical approach in cultures of fast-growing (FG) and slow-growing (SG) calli, as a model system. Our results revealed that addition of either ZmIGF or insulin to SG calli stimulated DNA synthesis and increased the growth rate through cell proliferation and increased the rate of ribosomal protein (RP) synthesis by the selective mobilization of RP mRNAs into polysomes. Furthermore, analysis of the phosphorylation status of the main TOR and S6K kinases from the TOR pathway revealed stimulation by ZmIGF or insulin, whereas rapamycin inhibited its activation. Remarkably, a putative maize insulin-like receptor was recognized by a human insulin receptor antibody, as demonstrated by immunoprecipitation from membrane protein extracts of maize callus. Furthermore, competition experiments between ZmIGF and insulin for the receptor site on maize protoplasts suggested structural recognition of the putative receptor by either effector. These data were confirmed by confocal immunolocalization within the cell membrane of callus cells. Taken together, these data indicate that cell growth and cell proliferation in maize depend on the activation of the TOR-S6K pathway through the interaction of an insulin-like growth factor and its receptor. This evidence suggests that higher plants as well as metazoans have conserved this biochemical pathway to regulate their growth, supporting the conclusion that it is a highly evolved conserved pathway.

Low Expression Of The FUCA1 Gene Is An Adverse Prognostic Factor In Myeloma and Combined With High Sialyltransferase Gene Expression Identifies Patients At Increased Risk Of Early Disease Progression and Death

BackgroundGlycosylation is a post translational modification which results in the addition of carbohydrate determinants to proteins and lipids. This has a major influence on the function of molecules known to be important in myeloma cell adhesion and trafficking such as integrins and selectins. We previously reported that ST3GAL6, a sialyltransferase involved in the synthesis of functional selectin ligands in humans, is over expressed in myeloma cells and associated with inferior survival in the MRC Myeloma IX study (MRC IX). We now extend our analysis of the MRC IX dataset to evaluate the potential prognostic significance of other glycosylation genes that are differentially regulated between normal and malignant plasma cells. MethodsAnalyzing publically available microarray transcriptomic datasets (Mayo Clinic GSE6477, University of Arkansas (UAMS) GSE24080, GSE2658) we first identified dysregulated glycosylation genes in MM. The prognostic significance of these candidate genes in MRCIX (singly and in combination) was analyzed using Kaplan Meier survival estimates for progression free survival and overall survival (PFS, OS). These results were validated in independent datasets (TT2 and TT3). Any genes significantly associated with survival were correlated with FISH abnormalities and ISS and multivariate analysis was performed to determine their independence as prognostic factors. QPCR was performed in cell lines to validate GEP findings. Matching SNP-based mapping array and methylation array data were analyzed. Membrane protein extracts from MM cell lines were applied directly to lectin microarrays following fluorescent labeling to generate cell surface glycan profiles. Immunohistochemistry for glycosylation enzymes and glycans was performed on patient bone marrow samples. ResultsHigh expression (top quartile as cut-off) of the sialyltransferase gene ST3GAL1 showed a trend towards inferior OS (median survival 35 mos .v. 45 mos, p=0.07) with significantly reduced PFS (19 .v. 14 mos, p=0.015). Increased expression of ST3GAL1 correlated with the presence of t(4;14) (p=0.009), del13q (p=0.001), +1q (p=0.01) and hypodiploidy (p=0.00001). Low expression (lower quartile by GEP) of the gene FUCA1, which encodes tissue alpha-L-fucosidase, was linked to inferior outcome (median OS 44 .v. 38 mos, p=0.025). On multivariate analysis low FUCA1 expression was independent of other important prognostic factors (HR=1.61, p=0.017). Patients with t(11;14) by FISH were more likely to have low FUCA1 than t(11;14) negative patients. There was no significant association between ISS and ST3GAL6, ST3GAL1 or FUCA1. We verified the effect of low FUCA1 in the GSE24080 dataset from TT2 and TT3 combined. Patients with low FUCA1 had significantly worse OS (p=0.0002). SNP mapping array and methylation array analysis did not show any evidence of correlation between copy number alterations or hypermethylation of FUCA1 and its low expression level. Since FUCA1 participates in N-glycan degradation with removal of fucose residues, this raises the possibility that a reduction in FUCA1 may lead to excessive fucosylation of cancer related glycans involved in adhesion and trafficking, such as sLex. Indeed, lectin arrays of MM cell lines revealed high levels of a-1,3/a-1,6 linked fucose, as determined by binding of Aleuria Aurantia Lectin (AAL). The HECA-452 antibody recognizes a functional trisaccharide domain shared by sialyl Lewis a and sLex and known to bind to E-selectin. Preliminary immunohistochemistry revealed increased HECA452 staining in bone marrow samples, which had both strong ST3GAL6 and low FUCA1 staining. Combining gene expression data showed that reduced expression of FUCA1 with increased expression of either ST3GAL6 or ST3GAL1 or both, identified a subgroup of patients (18%) in MRC Myeloma IX with particularly poor outcome (figure 1). Similar results were found in TT2 and TT3 with 17% of patients affected. [Display omitted] ConclusionsAltered glycosylation gene expression patterns may identify patients at high risk of disease progression and early death. Our data implicates sialyltransferases and selectin ligands as potential therapeutic targets in MM. Disclosures:Morgan:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Millenium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Johnson and Johnson: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

7th International Symposium on Enabling Technologies for Life Sciences (ETP)

7^th International Symposium on Enabling Technologies for Life Sciences (ETP)

Dangerous Liaisons between Detergents and Membrane Proteins. The Case of Mitochondrial Uncoupling Protein 2

The extraction of membrane proteins from their native environment by detergents is central to their biophysical characterization. Recent studies have emphasized that detergents may perturb the structure locally and modify the dynamics of membrane proteins. However, it remains challenging to determine whether these perturbations are negligible or could be responsible for misfolded conformations, altering the protein's function. In this work, we propose an original strategy combining functional studies and molecular simulations to address the physiological relevance of membrane protein structures obtained in the presence of detergents. We apply our strategy to a structure of isoform 2 of an uncoupling protein (UCP2) binding an inhibitor recently obtained in dodecylphosphocholine detergent micelles. Although this structure shares common traits with the ADP/ATP carrier, a member of the same protein family, its functional and biological significance remains to be addressed. In the present investigation, we demonstrate how dodecylphosphocholine severely alters the structure as well as the function of UCPs. The proposed original strategy opens new vistas for probing the physiological relevance of three-dimensional structures of membrane proteins obtained in non-native environments.

Improving the stability and function of purified ABCB1 and ABCA4: The influence of membrane lipids

ATP Binding Cassette (ABC) transporters play prominent roles in numerous cellular processes and many have been implicated in human diseases. Unfortunately, detailed mechanistic information on the majority of ABC transporters has not yet been elucidated. The slow rate of progress of molecular and high resolution structural studies may be attributed to the difficulty in the investigation of integral membrane proteins. These difficulties include the expression of functional, non-aggregated protein in heterologous systems. Furthermore, the extraction of membrane proteins from source material remains a major bottle-neck in the process since there are relatively few guidelines for selection of an appropriate detergent to achieve optimal extraction. Whilst affinity tag strategies have simplified the purification of membrane proteins; many challenges remain. For example, the chromatographic process and associated steps can rapidly lead to functional inactivation, random aggregation, or even precipitation of the target protein. Furthermore, optimisation of high yield and purity, does not guarantee successful structure determination. Based on this series of potential issues, any investigation into structure–function of membrane proteins requires a systematic evaluation of preparation quality. In particular, the evaluation should focus on function, homogeneity and mono-dispersity. The present investigation provides a detailed assessment of the quality of purified ATP Binding Cassette (ABC) transporters; namely ABCB1 (P-gp) and ABCA4 (ABCR). A number of suggestions are provided to facilitate the production of functional, homogeneous and mono-disperse preparations using the insect cell expression system. Finally, the ABCA4 samples have been used to provide structural insights into this essential photo-receptor cell protein.

Absolute measurement of species differences in sodium taurocholate cotransporting polypeptide (NTCP/Ntcp) and its modulation in cultured hepatocytes

Species differences among membrane transporters can be remarkable and difficult to properly assess by conventional methods. Herein, we employed the first use of stable isotope labeling in mammals or stable isotope-labeled peptides combined with mass spectrometry to identify species differences in sodium taurocholate cotransporting polypeptide (NTCP/Ntcp) protein expression in liver tissue and to characterize the modulation of protein expression in sandwich-cultured human (SCHH) and rat hepatocytes (SCRH). The lower limit of quantification was established to be 5 fmol on column with a standard curve that was linear up to 2000 fmol. The accuracy and precision were evaluated with three quality control samples and known amounts of synthetic proteotypic peptides that were spiked into the membrane protein extracts. The overall relative error and coefficient of variation were less than 10%. The expression of Ntcp in mouse and rat was significant higher than that in human (five-fold) and monkey (two-fold) and ranked as mouse > rat >> monkey > human. In the cultured hepatocytes, although significant downregulation of Ntcp expression in SCRH at day 5 after the culture was detected, NTCP expression in SCHH was comparable to the suspension hepatocytes. The results suggested that NTCP/Ntcp modulation in cultured hepatocytes is species specific. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3252–3263, 2013

Myocilin Mediates Myelination in the Peripheral Nervous System through ErbB2/3 Signaling

The glaucoma-associated gene, myocilin, is expressed in ocular and non-ocular tissues including the peripheral nervous system, but its functions in these tissues remain poorly understood. We demonstrate that in sciatic nerve, myocilin is expressed in Schwann cells with high concentrations at the nodes of Ranvier. There, myocilin interacts with gliomedin, neurofascin, and NrCAM, which are essential for node formation and function. Treatment of isolated dorsal root ganglion cultures with myocilin stimulates clustering of the nodal proteins neurofascin and sodium channel Nav1.2. Sciatic nerves of myocilin null mice express reduced levels of several myelin-associated and basal membrane proteins compared with those of wild-type littermates. They also demonstrate reduced myelin sheath thickness and partial disorganization of the nodes. Myocilin signaling through ErbB2/3 receptors may contribute to these observed effects. Myocilin binds to ErbB2/ErbB3, activates these receptors, and affects the downstream PI3K-AKT signaling pathway. These data implicate a role for myocilin in the development and/or maintenance of myelination and nodes of Ranvier in sciatic nerve.

2-Methyltetrahydrofuran and cyclopentylmethylether: Two green solvents for efficient purification of membrane proteins like FhuA

β-Barrel shaped membrane proteins are attractive hosts for hybrid catalysts in which reactions are controlled through space. Production and extraction of β-barrel shaped membrane proteins in gram scale is challenging due to their hydrophobicity. Solvent mixtures such as chloroform/methanol (CM) are widely used for membrane protein extraction but toxicity and mutagenicity were reported in several cases. 2-Methyltetrahydrofuran (2-MeTHF) and cyclopentylmethylether (CPME) are two green (reduction of solvent-related environmental damage in chemical production) and potentially efficient solvents for membrane protein purification. On the example of the ferric hydroxamate uptake protein component A (FhuA) a 4-Step method was developed to provide gram amounts of highly purified FhuA: cell disruption (Step 1), removal of membrane protein impurities with n-octyl-poly-oxyethylene (oPOE) (Step 2), dissolution of membranes and FhuA precipitation (Step 3), and refolding using urea and dialysis with polyethylene-polyethyleneglycol (PE-PEG; Step 4) resulted in high FhuA purity (95% 2-MeTHF, 80% CPME; 70mg FhuA per liter fermenter broth). Structural integrity of FhuA protein was confirmed by circular dichroism (CD) and a translocation functionality assay.