Podcasts about membrane proteins

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.02.551232v1?rss=1 Authors: Nguyen, T. P., Otani, T., Tsutsumi, M., Fujiwara, S., Nemoto, T., Fujimori, T., Furuse, M. Abstract: Epithelia must be able to resist mechanical force to preserve tissue integrity. While intercellular junctions are known to be important for the mechanical resistance of epithelia, the roles of tight junctions (TJs) remain to be established. We previously demonstrated that epithelial cells devoid of the TJ membrane proteins claudins and JAM-A completely lack TJs and exhibit focal breakages of their apical junctions. Here, we demonstrate that apical junctions undergo spontaneous fracture when claudin/JAM-A-deficient cells are exposed to mechanical stress. The junction fracture was accompanied by actin disorganization, and actin polymerization was required for apical junction integrity in the claudin/JAM-A-deficient cells. Further deletion of CAR resulted in the disruption of ZO-1 molecule ordering at cell junctions, accompanied by severe defects in apical junction integrity. These results demonstrate that TJ membrane proteins regulate the mechanical resistance of the apical junctional complex in epithelial cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

References J Biol Chem. 2021 Jul;297(1):100900 Immunol Rev. 2014 May; 259(1):88–102. --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message

Dr. Olaf Andersen is a Professor of Physiology and Biophysics at the Weill Medical College of Cornell University and Director of the Tri-Institutional MD-PhD Program in New York City. His research aims to understand all of the mechanisms by which small molecules can manipulate the functions of cells or whole organisms. How do these molecules work and what are they doing? These questions are particularly relevant for pharmacology and toxicity. When he's not doing science, Olaf keeps busy reading and brewing beer. His ambition as a brewer is to make a beer with a deep beer flavor but really low alcohol percentage. Olaf keeps a brewing diary that holds 20 years worth of notes on each batch he has ever brewed. He was awarded his MD from the University of Copenhagen in Denmark and completed postdoctoral research at the University of Copenhagen and Rockefeller University before joining the faculty at Cornell University. Olaf has received many awards and honors including being named a Foreign Member of The Royal Danish Academy of Sciences and Letters, receipt of the K. S. Cole Medal from the Biophysical Society, being named an Honorary Fellow of the Cornell University Weill Medical College Alumni Association, receipt Distinguished Service Award from the Biophysical Society, and receipt the Inaugural Bruce Ballard Mentoring Award. In this interview, Olaf shares more about his life and science. 

Mini Lecture 1 on Membrane Physiological Biochemistry. The amino acid sequence of membrane associated polypeptides obtains secondary structure, glycosylation, acylation and hydropathy which confer functionality (in part) by targeting specific membrane domains. --- Send in a voice message: https://anchor.fm/dr-daniel-j-guerra/message Support this podcast: https://anchor.fm/dr-daniel-j-guerra/support

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.22.351536v1?rss=1 Authors: Koehler Leman, J., Bonneau, R. Abstract: Structures of membrane proteins are challenging to determine experimentally and currently represent only about 2% of the structures in the ProteinDataBank. Because of this disparity, methods for modeling membrane proteins are fewer and of lower quality than those for modeling soluble proteins. However, better expression, crystallization, and cryo-EM techniques have prompted a recent increase in experimental structures of membrane proteins, which can act as templates to predict the structure of closely related proteins through homology modeling. Because homology modeling relies on a structural template, it is easier and more accurate than fold recognition methods or de novo modeling, which are used when the sequence similarity between the query sequence and the sequence of related proteins in structural databases is below 25%. In homology modeling, a query sequence is mapped onto the coordinates of a single template and refined. With the increase in available templates, several templates often cover overlapping segments of the query sequence. Multi-template modeling can be used to identify the best template for local segments and join them into a single model. Here we provide a protocol for modeling membrane proteins from multiple templates in the Rosetta software suite. This approach takes advantage of several integrated frameworks, namely RosettaScripts, RosettaCM, and RosettaMP with the membrane scoring function. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.28.316307v1?rss=1 Authors: Jin, F., Wang, Y., Wang, M., Sun, M., Hattori, M. Abstract: Membrane proteins play numerous physiological roles and are thus of tremendous interest in pharmacology. Nevertheless, stable and homogeneous sample preparation is one of the bottlenecks in biophysical and pharmacological studies of membrane proteins because membrane proteins are typically unstable and poorly expressed. To overcome such obstacles, GFP fusion-based Fluorescence-detection Size-Exclusion Chromatography (FSEC) has been widely employed for membrane protein expression screening for over a decade. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP and applied to a size-exclusion chromatography column attached to a fluorescence detector for FSEC analysis. FSEC-Nb enables one to evaluate the expression, monodispersity and thermostability of membrane proteins without the need of purification by utilizing the benefits of the GFP fusion-based FSEC method, but does not require direct GFP fusion to targeted proteins. We applied FSEC-Nb to screen zinc-activated ion channel (ZAC) family proteins in the Cys-loop superfamily and membrane proteins from SARS-CoV-2 as examples of the practical application of FSEC-Nb. We successfully identified a ZAC ortholog with high monodispersity but moderate expression levels that could not be identified with the previously developed GFP fusion-free FSEC method. Consistent with the results of FSEC-Nb screening, the purified ZAC ortholog showed monodispersed particles by both negative staining EM and cryo-EM. Furthermore, we identified two membrane proteins from SARS-CoV-2 with high monodispersity and expression level by FSEC-Nb, which may facilitate structural and functional studies of SARS-CoV-2. Overall, our results show FSEC-Nb as a powerful tool for membrane protein expression screening that can provide further opportunity to prepare well-behaved membrane proteins for structural and functional studies. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.16.299453v1?rss=1 Authors: Staritzbichler, R., Sarti, E., Yaklich, E., Stamm, M., Aleksandrova, A., Khafizov, K., Forrest, L. R. Abstract: The alignment of primary sequences is a fundamental step in the analysis of protein structure, function, and evolution. Integral membrane proteins pose a significant challenge for such sequence alignment approaches, because their evolutionary relationships can be very remote, and because a high content of hydrophobic amino acids reduces their complexity. Frequently, biochemical or biophysical data is available that informs the optimum alignment, for example, indicating specific positions that share common functional or structural roles. Currently, if those positions are not correctly aligned by a standard pairwise alignment procedure, the incorporation of such information into the alignment is typically addressed in an ad hoc manner, with manual adjustments. However, such modifications are problematic because they reduce the robustness and reproducibility of the alignment. An alternative approach is the use of restraints, or anchors, to incorporate such position-matching explicitly during alignment. Here we introduce position anchoring in the alignment tool AlignMe as an aid to pairwise sequence alignment of membrane proteins. Applying this approach to realistic scenarios involving distantly-related and low complexity sequences, we illustrate how the addition of even a single anchor can dramatically improve the accuracy of the alignments, while maintaining the reproducibility and rigor of the overall alignment. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.11.293043v1?rss=1 Authors: Orioli, S., Henning Hansen, C. G., Arleth, L. Abstract: We introduce a new software, called Marbles, that employs SAXS intensities to predict the shape of membrane proteins embedded into membrane nanodiscs. To gain computational speed and efficient convergence, the strategy is based on a hybrid approach that allows one to account for the nanodisc contribution to the SAXS intensity through a semi-analytical model, while the embedded membrane protein is treated as set of beads, similarly to well known ab-initio methods. The code, implemented in C++ with a Python user interface, provides a good performance and includes the possibility to systematically treat unstructured domains. We prove the reliability and flexibility of our approach by benchmarking the code on a toy model and two proteins of very different geometry and size. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.09.289488v1?rss=1 Authors: Kotani, N., Nakano, T. Abstract: COVID-19 represents a real threat to the global population, and understanding the biological features of the causative virus (SARS-CoV-2) is imperative to aid in mitigating this threat. Analyses of proteins such as primary receptors and co-receptors (co-factors) that are involved in SARS-CoV-2 entry into host cells will provide important clues to help control the virus. Here, we identified host cell membrane protein candidates that were present in proximity to the attachment sites of SARS-CoV-2 spike proteins through the use of proximity labeling and proteomics analysis. The identified proteins represent candidate key factors that may be required for viral entry. Our results indicated that a number of membrane proteins, including DPP4, Cadherin-17, and CD133, were identified to co-localize with cell membrane-bound SARS-CoV-2 spike proteins in Caco-2 cells that were used to expand the SARS-CoV-2 virion. We anticipate that the information regarding these protein candidates will be utilized for the future development of vaccines and antiviral agents against SARS-CoV-2. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.09.286310v1?rss=1 Authors: Gavins, G., Groeger, K., Bartoscheck, M. D., Wolf, P., Beck-Sickinger, A. G., Bultmann, S., Seitz, O. Abstract: DNA nanotechnology is an emerging field, which promises fascinating opportunities for the manipulation and imaging of proteins on a cell surface. The key to progress in the area is the ability to create the nucleic acid-protein junction in the context of living cells. Here we report a covalent labelling reaction, which installs a biostable peptide nucleic acid (PNA) tag. The reaction proceeds within minutes and is specific for proteins carrying a 2 kDa coiled coil peptide tag. Once installed the PNA label serves as a generic landing platform that enables the recruitment of fluorescent dyes via nucleic acid hybridization. We demonstrate the versatility of this approach by recruiting different fluorophores, assembling multiple fluorophores for increased brightness, and achieving reversible labelling by way of toehold mediated strand displacement. Additionally, we show that labelling can be carried out using two different coiled coil systems, with EGFR and ETBR, on both HEK293 and CHO cells. Finally, we apply the method to monitor internalization of EGFR on CHO cells. Copy rights belong to original authors. Visit the link for more info

Cheng shows how his laboratory has used Cryo-EM to study the atomic resolution of membrane proteins. It is challenging to use conventional methods to study membrane protein structure, given that the 3D structure of most membrane proteins is dependent on their interaction with the phospholipid bilayer. Cheng describes how his laboratory has overcome these challenges to successfully solve the protein structure of the TRPV1 ion channel in different conformations at atomic resolution. He describes the benefits of using of amphipols, lipid nanodiscs, and Fab-assisted approaches to facilitate structural studies.

Dr. Olaf Andersen is a Professor of Physiology and Biophysics at the Weill Medical College of Cornell University and Director of the Tri-Institutional MD-PhD Program in New York City. He was awarded his MD from the University of Copenhagen in Denmark and completed postdoctoral research at the University of Copenhagen and Rockefeller University before joining the faculty at Cornell University. Olaf has received many awards and honors including being named a Foreign Member of The Royal Danish Academy of Sciences and Letters, receipt of the K. S. Cole Medal from the Biophysical Society, being named an Honorary Fellow of the Cornell University Weill Medical College Alumni Association, receipt Distinguished Service Award from the Biophysical Society, and receipt the Inaugural Bruce Ballard Mentoring Award. Olaf is here with us today to tell us all about his journey through life and science.

How do molecular machines function in greasy membrane environments? Come learn why the challenges of this environment can facilitate Alzheimer's and Parkinson's disease.

Professor Kay Grunewald tells us how structural cell biology can help us understand virus infection. Cells constitute the smallest autonomous units of life. The tightly regulated structural and functional organisation is currently only rudimentary understood. Professor Kay Grünewald uses electron cryotomography in combination with other techniques to analyse virus' 'life cycle' in situ, which requires an understanding of its transient structures at the molecular level. Imaging techniques allow us to understand the communication between the virus and the components of the cell it is infecting, which can ultimately help to treat infectious diseases.

Dr Liz Carpenter talks about her research on membrane proteins and drug development. Membrane proteins are the gateways to our cells - with nutrients, waste products, and even DNA and proteins entering and leaving cells via these tightly controlled proteins. Drugs often target membrane proteins; therefore, understanding their molecular structure helps us design better drugs. Dr Liz Carpenter uses X-ray crystallography to solve membrane protein structures. This information is then used to improve treatments for heart disease and neurological diseases.

Professor Kay Grunewald tells us how structural cell biology can help us understand virus infection. Cells constitute the smallest autonomous units of life. The tightly regulated structural and functional organisation is currently only rudimentary understood. Professor Kay Grünewald uses electron cryotomography in combination with other techniques to analyse virus' 'life cycle' in situ, which requires an understanding of its transient structures at the molecular level. Imaging techniques allow us to understand the communication between the virus and the components of the cell it is infecting, which can ultimately help to treat infectious diseases.

Professor Kay Grunewald tells us how structural cell biology can help us understand virus infection. Cells constitute the smallest autonomous units of life. The tightly regulated structural and functional organisation is currently only rudimentary understood. Professor Kay Grünewald uses electron cryotomography in combination with other techniques to analyse virus' 'life cycle' in situ, which requires an understanding of its transient structures at the molecular level. Imaging techniques allow us to understand the communication between the virus and the components of the cell it is infecting, which can ultimately help to treat infectious diseases.

Professor Kay Grunewald tells us how structural cell biology can help us understand virus infection. Cells constitute the smallest autonomous units of life. The tightly regulated structural and functional organisation is currently only rudimentary understood. Professor Kay Grünewald uses electron cryotomography in combination with other techniques to analyse virus' 'life cycle' in situ, which requires an understanding of its transient structures at the molecular level. Imaging techniques allow us to understand the communication between the virus and the components of the cell it is infecting, which can ultimately help to treat infectious diseases.

Dr Liz Carpenter talks about her research on membrane proteins and drug development. Membrane proteins are the gateways to our cells - with nutrients, waste products, and even DNA and proteins entering and leaving cells via these tightly controlled proteins. Drugs often target membrane proteins; therefore, understanding their molecular structure helps us design better drugs. Dr Liz Carpenter uses X-ray crystallography to solve membrane protein structures. This information is then used to improve treatments for heart disease and neurological diseases.

Dr Liz Carpenter talks about her research on membrane proteins and drug development. Membrane proteins are the gateways to our cells - with nutrients, waste products, and even DNA and proteins entering and leaving cells via these tightly controlled proteins. Drugs often target membrane proteins; therefore, understanding their molecular structure helps us design better drugs. Dr Liz Carpenter uses X-ray crystallography to solve membrane protein structures. This information is then used to improve treatments for heart disease and neurological diseases.

Mon, 27 Aug 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15788/ https://edoc.ub.uni-muenchen.de/15788/1/Li_Nannan.pdf Li, Nannan ddc:570, ddc:500, Fakultät für Biologie

Thu, 21 Jun 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15496/ https://edoc.ub.uni-muenchen.de/15496/1/Spira_Felix.pdf Spira, Felix ddc:570, ddc:500, Fakultät für Biolog

Dr. Brown's research activities include: Nuclear magnetic resonance (NMR) is the most widely used spectroscopic method in chemistry and biochemistry – there many applications and research opportunities. Applications range from organic synthesis to protein structure elucidation in solution and fibrils or membranes, and to magnetic resonance imaging (MRI) of human subjects. Apart from its remarkable breadth and versatility, a unique feature of NMR is the ability to provide information about both structure and dynamics. Our research entails the development of NMR techniques through applications to materials science and biomolecular systems. A cross-disciplinary approach is taken to relate the properties of materials and molecular structure to function. Currently, we are actively engaged in research in several interrelated areas. Our aims in spectroscopy and analytical methods entail development and application of NMR methods to liquid-crystalline materials and biomolecular systems. In the area of biophysics, we are conducting structural studies of membrane proteins and membrane lipids. A common theme is to illuminate material or biochemical properties using molecular structural methods that advance our mechanistic understanding for applications. A range of chemical, physical, analytical, and biochemical methods are used, and provide the opportunity for broad-based cross-disciplinary training. Presented October 3, 2008.

Lecture 07: Kaplan discusses the various classes of membrane proteins and movement of molecules across the membrane.

Fri, 19 Oct 2007 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/9088/ https://edoc.ub.uni-muenchen.de/9088/1/Le_Bris_Anna.pdf Le Bris, Anna ddc:500, ddc:540, Fakultät für Chemie und Pharmazie

The filamentous cyanobacterium Anabaena sp. PCC 7120 (further referred to as Anabaena sp.) is a model system to study nitrogen fixation, cell differentiation, cell pattern formation and evolution of plastids. It is a multicellular photosynthetic microorganism consisting of two cell types, vegetative cells and nitrogen fixing heterocysts. This study focuses on the function and dynamics of the proteome of the Gram-negative outer membrane in Anabaena sp. with emphasis on cell differentiation and iron limitation. The newly developed methods for the membrane fractionation are presented, followed by analysis and comparison of the outer membrane proteomes of vegetative cells and heterocysts. The absence of major proteomic alterations in the outer membrane between two cell types, together with the presented data on GFP activity in mutant strains, experimentally support the previously proposed continuum of the outer membrane and the periplasm in Anabaena sp. filament. Also, somewhat different properties of the Anabaena sp. periplasm than in unicellular cyanobacteria are suggested. Furthermore, two common classes of the outer membrane -barrel proteins are analyzed closer. First, Alr2887 protein, as shown here, is a TolC homologue present in both cell types. Protein secretion through Alr2887 / TolC channel-tunnel is essential for the heterocysts maturation and the glycolipid layer formation. Furthermore, the inner membrane ABC transporter encoded by devBCA operon is proposed as component of the TolC efflux system in Anabaena sp. heterocysts. Second, phylogenetic analysis of the surprisingly abundant protein family of 24 TonB-dependent iron transporters in Anabaena sp. is presented. Five members of this family are detected in the outer membrane of vegetative cells under iron-repletion and two of them, All4026 and Alr0397, are explored closer. It is demonstrated that the function of these iron transporters is required for maintaining iron homeostasis of the filaments under iron-replete conditions. Consequently, their gene expression is constant and not enhanced by iron limitation. All4026 and Alr0397 have different specificity for siderophore substrates and in addition to iron transport, All4026 protein is capable of copper uptake and influence on copper homeostasis in Anabaena sp. as well.

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The mitochondrial outer membrane harbors different sub-classes of proteins that mediate numerous interactions between the mitochondrial metabolic and genetic system and the rest of the eukaryotic cell. Two classes of these proteins were investigated in this thesis. The first class, tail-anchored proteins, exposes a large domain to the cytosol and is anchored to the membrane by a single hydrophobic segment close to the C-terminus. This segment is usually flanked by positively–charged amino acids residues. In the first part of my study I could identify that the tail anchor domain fulfills four distinct functions: First, the tail anchor domain mediates the targeting to mitochondria in a process that probably requires the positive charges down stream the transmembrane segment. Second, the domain facilitates the insertion into the outer membrane. Third, the tail anchor domain is required for assembly of the proteins into the relevant complexes. Finally, it can stabilize such complexes. In the second part of my study I investigated the biogenesis of β-barrel proteins. These membrane proteins are unique for the outer membrane of mitochondria, chloroplast and gram-negative bacteria. Recently a complex which mediates the biogenesis of β-barrel proteins was identified and termed TOB (SAM) complex. At the beginning of this work, the TOB complex was known to consist of the peripheral associated membrane protein Mas37 and the essential membrane embedded component Tob55. Initially, we could identify Tob38 as a new component of the TOB complex. Tob38 is encoded by an essential gene and the protein associates with the TOB complex at the cytosolic side of mitochondria. It interacts with Mas37 and Tob55 and is associated with Tob55 even in the absence of Mas37. The Tob38–Tob55 core complex binds precursors of β-barrel proteins and facilitates their insertion into the outer membrane. The import of β-barrel precursors into Tob38-depleted mitochondria was demonstrated here to be dramatically reduced in comparison to wild type organelles. Notably, such an effect was not observed for other precursors of the outer membrane proteins and precursors distained to the various sub-mitochondrial compartments. Taken together, we conclude that Tob38 has a crucial function in the biogenesis of β-barrel proteins of mitochondria. Next, the import and the assembly pathways of Mas37 and Tob55 were analyzed in detail. Reduced insertion of the Tob55 precursor in the absence of Tom20 and Tom70 argues for initial recognition of the precursor of Tob55 by the import receptors. It is then transferred through the import channel formed by Tom40. Variants of the latter protein influenced the insertion of Tob55. Assembly of newly synthesized Tob55 into pre-existing TOB complexes, as analyzed by blue native gel electrophoresis, depended on pre-existing Tob55 and Tob38 but to a less extent on Mas37. In contrast, both the association of Mas37 precursor with mitochondria and its assembly into the TOB complex were not affected by mutation in the TOM complex. My results suggest that Mas37 assembled directly with the TOB core complex. Hence, the biogenesis of Mas37 represents a novel import pathway of mitochondrial proteins. Finally, the structure function relationships of Tob55 were investigated by combination of biochemical and genetic approaches. Tob55 exposes an N-terminal hydrophilic domain into the intermembrane space and is anchored in the outer membrane by its C-terminal β-barrel domain. Deletion of various lengths at the N-terminal domain did not affect the targeting of Tob55 precursor to mitochondria and its insertion into the outer membrane. Replacement of wild type Tob55 by these deletion variants resulted in reduced growth of cells. Mitochondria isolated from such cells contain reduced levels of β-barrel proteins and are impaired in their capacity to import newly synthesized β-barrel precursors. Finally, purified N-terminal domain of Tob55 was found to be able to bind β-barrel precursors in a specific manner. Taken together, these results demonstrate that the N-terminal domain of Tob55 has a function in recognizing precursors of β-barrel proteins. Furthermore, the receptor-like function of the N-terminal domain of Tob55 seems to have a role in coupling the translocation of β-barrel precursors across the TOM complex to their interaction with the TOB complex.

Channels of the plastid and mitochondrial outer membranes facilitate the turnover of molecules and ions via these membranes. Although channels have been studied many questions pertaining to the whole diversity of plastid and mitochondrial channels in Arabidopsis thaliana and Pisum sativum remain unanswered. In this thesis I studied OEP16, OEP37 and VDAC families in two model plants, in Arabidopsis and pea. The Arabidopsis OEP16 family represents four channels of α-helical structure, similar to the pea OEP16 protein. These channels are suggested to transport amino acids and compounds with primary amino groups. Immunoblot analysis, GFP/RFP protein fusion expression, as well as proteomic analysis showed that AtOEP16.1, AtOEP16.2 and AtOEP16.4 are located in the outer envelope membrane of plastids, while AtOEP16.3 is in mitochondria. The gene expression and immunoblot analyses revealed that AtOEP16.1 and AtOEP16.3 proteins are highly abundant and ubiquitous; expression of AtOEP16.1 is regulated by light and cold. AtOEP16.2 is highly expressed in pollen, seeds and seedlings. AtOEP16.4 is a low expressed housekeeping protein. Single knockout mutants of AtOEP16.1, AtOEP16.2 and AtOEP16.4, and double mutants of AtOEP16 gene family did not show any remarkable phenotype. However, macroarray analysis of Atoep16.1-p T-DNA mutant revealed 10 down-regulated and 6 up-regulated genes. In contrast to the α-helical OEP16 proteins, the OEP37 and VDAC proteins are of β-barrel structure. The PsOEP37 and AtOEP37 channel proteins form a selective barrier in the outer envelope of chloroplasts. Electrophysiological studies in lipid bilayer membranes showed that the PsOEP37 channel is permeable for cations. Specific expression profiles showed that AtOEP37 and PsOEP37 are highly expressed in the entire plant. The isolated PsVDAC gene encodes a protein, which is located in mitochondria. In Arabidopsis gene database, five Arabidopsis genes, which code for VDAC-like proteins were announced. One gene was not detected, whereas four of these genes expressed in leaves, roots, flower buds and pollen.

Background and aims: A number of Helicobacter pylori outer membrane proteins (OMPs) undergo phase variations. This study examined the relation between OMP phase variations and clinical outcome.Methods: Expression of H pylori BabA, BabB, SabA, and OipA proteins was determined by immunoblot. Multiple regression analysis was performed to determine the relation among OMP expression, clinical outcome, and mucosal histology.Results:H pylori were cultured from 200 patients (80 with gastritis, 80 with duodenal ulcer (DU), and 40 with gastric cancer). The most reliable results were obtained using cultures from single colonies of low passage number. Stability of expression with passage varied with OipA > BabA > BabB > SabA. OipA positive status was significantly associated with the presence of DU and gastric cancer, high H pylori density, and severe neutrophil infiltration. SabA positive status was associated with gastric cancer, intestinal metaplasia, and corpus atrophy, and negatively associated with DU and neutrophil infiltration. The Sydney system underestimated the prevalence of intestinal metaplasia/atrophy compared with systems using proximal and distal corpus biopsies. SabA expression dramatically decreased following exposure of H pylori to pH 5.0 for two hours.Conclusions: SabA expression frequently switched on or off, suggesting that SabA expression can rapidly respond to changing conditions in the stomach or in different regions of the stomach. SabA positive status was inversely related to the ability of the stomach to secrete acid, suggesting that its expression may be regulated by changes in acid secretion and/or in antigens expressed by the atrophic mucosa.

Background: The identification of beta-barrel membrane proteins out of a genomic/proteomic background is one of the rapidly developing fields in bioinformatics. Our main goal is the prediction of such proteins in genome/proteome wide analyses. Results: For the prediction of beta-barrel membrane proteins within prokaryotic proteomes a set of parameters was developed. We have focused on a procedure with a low false positive rate beside a procedure with lowest false prediction rate to obtain a high certainty for the predicted sequences. We demonstrate that the discrimination between beta-barrel membrane proteins and other proteins is improved by analyzing a length limited region. The developed set of parameters is applied to the proteome of E. coli and the results are compared to four other described procedures. Conclusion: Analyzing the beta-barrel membrane proteins revealed the presence of a defined membrane inserted beta-barrel region. This information can now be used to refine other prediction programs as well. So far, all tested programs fail to predict outer membrane proteins in the proteome of the prokaryote E. coli with high reliability. However, the reliability of the prediction is improved significantly by a combinatory approach of several programs. The consequences and usability of the developed scores are discussed.

Glycosyl-phosphatidylinositol-anchored membrane proteins (GPI-proteins) are normally identified either by cleavage of the lipid anchor using (glycosyl)phosphatidylinositol-specific phospholipases C or D (GPI-PLs) or by metabolic labeling of the lipid moiety with specific building blocks. Therefore, methods for discrimination between transmembrane proteins and GPI-proteins on the basis of their physicochemical properties are desirable. Here we are presenting a selective extraction method for typical well-characterized mammalian GPI-proteins, e.g., acetylcholine esterase, alkaline phosphatase, 5′-nucleotidase, and lipoprotein lipase, using a derivative of taurocholate. The results were compared to those obtained with well-characterized transmembrane proteins, e.g., insulin receptor and hydroxymethyl glutaryl coenzyme A-reductase, glucose transporters, or aminopeptidase M and several commercially available detergents. With regard to total membrane proteins, it was possible to selectively enrich GPI-proteins up to 8- to 14-fold by using concentrations between 0.1 and 0.3% of 4′-NH2-amino-7β-benzamido-taurocholic acid (BATC). In addition, the cleavage specificity and efficiency of (G)PI-PLs were increased in the presence of identical concentrations of BATC compared to commonly used detergents, e.g., Nonidet P-40. Therefore, the present study shows that the use of BATC facilitates the identification of glycosyl-phosphatidylinositol-anchored membrane proteins.

Sulphonylurea drugs stimulate glucose transport and metabolism in muscle and fat cells in vitro. The molecular basis for the insulin-mimetic extrapancreatic effects of these oral antidiabetic therapeutic agents is unknown at present. Here we demonstrate that incubation of 3T3 adipocytes with the novel sulphonylurea, glimepiride, causes a time- and concentration-dependent release of the glycosylphosphatidylinositol (GPI)-anchored ecto-proteins, 5'-nucleotidase, lipoprotein lipase and a 62 kDa cyclic AMP (cAMP)-binding protein from the plasma membrane into the culture medium. The change in the localization is accompanied by conversion of the membrane-anchored amphiphilic proteins into their soluble hydrophilic versions, as judged by pulse-chase experiments and Triton X-114 partitioning, and by appearance of anti-cross-reacting determinant (CRD) immunoreactivity of the released proteins as shown by Western blotting. Metabolic labelling of cells with myo-[14C]inositol demonstrates that inositol is retained in the major portion of released lipoprotein lipase and cAMP-binding ectoprotein. The identification of inositol phosphate after deamination of these proteins with nitrous acid suggests cleavage of their GPI membrane anchor by a GPI-specific phospholipase C. However, after longer incubation with glimepiride the amount of soluble versions of the GPI-proteins lacking inositol and anti-CRD immunoreactivity increases, which may be caused by additional drug-stimulated hydrolytic events within their GPI structure or C-termini. Since insulin also stimulates membrane release of these GPI-modified proteins, and in combination with glimepiride in a synergistic manner, sulphonylurea drugs may exert their peripheral actions in adipose tissue by using (part of) the insulin postreceptor signalling cascade at the step of activation of a GPI-specific phospholipase C.

Two distinct pathways of sorting and assembly of nuclear-encoded mitochondrial inner membrane proteins are described. In the first pathway, precursor proteins that carry amino-terminal targeting signals are initially translocated via contact sites between both mitochondrial membranes into the mitochondrial matrix. They become proteolytically processed, interact with the 60-kDa heat-shock protein hsp60 in the matrix and are retranslocated to the inner membrane. The sorting of subunit 9 of Neurospora crassa Fo-ATPase has been studied as an example. Fo subunit 9 belongs to that class of nuclear-encoded mitochondrial proteins which are evolutionarily derived from a prokaryotic ancestor according to the endosymbiont hypothesis. We suggest that after import into mitochondria, these proteins follow the ancestral sorting and assembly pathways established in prokäryotes (conservative sorting). On the other hand, ADP/ATP carrier was found not to require interaction with hsp60 for import and assembly. This agrees with previous findings that the ADP/ATP carrier possesses non-amino-terminal targeting signals and uses a different import receptor to other mitochondrial precursor proteins. It is proposed that the ADP/ATP carrier represents a class of mitochondrial inner membrane proteins which do not have a prokaryotic equivalent and thus appear to follow a non-conservative sorting pathway.

Fri, 1 Jan 1988 12:00:00 +0100 http://epub.ub.uni-muenchen.de/8575/ http://epub.ub.uni-muenchen.de/8575/1/8575.pdf Zimmermann, Richard; Sagstetter, Maria; Schlenstedt, Gabriel; Wiech, Hans; Kaßeckert, Brigitta; Müller, Günter Kamp, Jos A. F. op den (Hrsg.) (1988): Import of Small Secretory and Membrane Proteins into the Endoplasmic Reticulum. NATO Advanced Study Inst. on New Perspectives in the Dynamics of Assembly of Biomembranes, 24.08.-04.09.1987, Cargèse, Korsica, Frankreich. Chemie und Ph

Fri, 1 Jan 1988 12:00:00 +0100 https://epub.ub.uni-muenchen.de/7514/1/7514.pdf Neupert, Walter; Harmey, Matthew A.; Nicholson, Donald W.; Stuart, Rosemary A. ddc:610, Medizin

Sat, 1 Jan 1983 12:00:00 +0100 https://epub.ub.uni-muenchen.de/7399/1/7399.pdf Neupert, Walter; Zimmermann, Richard

Fri, 1 Jan 1982 12:00:00 +0100 https://epub.ub.uni-muenchen.de/7403/1/7403.pdf Neupert, Walter; Schmidt, Bernd; Schleyer, Manfred; Hennig, Bernd; Teintze, Martin ddc:610, Medizin

Outer and inner membranes of Neurospora crassa mitochondria were separated by the combined swelling, shrinking, sonication procedure. Membranes were characterized by electron microscopy and by marker enzyme activities. A red carotenoid pigment was found to be concentrated in the outer membrane. The inner mitochondrial membrane was resolved into about 20 protein bands on polyacrylamide gel electrophoresis, whereas the outer membrane shows essentially one single protein band. Only negligible incorporation of radioactive amino acids occurs into outer membrane when isolated mitochondria are synthesizing polypeptide chains. In agreement with this observation labeling of outer membrane protein is almost entirely blocked, when whole Neurospora cells are incubated with radioactive amino acids in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Finally, the essential electrophoretic protein band from outer membrane does not become labeled when mitochondria are incubated with radioactive amino acids either in vitro or in vivo in the presence of cycloheximide. It is concluded that the vast majority, if not all, of the outer membrane protein is synthesized by the cytoplasmic system and that polypeptide chains formed by the mitochondrial ribosomes are integrated into the inner mitochondrial membrane.

Thu, 1 Aug 1968 12:00:00 +0100 http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T36-447MYSB-1G&_user=616146&_coverDate=08%2F31%2F1968&_rdoc=8&_fmt=high&_orig=browse&_srch=doc-info(%23toc%234938%231968%23999989996%23268412%23FLP%23display%23Volume)&_cdi=4938&_sort=d&_docanchor https://epub.ub.uni-muenchen.de/7156/1/Neupert_Walter_7156.pdf Neupert, Walter; Brunner, G. ddc:610, Medi