Molecular Membrane Biology最新文献_第9页

Exploiting Leishmania tarentolae cell-free extracts for the synthesis of human solute carriers. 利用利什曼原虫无细胞提取物合成人溶质载体。

Molecular Membrane Biology Pub Date : 2013-07-01 Epub Date: 2013-06-26 DOI: 10.3109/09687688.2013.807362

Suzan Ruehrer, Hartmut Michel

{"title":"Exploiting Leishmania tarentolae cell-free extracts for the synthesis of human solute carriers.","authors":"Suzan Ruehrer, Hartmut Michel","doi":"10.3109/09687688.2013.807362","DOIUrl":"https://doi.org/10.3109/09687688.2013.807362","url":null,"abstract":"Cell-free protein production offers a versatile alternative to complement in vivo expression systems. However, usage of prokaryotic cell-free systems often leads to non-functional proteins. We modified a previously designed cell-free system based on the protozoan Leishmania tarentolae, a parasite of the Moorish gecko Tarentola mauritanica, together with a species-independent translational sequences-based plasmid to produce human membrane proteins in 2 hours reaction time. We successfully established all four commonly used expression modes for cell-free synthesis of membrane proteins with a human organic anion transporter, SLC17A3, as a model membrane protein: (i) As precipitates without the addition of any hydrophobic environment, (ii) in the presence of detergents, (iii) with the addition of liposomes, and (iv) supplemented with nanodiscs. We utilized this adapted system to synthesize 22 human solute carriers from 20 different families. Our results demonstrate the capability of the Leishmania tarentolae cell-free system for the production of a huge variety of human solute carriers in the precipitate mode. Furthermore, purified SLC17A3 shows the formation of an oligomeric state.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 4","pages":"288-302"},"PeriodicalIF":0.0,"publicationDate":"2013-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2013.807362","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31535554","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 10

Hydrophobic segment of dengue virus C protein. Interaction with model membranes. 登革病毒C蛋白疏水片段。与模型膜的相互作用。

Molecular Membrane Biology Pub Date : 2013-07-01 Epub Date: 2013-06-07 DOI: 10.3109/09687688.2013.805835

Henrique Nemésio, M Francisca Palomares-Jerez, José Villalaín

{"title":"Hydrophobic segment of dengue virus C protein. Interaction with model membranes.","authors":"Henrique Nemésio, M Francisca Palomares-Jerez, José Villalaín","doi":"10.3109/09687688.2013.805835","DOIUrl":"https://doi.org/10.3109/09687688.2013.805835","url":null,"abstract":"Dengue virus (DENV) C protein is essential for viral assembly. DENV C protein associates with intracellular membranes through a conserved hydrophobic domain and accumulates around endoplasmic reticulum-derived lipid droplets which could provide a platform for capsid formation during assembly. In a previous work we described a region in DENV C protein which induced a nearly complete membrane rupture of several membrane model systems, which was coincident with the theoretically predicted highly hydrophobic region of the protein. In this work we have carried out a study of the binding to and interaction with model biomembranes of a peptide corresponding to this DENV C region, DENV2C6. We show that DENV2C6 partitions into phospholipid membranes, is capable of rupturing membranes even at very low peptide-to-lipid ratios and its membrane-activity is modulated by lipid composition. These results identify an important region in the DENV C protein which might be directly implicated in the DENV life cycle through the modulation of membrane structure.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 4","pages":"273-87"},"PeriodicalIF":0.0,"publicationDate":"2013-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2013.805835","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31488317","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 14

Rab1a and Rab5a preferentially bind to binary lipid compositions with higher stored curvature elastic energy. Rab1a和Rab5a优先结合具有较高曲率弹性能的二元脂质组合物。

Molecular Membrane Biology Pub Date : 2013-07-01 DOI: 10.3109/09687688.2013.818725

Marie L Kirsten, Rudi A Baron, Miguel C Seabra, Oscar Ces

{"title":"Rab1a and Rab5a preferentially bind to binary lipid compositions with higher stored curvature elastic energy.","authors":"Marie L Kirsten, Rudi A Baron, Miguel C Seabra, Oscar Ces","doi":"10.3109/09687688.2013.818725","DOIUrl":"https://doi.org/10.3109/09687688.2013.818725","url":null,"abstract":"Abstract Rab proteins are a large family of GTP-binding proteins that regulate cellular membrane traffic and organelle identity. Rab proteins cycle between association with membranes and binding to RabGDI. Bound on membranes, each Rab has a very specific cellular location and it is this remarkable degree of specificity with which Rab GTPases recognize distinct subsets of intracellular membranes that forms the basis of their ability to act as key cellular regulators, determining the recruitment of downstream effectors to the correct membrane at the correct time. The molecular mechanisms controlling Rab localization remain poorly understood. Here, we present a fluorescence-based assay to investigate Rab GTPase membrane extraction and delivery by RabGDI. Using EGFP-Rab fusion proteins the amount of Rab:GDI complex obtained by GDI extraction of Rab proteins from HEK293 membranes could be determined, enabling control of complex concentration. Subsequent partitioning of the Rab GTPases into vesicles made up of artificial binary lipid mixtures showed for the first time, that the composition of the target membrane plays a key role in the localization of Rab proteins by sensing the stored curvature elastic energy in the membrane.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 4","pages":"303-14"},"PeriodicalIF":0.0,"publicationDate":"2013-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2013.818725","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31546885","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 10

Close allies in membrane protein research: cell-free synthesis and nanotechnology. 膜蛋白研究的紧密盟友:无细胞合成和纳米技术。

Molecular Membrane Biology Pub Date : 2013-05-01 Epub Date: 2013-01-24 DOI: 10.3109/09687688.2012.762125

Nadim Shadiac, Yagnesh Nagarajan, Shane Waters, Maria Hrmova

{"title":"Close allies in membrane protein research: cell-free synthesis and nanotechnology.","authors":"Nadim Shadiac, Yagnesh Nagarajan, Shane Waters, Maria Hrmova","doi":"10.3109/09687688.2012.762125","DOIUrl":"https://doi.org/10.3109/09687688.2012.762125","url":null,"abstract":"Membrane proteins control fundamental processes that are inherent to nearly all forms of life such as transport of molecules, catalysis, signaling, vesicle fusion, sensing of chemical and physical stimuli from the environment, and cell-cell interactions. Membrane proteins are harbored within a non-equilibrium fluid-like environment of biological membranes that separate cellular and non-cellular environments, as well as in compartmentalized cellular organelles. One of the classes of membrane proteins that will be specifically treated in this article are transport proteins of plant origin, that facilitate material and energy transfer at the membrane boundaries. These proteins import essential nutrients, export cellular metabolites, maintain ionic and osmotic equilibriums and mediate signal transduction. The aim of this article is to report on the progress of membrane protein functional and structural relationships, with a focus on producing stable and functional proteins suitable for structural and biophysical studies. We interlink membrane protein production primarily through wheat-germ cell-free protein synthesis (WG-CFPS) with the growing repertoire of membrane mimicking environments in the form of lipids, surfactants, amphipathic surfactant polymers, liposomes and nanodiscs that keep membrane proteins soluble. It is hoped that the advancements in these fields could increase the number of elucidated structures, in particular those of plant membrane proteins, and contribute to bridging of the gap between structures of soluble and membrane proteins, the latter being comparatively low.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 3","pages":"229-45"},"PeriodicalIF":0.0,"publicationDate":"2013-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2012.762125","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31182157","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 21

The identification of novel, high affinity AQP9 inhibitors in an intracellular binding site. 在细胞内结合位点鉴定新的高亲和力AQP9抑制剂。

Molecular Membrane Biology Pub Date : 2013-05-01 Epub Date: 2013-03-01 DOI: 10.3109/09687688.2013.773095

Sören J Wacker, Camilo Aponte-Santamaría, Per Kjellbom, Søren Nielsen, Bert L de Groot, Michael Rützler

{"title":"The identification of novel, high affinity AQP9 inhibitors in an intracellular binding site.","authors":"Sören J Wacker, Camilo Aponte-Santamaría, Per Kjellbom, Søren Nielsen, Bert L de Groot, Michael Rützler","doi":"10.3109/09687688.2013.773095","DOIUrl":"https://doi.org/10.3109/09687688.2013.773095","url":null,"abstract":"Background: The involvement of aquaporin (AQP) water and small solute channels in the etiology of several diseases, including cancer, neuromyelitis optica and body fluid imbalance disorders, has been suggested previously. Furthermore, results obtained in a mouse model suggested that AQP9 function contributes to hyperglycemia in type-2 diabetes. In addition, the physiological role of several AQP family members remains poorly understood. Small molecule inhibitors of AQPs are therefore desirable to further study AQP physiological and pathophysiological functions.Methods: The binding of recently established AQP9 inhibitors to a homology model of AQP9 was investigated by molecular dynamics simulations and molecular docking. Putative inhibitor binding sites identified with this procedure were modified by site-directed mutagenesis. Active compounds were measured in a mammalian cell water permeability assay of mutated AQP9 isoforms and tested for changes in inhibitory effects.Controls: Three independent cell lines were established for each mutated AQP9 isoform and functionality of mutant isoforms was established.Principal findings: We have identified putative binding sites of recently established AQP9 inhibitors. This information facilitated successful identification of novel AQP9 inhibitors with low micromolar IC50 values in a cell based assay by in silico screening of a compound library targeting specifically this binding site.Significance: We have established a successful strategy for AQP small molecule inhibitor identification. AQP inhibitors may be relevant as experimental tools, to enhance our understanding of AQP function, and in the treatment of various diseases.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 3","pages":"246-60"},"PeriodicalIF":0.0,"publicationDate":"2013-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2013.773095","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31271243","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 31

Permeabilization of enterocytes induced by absorption of dietary fat. 饮食脂肪吸收诱导肠细胞通透性。

Molecular Membrane Biology Pub Date : 2013-05-01 Epub Date: 2013-03-26 DOI: 10.3109/09687688.2013.780642

Erik Michael Danielsen, Gert H Hansen, Karina Rasmussen, Lise-Lotte Niels-Christiansen

{"title":"Permeabilization of enterocytes induced by absorption of dietary fat.","authors":"Erik Michael Danielsen, Gert H Hansen, Karina Rasmussen, Lise-Lotte Niels-Christiansen","doi":"10.3109/09687688.2013.780642","DOIUrl":"https://doi.org/10.3109/09687688.2013.780642","url":null,"abstract":"Absorption of dietary fat in the small intestine involves epithelial exposure to potentially harmful molecules such as bile salts and free fatty acids. We used organ culture of porcine jejunal explants incubated with a pre-digested mixture of fat (plant oil), bile and pancreatin to mimick the physiological process of dietary fat absorption, and short exposures to the fat mixture caused fat droplet accumulation within villus enterocytes. Lucifer yellow (LY), a fluorescent membrane-impermeable polar tracer was included to monitor epithelial integrity. Both in controls and during fat absorption LY penetrated the epithelium and accumulated in the basal lamina and the lamina propria. LY was also seen in the paracellular space, whereas villus enterocytes were generally only weakly labeled except for small amounts taken up by apical endocytosis. In the crypts, however, fat absorption induced cell permeabilization with LY accumulating in the cytosol and nucleus. Morphologically, both apical and basolateral membranes appeared intact, indicating that the leakiness was caused by minor lesions in the membrane. Albeit to a lesser extent, bile alone was capable of permeabilizing crypt cells, implying that the surfactant properties of bile salts are involved in the process. In addition to LY, crypt enterocytes also became permeable for albumin, ovalbumin and insulin. In conclusion, during fat absorption the permeability of the gut epithelium is increased mainly in the crypts. A possible explanation is that cell membranes of immature crypt cells, lacking detergent-resistant lipid raft microdomains, are less resistant to the deleterious effects of bile salts and free fatty acids.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 3","pages":"261-72"},"PeriodicalIF":0.0,"publicationDate":"2013-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2013.780642","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31426661","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 15

The substrate specificity of the human ADP/ATP carrier AAC1. 人ADP/ATP载体AAC1的底物特异性。

Molecular Membrane Biology Pub Date : 2013-03-01 Epub Date: 2012-11-23 DOI: 10.3109/09687688.2012.745175

John Mifsud, Stéphanie Ravaud, Eva-Maria Krammer, Chris Chipot, Edmund R S Kunji, Eva Pebay-Peyroula, Francois Dehez

{"title":"The substrate specificity of the human ADP/ATP carrier AAC1.","authors":"John Mifsud, Stéphanie Ravaud, Eva-Maria Krammer, Chris Chipot, Edmund R S Kunji, Eva Pebay-Peyroula, Francois Dehez","doi":"10.3109/09687688.2012.745175","DOIUrl":"10.3109/09687688.2012.745175","url":null,"abstract":"The mitochondrial ADP/ATP carrier imports ADP from the cytosol into the mitochondrial matrix for its conversion to ATP by ATP synthase and exports ATP out of the mitochondrion to replenish the eukaryotic cell with chemical energy. Here the substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches. In the first the protein was functionally expressed in Escherichia coli membranes as a fusion protein with maltose binding protein and the effect of excess of unlabeled compounds on the uptake of [(32)P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [(14)C]-ADP in whole cells was measured in the presence of excess of unlabeled compounds and in fused membrane vesicles loaded with unlabeled compounds to demonstrate their transport. A large number of nucleotides were tested, but only ADP and ATP are suitable substrates for human AAC1, demonstrating a very narrow specificity. Next we tried to understand the molecular basis of this specificity by carrying out molecular-dynamics simulations with selected nucleotides, which were placed at the entrance of the central cavity. The binding of the phosphate groups of guanine and adenine nucleotides is similar, yet there is a low probability for the base moiety to be bound, likely to be rooted in the greater polarity of guanine compared to adenine. AMP is unlikely to engage fully with all contact points of the substrate binding site, suggesting that it cannot trigger translocation.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 2","pages":"160-8"},"PeriodicalIF":0.0,"publicationDate":"2013-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2012.745175","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31068696","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 51

Ligand binding properties of human galanin receptors. 人丙氨酸受体的配体结合特性。

Molecular Membrane Biology Pub Date : 2013-03-01 Epub Date: 2012-12-13 DOI: 10.3109/09687688.2012.750384

Wiktor Jurkowski, Samira Yazdi, Arne Elofsson

引用次数: 20

The C-terminal cavity of the Na,K-ATPase analyzed by docking and electrophysiology. 通过对接和电生理分析Na, k - atp酶的c端空腔。

Molecular Membrane Biology Pub Date : 2013-03-01 Epub Date: 2012-08-22 DOI: 10.3109/09687688.2012.713520

Peter Aasted Paulsen, Wiktor Jurkowski, Rossen Apostolov, Erik Lindahl, Poul Nissen, Hanne Poulsen

{"title":"The C-terminal cavity of the Na,K-ATPase analyzed by docking and electrophysiology.","authors":"Peter Aasted Paulsen, Wiktor Jurkowski, Rossen Apostolov, Erik Lindahl, Poul Nissen, Hanne Poulsen","doi":"10.3109/09687688.2012.713520","DOIUrl":"https://doi.org/10.3109/09687688.2012.713520","url":null,"abstract":"The Na,K-ATPase is essential to all animals, since it maintains the electrochemical gradients that energize the plasma membrane. Naturally occurring inhibitors of the pump from plants have been used pharmaceutically in cardiac treatment for centuries. The inhibitors block the pump by binding on its extracellular side and thereby locking it. To explore the possibilities for designing an alternative way of targeting the pump function, we have examined the structural requirements for binding to a pocket that accommodates the two C-terminal residues, YY, in the crystal structures of the pump. To cover the sample space of two residues, we first performed docking studies with the 400 possible dipeptides. For validation of the in silico predictions, pumps with 13 dipeptide sequences replacing the C-terminal YY were expressed in Xenopus laevis oocytes and examined with electrophysiology. Our data show a significant correlation between the docking scores from two different methods and the experimentally determined sodium affinities, which strengthens the previous hypothesis that sodium binding is coupled to docking of the C-terminus. From the dipeptides that dock the best and better than wild-type YY, it may therefore be possible to develop specific drugs targeting a previously unexplored binding pocket in the sodium pump.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 2","pages":"195-205"},"PeriodicalIF":0.0,"publicationDate":"2013-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2012.713520","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30851124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 6

Probing the contacts of a low-affinity substrate with a membrane-embedded transport protein using 1H-13C cross-polarisation magic-angle spinning solid-state NMR. 利用1H-13C交叉极化魔角自旋固体核磁共振探测低亲和底物与膜嵌入转运蛋白的接触。

Molecular Membrane Biology Pub Date : 2013-03-01 Epub Date: 2012-11-23 DOI: 10.3109/09687688.2012.743193

Simon G Patching, Peter J F Henderson, David J Sharples, David A Middleton

{"title":"Probing the contacts of a low-affinity substrate with a membrane-embedded transport protein using 1H-13C cross-polarisation magic-angle spinning solid-state NMR.","authors":"Simon G Patching, Peter J F Henderson, David J Sharples, David A Middleton","doi":"10.3109/09687688.2012.743193","DOIUrl":"https://doi.org/10.3109/09687688.2012.743193","url":null,"abstract":"Solid-state NMR combined with sample deuteration was used to probe the proximity of the low-affinity substrate D-glucose to its binding site within the Escherichia coli sugar transport protein GalP. Samples of E. coli inner membranes with amplified expression of GalP were incubated in D(2)O with D-[(13)C(6)]glucose and (13)C NMR signals from the substrate were assigned in two-dimensional dipolar-assisted rotational resonance (DARR) spectra. The signals were confirmed as representing D-glucose bound to GalP as the peaks were abolished after the substrate was displaced from the specific site with the inhibitor forskolin. The (13)C chemical shift values for D-[(13)C(6)]glucose in solution revealed some differences compared to those for ligand bound to GalP, the differences being most pronounced for positions C1 and C2, and especially for C1 in the α-anomer. (13)C cross-polarization build-up was measured for C1 and C2 of D-[(13)C(6)]glucose and D-[(2)H(7), (13)C(6)]glucose in GalP membranes suspended in D(2)O. The build-up curves for the deuterated substrate reflect intermolecular (1)H-(13)C interactions between the protein and the fully deuterated substrate; the signal build-up suggests that the α-anomer is situated closer to the protein binding site than is the β-anomer, consistent with its relatively high signal intensities and more pronounced chemical shift changes in the 2D-correlation spectra. These results demonstrate the utility of solid-state NMR combined with sample deuteration for mapping the binding interface of low affinity ligands with membrane proteins.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 2","pages":"129-37"},"PeriodicalIF":0.0,"publicationDate":"2013-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2012.743193","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31064985","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 10