Molecular Membrane Biology最新文献_第6页

Purification and interaction analyses of two human lysosomal vitamin B12 transporters: LMBD1 and ABCD4. 两种人溶酶体维生素B12转运体LMBD1和ABCD4的纯化和相互作用分析。

Molecular Membrane Biology Pub Date : 2014-11-01 DOI: 10.3109/09687688.2014.990998

Justin C Deme, Mark A Hancock, Xiaobing Xia, Chitra A Shintre, Maria Plesa, Jaeseung C Kim, Elisabeth P Carpenter, David S Rosenblatt, James W Coulton

{"title":"Purification and interaction analyses of two human lysosomal vitamin B12 transporters: LMBD1 and ABCD4.","authors":"Justin C Deme, Mark A Hancock, Xiaobing Xia, Chitra A Shintre, Maria Plesa, Jaeseung C Kim, Elisabeth P Carpenter, David S Rosenblatt, James W Coulton","doi":"10.3109/09687688.2014.990998","DOIUrl":"https://doi.org/10.3109/09687688.2014.990998","url":null,"abstract":"Mutations in human LMBRD1 and ABCD4 prevent lysosomal export of vitamin B(12) to the cytoplasm, impairing the vitamin B(12)-dependent enzymes methionine synthase and methylmalonyl-CoA mutase. The gene products of LMBRD1 and ABCD4 are implicated in vitamin B(12) transport at the lysosomal membrane and are proposed to act in complex. To address the mechanism for lysosomal vitamin B(12) transport, we report the novel recombinant production of LMBD1 and ABCD4 for detailed biophysical analyses. Using blue native PAGE, chemical crosslinking, and size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), we show that both detergent-solubilized LMBD1 and detergent-solubilized ABCD4 form homodimers. To examine the functional binding properties of these proteins, label-free surface plasmon resonance (SPR) provides direct in vitro evidence that: (i) LMBD1 and ABCD4 interact with low nanomolar affinity; and (ii) the cytoplasmic vitamin B(12)-processing protein MMACHC also interacts with LMBD1 and ABCD4 with low nanomolar affinity. Accordingly, we propose a model whereby membrane-bound LMBD1 and ABCD4 facilitate the vectorial delivery of lysosomal vitamin B(12) to cytoplasmic MMACHC, thus preventing cofactor dilution to the cytoplasmic milieu and protecting against inactivating side reactions.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"31 7-8","pages":"250-61"},"PeriodicalIF":0.0,"publicationDate":"2014-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2014.990998","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32929154","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: 24

Bacterial lipid modification of proteins requires appropriate secretory signals even for expression - implications for biogenesis and protein engineering. 细菌对蛋白质的脂质修饰甚至需要适当的分泌信号来表达-这对生物发生和蛋白质工程的影响。

Molecular Membrane Biology Pub Date : 2014-09-01 Epub Date: 2014-08-26 DOI: 10.3109/09687688.2014.943819

Subramani Kumar, M M Balamurali, Krishnan Sankaran

{"title":"Bacterial lipid modification of proteins requires appropriate secretory signals even for expression - implications for biogenesis and protein engineering.","authors":"Subramani Kumar, M M Balamurali, Krishnan Sankaran","doi":"10.3109/09687688.2014.943819","DOIUrl":"https://doi.org/10.3109/09687688.2014.943819","url":null,"abstract":"Sec- and Tat-mediated bacterial lipid modification of proteins are important posttranslational processes owing to their vital roles in cellular functions, membrane targeting and biotechnological applications like ELISA, biosensor, adjuvant-free vaccines, liposomal drug delivery etc. However a better understanding of the tight coupling of secretory and lipid modification machineries and the processes associated will help unravel this essential biological event and utilize it for engineering applications. Further, there is a need for a systematic and convincing investigation into membrane targeting, solubilization and ease-of-purification of engineered lipoproteins to facilitate scientists in readily applying this new protein engineering tool. Therefore, in this study, we have investigated systematically recombinant expression, translocation, solubilization and purification of three White Spot Syndrome Viral (WSSV) proteins, ICP11, VP28 and VP281. Our study shows that the lipid modification and secretion processes are tightly coupled to the extent that mismatch between folding kinetics and signal sequence of target proteins could lead to transcriptional-translational uncoupling or aborted translation. The proteins expressed as lipoproteins through Tat-pathway were targeted to the inner membrane achieving considerable enrichment. These His-tagged proteins were then purified to apparent homogeneity in detergent-free form using single-step Immobilized Metal Affinity Chromatography. This study has interesting findings in lipoprotein biogenesis enhancing the scope of this unique post-translational protein engineering tool for obtaining pure detergent-free, membrane or hydrophobic surface-associating diagnostic targets and vaccine candidates for WSSV.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"31 6","pages":"183-94"},"PeriodicalIF":0.0,"publicationDate":"2014-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2014.943819","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32613970","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

Topology mapping to characterize cyanobacterial bicarbonate transporters: BicA (SulP/SLC26 family) and SbtA. 表征蓝藻碳酸氢盐转运体的拓扑映射:BicA (SulP/SLC26家族)和SbtA。

Molecular Membrane Biology Pub Date : 2014-09-01 DOI: 10.3109/09687688.2014.953222

G Dean Price, Susan M Howitt

{"title":"Topology mapping to characterize cyanobacterial bicarbonate transporters: BicA (SulP/SLC26 family) and SbtA.","authors":"G Dean Price, Susan M Howitt","doi":"10.3109/09687688.2014.953222","DOIUrl":"https://doi.org/10.3109/09687688.2014.953222","url":null,"abstract":"This mini-review addresses advances in understanding the transmembrane topologies of two unrelated, single-subunit bicarbonate transporters from cyanobacteria, namely BicA and SbtA. BicA is a Na(+)-dependent bicarbonate transporter that belongs to the SulP/SLC26 family that is widespread in both eukaryotes and prokaryotes. Topology mapping of BicA via the phoA/lacZ fusion reporter method identified 12 transmembrane helices with an unresolved hydrophobic region just beyond helix 8. Re-interpreting this data in the light of a recent topology study on rat prestin leads to a consensus topology of 14 transmembrane domains with a 7+7 inverted repeat structure. SbtA is also a Na(+)-dependent bicarbonate transporter, but of considerably higher affinity (Km 2-5 μM versus >100 μM for BicA). Whilst SbtA is widespread in cyanobacteria and a few bacteria, it appears to be absent from eukaryotes. Topology mapping of SbtA via the phoA/lacZ fusion reporter method identified 10 transmembrane helices. The topology consists of a 5+5 inverted repeat, with the two repeats separated by a large intracellular loop. The unusual location of the N and C-termini outside the cell raises the possibility that SbtA forms a novel fold, not so far identified by structural and topological studies on transport proteins.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"31 6","pages":"177-82"},"PeriodicalIF":0.0,"publicationDate":"2014-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2014.953222","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32669629","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: 11

Brij detergents reveal new aspects of membrane microdomain in erythrocytes. Brij洗涤剂揭示了红细胞膜微结构域的新方面。

Molecular Membrane Biology Pub Date : 2014-09-01 DOI: 10.3109/09687688.2014.949319

Bruna Renata Casadei, Patrícia De Oliveira Carvalho, Karin A Riske, Raquel De Melo Barbosa, Eneida De Paula, Cleyton Crepaldi Domingues

{"title":"Brij detergents reveal new aspects of membrane microdomain in erythrocytes.","authors":"Bruna Renata Casadei, Patrícia De Oliveira Carvalho, Karin A Riske, Raquel De Melo Barbosa, Eneida De Paula, Cleyton Crepaldi Domingues","doi":"10.3109/09687688.2014.949319","DOIUrl":"https://doi.org/10.3109/09687688.2014.949319","url":null,"abstract":"Membrane microdomains enriched in cholesterol, sphingolipids (rafts), and specific proteins are involved in important physiological functions. However their structure, size and stability are still controversial. Given that detergent-resistant membranes (DRMs) are in the liquid-ordered state and are rich in raft-like components, they might correspond to rafts at least to some extent. Here we monitor the lateral order of biological membranes by characterizing DRMs from erythrocytes obtained with Brij-98, Brij-58, and TX-100 at 4 °C and 37 °C. All DRMs were enriched in cholesterol and contained the raft markers flotillin-2 and stomatin. However, sphingomyelin (SM) was only found to be enriched in TX-100-DRMs - a detergent that preferentially solubilizes the membrane inner leaflet - while Band 3 was present solely in Brij-DRMs. Electron paramagnetic resonance spectra showed that the acyl chain packing of Brij-DRMs was lower than TX-100-DRMs, providing evidence of their diverse lipid composition. Fatty acid analysis revealed that the SM fraction of the DRMs was enriched in lignoceric acid, which should specifically contribute to the resistance of SM to detergents. These results indicate that lipids from the outer leaflet, particularly SM, are essential for the formation of the liquid-ordered phase of DRMs. At last, the differential solubilization process induced by Brij-98 and TX-100 was monitored using giant unilamellar vesicles. This study suggests that Brij and TX-100-DRMs reflect different degrees of lateral order of the membrane microdomains. Additionally, Brij DRMs are composed by both inner and outer leaflet components, making them more physiologically relevant than TX-100-DRMs to the studies of membrane rafts.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"31 6","pages":"195-205"},"PeriodicalIF":0.0,"publicationDate":"2014-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2014.949319","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32669630","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: 16

Microscopy approaches to investigate protein dynamics and lipid organization. 显微镜方法研究蛋白质动力学和脂质组织。

Molecular Membrane Biology Pub Date : 2014-08-01 Epub Date: 2014-07-21 DOI: 10.3109/09687688.2014.937469

Joanna M Kwiatek, Elizabeth Hinde, Katharina Gaus

引用次数: 7

The blood-brain barrier: structure, function and therapeutic approaches to cross it. 血脑屏障:结构、功能和跨越它的治疗方法。

Molecular Membrane Biology Pub Date : 2014-08-01 Epub Date: 2014-07-21 DOI: 10.3109/09687688.2014.937468

Marta Tajes, Eva Ramos-Fernández, Xian Weng-Jiang, Mònica Bosch-Morató, Biuse Guivernau, Abel Eraso-Pichot, Bertrán Salvador, Xavier Fernàndez-Busquets, Jaume Roquer, Francisco J Muñoz

{"title":"The blood-brain barrier: structure, function and therapeutic approaches to cross it.","authors":"Marta Tajes, Eva Ramos-Fernández, Xian Weng-Jiang, Mònica Bosch-Morató, Biuse Guivernau, Abel Eraso-Pichot, Bertrán Salvador, Xavier Fernàndez-Busquets, Jaume Roquer, Francisco J Muñoz","doi":"10.3109/09687688.2014.937468","DOIUrl":"https://doi.org/10.3109/09687688.2014.937468","url":null,"abstract":"The blood-brain barrier (BBB) is constituted by a specialized vascular endothelium that interacts directly with astrocytes, neurons and pericytes. It protects the brain from the molecules of the systemic circulation but it has to be overcome for the proper treatment of brain cancer, psychiatric disorders or neurodegenerative diseases, which are dramatically increasing as the population ages. In the present work we have revised the current knowledge on the cellular structure of the BBB and the different procedures utilized currently and those proposed to cross it. Chemical modifications of the drugs, such as increasing their lipophilicity, turn them more prone to be internalized in the brain. Other mechanisms are the use of molecular tools to bind the drugs such as small immunoglobulins, liposomes or nanoparticles that will act as Trojan Horses favoring the drug delivery in brain. This fusion of the classical pharmacology with nanotechnology has opened a wide field to many different approaches with promising results to hypothesize that BBB will not be a major problem for the new generation of neuroactive drugs. The present review provides an overview of all state-of-the-art of the BBB structure and function, as well as of the classic strategies and these appeared in recent years to deliver drugs into the brain for the treatment of Central Nervous System (CNS) diseases.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"31 5","pages":"152-67"},"PeriodicalIF":0.0,"publicationDate":"2014-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2014.937468","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32523330","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: 191

Generating thermostabilized agonist-bound GPR40/FFAR1 using virus-like particles and a label-free binding assay. 使用病毒样颗粒和无标记结合试验生成热稳定的激动剂结合GPR40/FFAR1。

Molecular Membrane Biology Pub Date : 2014-08-01 DOI: 10.3109/09687688.2014.923588

Yoshihiko Hirozane, Takashi Motoyaji, Takamitsu Maru, Kengo Okada, Naoki Tarui

{"title":"Generating thermostabilized agonist-bound GPR40/FFAR1 using virus-like particles and a label-free binding assay.","authors":"Yoshihiko Hirozane, Takashi Motoyaji, Takamitsu Maru, Kengo Okada, Naoki Tarui","doi":"10.3109/09687688.2014.923588","DOIUrl":"https://doi.org/10.3109/09687688.2014.923588","url":null,"abstract":"Elucidating the detailed mechanism of activation of membrane protein receptors and their ligand binding is essential for structure-based drug design. Membrane protein crystal structure analysis successfully aids in understanding these fundamental molecular interactions. However, protein crystal structure analysis of the G-protein-coupled receptor (GPCR) remains challenging, even for the class of GPCRs which have been included in the majority of structure analysis reports among membrane proteins, due to the substantial instability of these receptors when extracted from lipid bilayer membranes. It is known that increased thermostability tends to decrease conformational flexibility, which contributes to the generation of diffraction quality crystals. However, this is still not straightforward, and significant effort is required to identify thermostabilized mutants that are optimal for crystallography. To address this issue, a versatile screening platform based on a label-free ligand binding assay combined with transient overexpression in virus-like particles was developed. This platform was used to generate thermostabilized GPR40 [also known as free fatty acid receptor 1 (FFAR1)] for fasiglifam (TAK-875). This demonstrated that the thermostabilized mutant GPR40 (L42A/F88A/G103A/Y202F) was successfully used for crystal structure analysis.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"31 5","pages":"168-75"},"PeriodicalIF":0.0,"publicationDate":"2014-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2014.923588","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32540027","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: 12

The role of the endosomal sorting complexes required for transport (ESCRT) in tumorigenesis. 运输所需的内体分选复合物(ESCRT)在肿瘤发生中的作用。

Molecular Membrane Biology Pub Date : 2014-06-01 Epub Date: 2014-03-18 DOI: 10.3109/09687688.2014.894210

Claudia Mattissek, David Teis

引用次数: 42

Modeling FRET to investigate the selectivity of lactose permease of Escherichia coli for lipids. 建立FRET模型，研究大肠杆菌乳糖渗透酶对脂质的选择性。

Molecular Membrane Biology Pub Date : 2014-06-01 Epub Date: 2014-05-15 DOI: 10.3109/09687688.2014.915351

Carme Suárez-Germà, Jordi Hernández-Borrell, Manuel Prieto, Luís M S Loura

{"title":"Modeling FRET to investigate the selectivity of lactose permease of Escherichia coli for lipids.","authors":"Carme Suárez-Germà, Jordi Hernández-Borrell, Manuel Prieto, Luís M S Loura","doi":"10.3109/09687688.2014.915351","DOIUrl":"https://doi.org/10.3109/09687688.2014.915351","url":null,"abstract":"Förster resonance energy transfer (FRET) is a photophysical process by which a donor (D) molecule in an electronic excited state transfers its excitation energy to a second species, the acceptor (A). Since FRET efficiency depends on D-A separation, the measurement of donor fluorescence in presence and absence of the acceptor allows determination of this distance, and therefore FRET has been extensively used as a \"spectroscopic ruler\". In membranes, interpretation of FRET is more complex, since one D may be surrounded by many A molecules. Such is the case encountered with membrane proteins and lipids in the bilayer. This paper reviews the application of a model built to analyze FRET data between a single tryptophan mutant of the transmembrane protein lactose permease (W151/C154G of LacY), the sugar/H(+) symporter from Escherichia coli, and different pyrene-labeled phospholipids. Several variables of the system with biological implication have been investigated: The selectivity of LacY for different species of phospholipids, the enhancement of the sensitivity of the FRET modeling, and the mutation of a particular aminoacid (D68C) of the protein. The results obtained support: (i) Preference of LacY for phosphatidylethanolamine (PE) over phosphatidylglycerol (PG); (ii) affinity of LacY for fluid (L(α)) phases; and (iii) importance of the aspartic acid in position 68 in the sequence of LacY regarding the interaction with the phospholipid environment. Besides, by exploring the enhancement of the sensitivity by using pure lipid matrices with higher mole fractions of labelled-phospholipid, the dependence on acyl chain composition is unveiled.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"31 4","pages":"120-30"},"PeriodicalIF":0.0,"publicationDate":"2014-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2014.915351","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32340386","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: 8

TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor. TROSY核磁共振与52 kDa糖转运蛋白和结合的小分子抑制剂。

Molecular Membrane Biology Pub Date : 2014-06-01 Epub Date: 2014-05-07 DOI: 10.3109/09687688.2014.911980

Arnout P Kalverda, James Gowdy, Gary S Thompson, Steve W Homans, Peter J F Henderson, Simon G Patching

{"title":"TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor.","authors":"Arnout P Kalverda, James Gowdy, Gary S Thompson, Steve W Homans, Peter J F Henderson, Simon G Patching","doi":"10.3109/09687688.2014.911980","DOIUrl":"https://doi.org/10.3109/09687688.2014.911980","url":null,"abstract":"Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [(15)N-(1)H]- and [(13)C-(1)H]-methyl-TROSY NMR spectra with a 52 kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48 h at a temperature of 25 °C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan (15)N backbone positions and also resolved signals for (15)N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [(13)C-(1)H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"31 4","pages":"131-40"},"PeriodicalIF":0.0,"publicationDate":"2014-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2014.911980","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32321640","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: 13