WorldWideScience

Sample records for basement membrane protein

  1. Ultrastructural localization of the core protein of a basement membrane-specific chondroitin sulfate proteoglycan in adult rat skin

    DEFF Research Database (Denmark)

    McCarthy, K J; Horiguchi, Y; Couchman, J R;

    1990-01-01

    Basement membranes are complex extracellular matrices present at epithelial/mesenchymal interfaces of tissues. The dermal-epidermal junction has been shown to contain numerous components, some of the most well known being laminin, types IV and VII collagens, heparan sulfate proteoglycan, fibronec......Basement membranes are complex extracellular matrices present at epithelial/mesenchymal interfaces of tissues. The dermal-epidermal junction has been shown to contain numerous components, some of the most well known being laminin, types IV and VII collagens, heparan sulfate proteoglycan......, fibronectin, and entactin/nidogen. IN this paper we show, using core protein-specific antibodies, the presence of a newly described basement membrane-specific chondroitin sulfate proteoglycan at the epithelial/mesenchymal interface of adult rat skin. Ultrastructurally, this antigen was proven to reside...... primarily within the basal lamina, apparently concentrated in the lamina densa. In addition, some of the proteoglycan was also present beneath the lamina densa, associated with the reticular lamina collagen fibrils....

  2. Immunohistochemical expression of basement membrane proteins of verrucous carcinoma of the oral mucosa.

    Science.gov (United States)

    Arduino, Paolo G; Carrozzo, Marco; Pagano, Marco; Broccoletti, Roberto; Scully, Crispian; Gandolfo, Sergio

    2010-06-01

    Squamous cell carcinoma (SCC) of the oral cavity is an extremely invasive tumour of stratified squamous epithelium that spreads throughout degradation of the basement membrane (BM) and extra-cellular matrix. Oral verrucous carcinoma (VC) is a rare low-grade variant of oral SCC that penetrates into the subepithelial connective tissue. It also has a different clinical behaviour from classical oral SCC. We investigated the immunohistochemical expression of laminin, laminin-5, collagen IV and fibronectin in VC, severe epithelial dysplasia (SED) and SCC in order to analyse if the pattern of these molecules expression contributes to the differences in the biological behaviour of these diseases. The staining pattern of laminin was less intensive in SCC compared with SED and VC, and collagen IV expression was increased in VC compared with SED. Discontinuities of laminin, collagen IV and fibronectin were more evident in SED than in VC. This study indicates that VC has a biological behaviour different from SED or SCC, observable by immunohistochemistry in the BM zone.

  3. The major basement membrane components localize to the chondrocyte pericellular matrix--a cartilage basement membrane equivalent?

    DEFF Research Database (Denmark)

    Kvist, Alexander J.; Nyström, Alexander; Hultenby, Kjell;

    2007-01-01

    In this study, we demonstrate that articular cartilage chondrocytes are surrounded by the defining basement membrane proteins laminin, collagen type IV, nidogen and perlecan, and suggest that these form the functional equivalent of a basement membrane. We found by real-time PCR that mouse chondro...

  4. Basement membrane chondroitin sulfate proteoglycans: localization in adult rat tissues

    DEFF Research Database (Denmark)

    McCarthy, K J; Couchman, J R

    1990-01-01

    and characterization of core protein-specific monoclonal antibodies (MAb) against a chondroitin sulfate proteoglycan (CSPG) present in Reichert's membrane, a transient extra-embryonic structure of rodents. This CSPG was also demonstrated to be present in adult rat kidney. We report here the tissue distribution...... of epitopes recognized by these MAb. The ubiquitous presence of these epitopes in the basement membranes of nearly all adult rat tissues demonstrates that at least one CSPG is a constituent of most basement membranes, and by virtue of its unique distribution is distinct from other chondroitin and dermatan...

  5. Synthesis and deposition of basement membrane proteins by primary brain capillary endothelial cells in a murine model of the blood-brain barrier

    DEFF Research Database (Denmark)

    Thomsen, Maj Schneider; Birkelund, Svend; Burkhart, Annette;

    2016-01-01

    The brain vascular basement membrane is important for both blood-brain barrier (BBB) development, stability, and barrier integrity and the contribution hereto from brain capillary endothelial cells (BCECs), pericytes, and astrocytes of the BBB is probably significant. The aim of the present study......-culture, in co-culture with pericytes or mixed glial cells, or as a triple-culture with both pericytes and mixed glial cells. The integrity of the BBB models was validated by measures of transendothelial electrical resistance (TEER) and passive permeability to mannitol. The expression of basement membrane...... proteins was analysed using RT-qPCR, mass spectrometry, and immunocytochemistry. Co-culturing mBCECs with pericytes, mixed glial cells, or both significantly increased the TEER compared to the mono-culture, and a low passive permeability was correlated with high TEER. The mBCECs expressed all major...

  6. Still more complexity in mammalian basement membranes

    DEFF Research Database (Denmark)

    Erickson, A C; Couchman, J R

    2000-01-01

    At the epithelial/mesenchymal interface of most tissues lies the basement membrane (BM). These thin sheets of highly specialized extracellular matrix vary in composition in a tissue-specific manner, and during development and repair. For about two decades it has been apparent that all BMs contain...

  7. Basement membrane proteoglycans are of epithelial origin in rodent skin

    DEFF Research Database (Denmark)

    Yamane, Y; Yaoita, H; Couchman, J R

    1996-01-01

    Basement membrane proteoglycans in mammalian skin comprise at least one chondroitin sulfate proteoglycan and heparan sulfate proteoglycans, including perlecan. In this study, the origins of basement membrane chondroitin sulfate proteoglycan and perlecan were investigated both in vivo and in vitro...

  8. Atypical anti-glomerular basement membrane disease

    OpenAIRE

    Troxell, Megan L.; Donald C Houghton

    2015-01-01

    Background Anti-glomerular basement membrane (anti-GBM) disease classically presents with aggressive necrotizing and crescentic glomerulonephritis, often with pulmonary hemorrhage. The pathologic hallmark is linear staining of GBMs for deposited immunoglobulin G (IgG), usually accompanied by serum autoantibodies to the collagen IV alpha-3 constituents of GBMs. Methods Renal pathology files were searched for cases with linear anti-GBM to identify cases with atypical or indolent course. Histopa...

  9. Coarctation induces alterations in basement membranes in the cardiovascular system

    DEFF Research Database (Denmark)

    Lipke, D W; McCarthy, K J; Elton, T S;

    1993-01-01

    ventricular hypertrophy was maximal within 5 days. In immunohistochemical studies, fibronectin and laminin were increased and the basement membrane chondroitin sulfate proteoglycan decreased in both the subendothelial space and smooth muscle cell basement membranes of the aorta above the clip compared...... membrane components in the heart and vasculature peaked before maximal cardiac hypertrophy (5 days). These studies indicate that alterations in basement membrane component deposition in the hypertrophied vasculature occur at both transcriptional and translational levels and suggest that the cell attachment...

  10. Regulation of the basement membrane by epithelia generated forces

    Science.gov (United States)

    Tanner, Kandice

    2012-12-01

    Tumor metastasis involves a progressive loss of tissue architecture and dissolution of structural boundaries between the epithelium and connective tissue. The basement membrane (BM), a specialized network of extracellular matrix proteins forms a barrier that physically restricts pre-invasive lesions such that they remain as local insults. The BM is not a static structure, but one that is constantly regenerated and remodeled in the adult organism. Matrix organization also regulates cell function. Thus alterations in the balance of synthesis, remodeling and proteolytic degradation of the extracellular matrix proteins may contribute to a loss of structural integrity. However, the de novo assembly and maintenance of the complex structural properties of in vivo basement membranes remain elusive. Here, this paper highlights the current understanding on the structural properties and the establishment of the BM, and discusses the potential role of self-generated forces in adult tissue remodeling and the maintenance of the BM as a malignancy suppressor.

  11. Genome wide analysis indicates genes for basement membrane and cartilage matrix proteins as candidates for hip dysplasia in Labrador Retrievers.

    Directory of Open Access Journals (Sweden)

    Ineke C M Lavrijsen

    Full Text Available Hip dysplasia, an abnormal laxity of the hip joint, is seen in humans as well as dogs and is one of the most common skeletal disorders in dogs. Canine hip dysplasia is considered multifactorial and polygenic, and a variety of chromosomal regions have been associated with the disorder. We performed a genome-wide association study in Dutch Labrador Retrievers, comparing data of nearly 18,000 single nucleotide polymorphisms (SNPs in 48 cases and 30 controls using two different statistical methods. An individual SNP analysis based on comparison of allele frequencies with a χ(2 statistic was used, as well as a simultaneous SNP analysis based on Bayesian variable selection. Significant association with canine hip dysplasia was observed on chromosome 8, as well as suggestive association on chromosomes 1, 5, 15, 20, 25 and 32. Next-generation DNA sequencing of the exons of genes of seven regions identified multiple associated alleles on chromosome 1, 5, 8, 20, 25 and 32 (p<0.001. Candidate genes located in the associated regions on chromosomes 1, 8 and 25 included LAMA2, LRR1 and COL6A3, respectively. The associated region on CFA20 contained candidate genes GDF15, COMP and CILP2. In conclusion, our study identified candidate genes that might affect susceptibility to canine hip dysplasia. These genes are involved in hypertrophic differentiation of chondrocytes and extracellular matrix integrity of basement membrane and cartilage. The functions of the genes are in agreement with the notion that disruptions in endochondral bone formation in combination with soft tissue defects are involved in the etiology of hip dysplasia.

  12. Glomerular Basement Membrane Type IV Collagen in Health and Disease

    OpenAIRE

    Fish, Alfred J.; Kashtan, Clifford E.; Matsukura, Hiro; Butkowski, Ralph J.

    1991-01-01

    Glomerular basement membrane is the major supporting structural element of the glomerular capillary wall. This is a highly complex locus which functionally serves as a filtration barrier, and has been the subject of detailed investigation. The composition of whole glomerular basement membrane suggests that collagen is a major component. Isolation and characterization of the collagenous domains has revealed that glomerular basement membrane is chiefly composed of type IV collagen. This molecul...

  13. Immunological characterization of a basement membrane-specific chondroitin sulfate proteoglycan

    DEFF Research Database (Denmark)

    McCarthy, K J; Accavitti, M A; Couchman, J R

    1989-01-01

    Reichert's membrane, an extraembryonic membrane present in developing rodents, has been proposed as an in vivo model for the study of basement membranes. We have used this membrane as a source for isolation of basement membrane proteoglycans. Reichert's membranes were extracted in a guanidine/3...... (Mr = 5-6 x 10(5)), with a core protein of Mr = approximately 1.5-1.6 x 10(5) and composed exclusively of chondroitin sulfate chains with an average Mr = 1.6-1.8 x 10(4). In addition, a CSPG was purified from adult rat kidney, whose core protein was also Mr = 1.6 x 10(5). The proteoglycan and its core...... protein were also recognized by all four mAbs. Indirect immunofluorescence of rat tissue sections stained with these antibodies reveal a widespread distribution of this proteoglycan, localized specifically to Reichert's membrane and nearly all basement membranes of rat tissues. In addition to heparan...

  14. Symposium: Role of the extracellular matrix in mammary development. Regulation of milk protein and basement membrane gene expression: The influence of the extracellular matrix

    Energy Technology Data Exchange (ETDEWEB)

    Aggeler, J.; Park, C.S.; Bissell, M.J.

    1988-10-01

    Synthesis and secretion of milk proteins ({alpha}-casein, {beta}-casein, {gamma}-casein, and transferrin) by cultured primary mouse mammary epithelial cells is modulated by the extracellular matrix. In cells grown on released or floating type I collagen gels, mRNA for {beta}-casein and transferrin is increased as much as 30-fold over cells grown on plastic. Induction of {beta}-casein expression depends strongly on the presence of lactogenic hormones, especially prolactin, in the culture. When cells are plated onto partially purified reconstituted basement membrane, dramatic changes in morphology and milk protein gene expression are observed. Cells cultured on the matrix for 6 to 8 d in the presence of prolactin, insulin, and hydrocortisone form hollow spheres and duct-like structures that are completely surrounded by matrix. The cells lining these spheres appear actively secretory and are oriented with their apices facing the lumen. Hybridization experiments indicate that mRNA for {beta}-casein can be increased as much as 70-fold in these cultures. Because > 90% of the cultured cells synthesize immunoreactive {beta}-casein, as compared with only 40% of cells in the late pregnant gland, the matrix appears to be able to induce protein expression in previously silent cells. Synthesis of laminin and assembly of a mammary-specific basal lamina by cells cultured on different extracellular matrices also appears to depend on the presence of lactogenic hormones. These studies provide support for the concept of dynamic reciprocity in which complex interactions between extracellular matrix and the cellular cytoskeleton contribute to the induction and maintenance of tissue-specific gene expression in the mammary gland.

  15. Expression of VLA-integrins and their related basement membrane ligands in gingiva from patients of various periodontitis categories

    DEFF Research Database (Denmark)

    Gürses, N.; Thorup, Alis Karabulut; Reibel, J.;

    1999-01-01

    integrins, basement membrane, gingiva, periodontitis, periodontal disease activity immunofluorescence......integrins, basement membrane, gingiva, periodontitis, periodontal disease activity immunofluorescence...

  16. The bi-functional organization of human basement membranes.

    Science.gov (United States)

    Halfter, Willi; Monnier, Christophe; Müller, David; Oertle, Philipp; Uechi, Guy; Balasubramani, Manimalha; Safi, Farhad; Lim, Roderick; Loparic, Marko; Henrich, Paul Bernhard

    2013-01-01

    The current basement membrane (BM) model proposes a single-layered extracellular matrix (ECM) sheet that is predominantly composed of laminins, collagen IVs and proteoglycans. The present data show that BM proteins and their domains are asymmetrically organized providing human BMs with side-specific properties: A) isolated human BMs roll up in a side-specific pattern, with the epithelial side facing outward and the stromal side inward. The rolling is independent of the curvature of the tissue from which the BMs were isolated. B) The epithelial side of BMs is twice as stiff as the stromal side, and C) epithelial cells adhere to the epithelial side of BMs only. Side-selective cell adhesion was also confirmed for BMs from mice and from chick embryos. We propose that the bi-functional organization of BMs is an inherent property of BMs and helps build the basic tissue architecture of metazoans with alternating epithelial and connective tissue layers.

  17. The bi-functional organization of human basement membranes.

    Directory of Open Access Journals (Sweden)

    Willi Halfter

    Full Text Available The current basement membrane (BM model proposes a single-layered extracellular matrix (ECM sheet that is predominantly composed of laminins, collagen IVs and proteoglycans. The present data show that BM proteins and their domains are asymmetrically organized providing human BMs with side-specific properties: A isolated human BMs roll up in a side-specific pattern, with the epithelial side facing outward and the stromal side inward. The rolling is independent of the curvature of the tissue from which the BMs were isolated. B The epithelial side of BMs is twice as stiff as the stromal side, and C epithelial cells adhere to the epithelial side of BMs only. Side-selective cell adhesion was also confirmed for BMs from mice and from chick embryos. We propose that the bi-functional organization of BMs is an inherent property of BMs and helps build the basic tissue architecture of metazoans with alternating epithelial and connective tissue layers.

  18. Abnormal glomerular basement membrane in idiopathic multicentric osteolysis

    NARCIS (Netherlands)

    Bakker, SJL; Vos, GD; Verschure, PDMM; Mulder, AH; Tiebosch, TMG

    1996-01-01

    The primary cause of nephropathy in idiopathic multicentric osteolysis is as yet unknown. We report a young girl with idiopathic multicentric osteolysis and nephropathy. An abnormal glomerular basement membrane was the only abnormality found in a renal biopsy taken 2 years before the development of

  19. Ultrastructure of basement membranes in developing shark tooth.

    Science.gov (United States)

    Sawada, T; Inoue, S

    2003-01-01

    Based on studies of the tooth of largely mammalian species, the dental basement membranes are shown to be specialized for various roles significant in the development and maintenance of the tooth. Comparative studies with the nonmammalian tooth will facilitate further clarification of the mechanisms of mammalian tooth formation. In this study, basement membranes of the shark tooth in successive developmental stages was ultrastructurally examined for elucidation of their roles in odontogenesis. Teeth of a shark, Cephaloscyllium umbratile, were processed for thin section electron microscopy. Throughout the developmental stages the lamina densa of the basement membrane was made up of a fine network of "cords," irregular anastomosing strands known to be the major component of mammalian basement membranes. In the presecretory stage of the shark tooth, dental papilla cells were immobilized for their differentiation into odontoblasts by means of the binding of their processes to numerous narrow extensions of the lamina densa of the inner dental epithelium. In the secretory stage, a number of cords of the widened lamina densa were extended towards and bound to tubular vesicles of the forming enameloid. During the mineralization stage, fragments of the degrading enameloid matrix appeared to be moving through the lamina densa to the epithelial cells for processing. In the maturation stage, half of the lamina densa facing the enameloid was mineralized forming an advancing edge of mineralization of the enameloid. It provided strong binding and smooth transition of organic to mineral phase which may allow transportation of substances across the phases for enameloid maturation in a way similar to that reported in the mammalian tooth. These observations indicate that basement membranes of the developing shark tooth, as those in the mammalian tooth, play various roles, including anchoring, firm binding, and possible mediation of the transport of substances that are known to be

  20. Isotropic Versus Bipolar Functionalized Biomimetic Artificial Basement Membranes and Their Evaluation in Long-Term Human Cell Co-Culture.

    Science.gov (United States)

    Rossi, Angela; Wistlich, Laura; Heffels, Karl-Heinz; Walles, Heike; Groll, Jürgen

    2016-08-01

    In addition to dividing tissues into compartments, basement membranes are crucial as cell substrates and to regulate cellular behavior. The development of artificial basement membranes is indispensable for the ultimate formation of functional engineered tissues; however, pose a challenge due to their complex structure. Herein, biodegradable electrospun polyester meshes are presented, exhibiting isotropic or bipolar bioactivation as a biomimetic and biofunctional model of the natural basement membrane. In a one-step preparation process, reactive star-shaped prepolymer additives, which generate a hydrophilic fiber surface, are electrospun with cell-adhesion-mediating peptides, derived from major components of the basement membrane. Human skin cells adhere to the functionalized meshes, and long-term co-culture experiments confirm that the artificial basement membranes recapitulate and preserve tissue specific functions. Several layers of immortalized human keratinocytes grow on the membranes, differentiating toward the surface and expressing typical epithelial markers. Fibroblasts migrate into the reticular lamina mimicking part of the mesh. Both cells types begin to produce extracellular matrix proteins and to remodel the initial membrane. It is shown at the example of skin that the artificial basement membrane design provokes biomimetic responses of different cell types and can thus be used as basis for the future development of basement membrane containing tissues. PMID:27283510

  1. Ultrastructural appearance of renal and other basement membranes in the Bull terrier model of autosomal dominant hereditary nephritis.

    Science.gov (United States)

    Hood, J C; Savige, J; Seymour, A E; Dowling, J; Martinello, P; Colville, D; Sinclair, R; Naito, I; Jennings, G; Huxtable, C

    2000-08-01

    Bull terrier hereditary nephritis may represent a model for autosomal dominant Alport's syndrome because affected dogs have the typically lamellated glomerular basement membrane (GBM) and father-to-son disease transmission occurs. This study examined the ultrastructural appearance of the renal and extrarenal basement membranes and their composition in affected Bull terriers. Affected stillborn animals and puppies had subepithelial frilling and vacuolation of the GBM. In adult dogs, lamellation was common, and subepithelial frilling and vacuolation were less prominent. Foot-process effacement and mesangial matrix expansion occurred frequently. Basement membranes in the glomeruli, tubules, and Bowman's capsule were significantly thickened and often mineralized. Immunohistochemical examination showed alpha 1(IV) and alpha 2(IV) collagen chains in all renal basement membranes; alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains in the GBM, distal tubular basement membrane, and Bowman's capsule; and the alpha 6(IV) chain in Bowman's capsule. Conversely, the basement membranes from the affected Bull terrier cornea, lens capsule, retina, skin, lung, and muscle had a normal ultrastructural appearance and were not thickened compared with membranes in normal age-matched dogs. The distribution of basement membrane abnormalities in Bull terrier hereditary nephritis may occur because the defective protein is present exclusively or more abundantly in the kidney and is structurally more important in the kidney or because of local intrarenal stresses. PMID:10922317

  2. Antiglomerular basement membrane antibody-crescentic glomerulonephritis complicating chronic bronchiectasis.

    Science.gov (United States)

    Enríquez, R; Cabezuelo, J B; Sirvent, A E; Andrada, E; Amorós, F; Orti, C

    2001-04-01

    A 68-year-old woman with chronic bronchiectasis presented with haematuria and severe oligoanuric renal failure with no other serious systemic manifestation. Antiglomerular basement membrane (anti-GBM) antibodies and anti-myeloperoxidase antibodies were positive. Renal biopsy revealed anti-GBM crescentic glomerulonephritis. A conservative approach was followed and the patient is stable on chronic haemodialysis 6 months later. To the authors' knowledge, there has only been one previous report of anti-GBM disease complicating bronchiectasis.

  3. Identification of the cutaneous basement membrane zone antigen and isolation of antibody in linear immunoglobulin A bullous dermatosis.

    OpenAIRE

    Zone, J J; Taylor, T B; Kadunce, D P; Meyer, L J

    1990-01-01

    Linear IgA bullous dermatosis (LABD) is a rare blistering skin disease characterized by basement membrane zone deposition of IgA. This study identifies a tissue antigen detected by patient serum and then isolates the autoantibody using epidermis and protein bands blotted on nitrocellulose as immunoabsorbents. Sera from 10 patients (9 with cutaneous disease and 1 with cicatrizing conjunctivitis) were evaluated. Indirect immunofluorescence revealed an IgA anti-basement membrane antibody in 6 of...

  4. Autoantibodies against basement membrane collagen type IV are associated with myocardial infarction

    OpenAIRE

    Olga McLeod; Pontus Dunér; Ann Samnegård; Per Tornvall; Jan Nilsson; Anders Hamsten; Eva Bengtsson

    2015-01-01

    Background: Collagen type IV is the major constituent of basement membranes underlying endothelial cells and is important for endothelial cell attachment and function. Autoantibodies against native collagen type IV have been found in various autoimmune diseases. Oxidation of LDL in the vascular wall results in the formation of reactive aldehydes, which could modify surrounding matrix proteins. Like oxidized LDL, these modified matrix proteins are likely to induce immune responses. We examined...

  5. Effects of radiation on the permeability of human basement membranes

    Science.gov (United States)

    Fan, B.-T.; Achour, S.; Simmonet, F.; Guerin, D.

    1999-02-01

    The influence of radiation on the permeability properties of human basement membrane was investigated by measuring the diffusion rate of several organic compounds (glycine, proline, glucose, urea and insulin) through human anterior lens capsules. The basement membranes borne an γ-irradiation treatment change significantly their permeability vis-a-vis studied organic substances. This modification in physico-chemical properties is probably due to the radiation, which alters or degrades the complex structure (or architecture) of basement membranes. Moreover the change in permeability is dependent upon the diffusing compounds. An increase in diffusion has been observed for glucose, glycine and urea. However for insulin and proline, a decrease in diffusion rate was observed. L'influence de radiation sur la perméabilité de la membrane basale a été étudiée par la mesure de la vitesse de diffusion de plusieurs composés organiques d'intérêt biologique (glycine, proline, glucose, urée et insuline) à travers la lame basale antérieure du cristallin de l'oil humain. Les membranes basales qui sont traitées avec l'irradiation γ changent significativement leur perméabilité vis-à-vis des substances organiques. Ce changement de propriétés physico-chimiques est probablement dû à l'altération ou la dégradation de la structure (ou de l'architecture) de la membrane basale entraînée par l'irradiation. De plus, la modification de la perméabilité de la membrane basale est dépendante des composés diffusants. Une augmentation de la vitesse de diffusion a été observée pour le glucose, le glycine et l'urée. Par contre, dans les cas de l'insuline et de la proline, on a observé une diminution de la vitesse de diffusion.

  6. Basement membrane changes in capillaries of the ageing human retina

    Science.gov (United States)

    Powner, Michael B; Scott, Andrew; Zhu, Meidong; Munro, Peter M G; Foss, Alexander J E; Hageman, Gregory S; Gillies, Mark C; Fruttiger, Marcus

    2014-01-01

    Objectives The ultrastructural appearance of retinal capillaries can yield important information about disease mechanisms, but is not well characterised in human post mortem samples. We therefore aimed to create a baseline for the appearance of capillaries and establish how this is influenced by post mortem fixation delays and donor age. Methods Electron microscopy was used to characterise retinal capillaries in 20 anonymous donors (with no known eye diseases) of various ages and with various post mortem fixation delays. In addition, samples from six patients with conditions that are known to affect the retinal vasculature (four cases of type 2 diabetes without diabetic retinopathy, one case of diabetic retinopathy and one case of macular telangiectasia type 2) were analysed. Results Vacuoles were found in capillary basement membranes at the vessel—glia interface in all samples, from both the normal and disease cases. Vacuole frequency increased with donor age but was not influenced by post mortem fixation delays. Conclusion Vacuoles in the basement membrane are a normal feature of adult human retinal capillaries and do not indicate disease. Their incidence increases with age and might be a contributing factor to late-onset pathologies of the retinal vasculature. PMID:21606466

  7. Immunohistochemical localization of basement membrane components during hair follicle morphogenesis

    DEFF Research Database (Denmark)

    Westgate, G E; Shaw, D A; Harrap, G J;

    1984-01-01

    Specific antisera were used to investigate the distributions of several basement membrane zone (BMZ) components, namely, bullous pemphigoid antigen (BPA), heparan sulfate proteoglycan (HSPG), laminin, and type IV collagen, during the development of hair follicles in late embryo rats. BPA was not ......Specific antisera were used to investigate the distributions of several basement membrane zone (BMZ) components, namely, bullous pemphigoid antigen (BPA), heparan sulfate proteoglycan (HSPG), laminin, and type IV collagen, during the development of hair follicles in late embryo rats. BPA...... of the elongating follicle. HSPG was associated with the basal cell layer prior to the appearance of hair follicle primordia and became BMZ-associated before birth but after follicle buds were first observed. HSPG was also found to be associated with the basal cell surfaces in the epidermis, but not in the hair...... follicle. Laminin and type IV collagen were continually present in epidermal and follicular BMZ both before and during development of hair follicles and were later present in the dermal papilla matrix. From these observations we conclude that (1) laminin and type IV collagen are functionally important...

  8. Basement membrane abnormalities in human eyes with diabetic retinopathy

    DEFF Research Database (Denmark)

    Ljubimov, A V; Burgeson, R E; Butkowski, R J;

    1996-01-01

    Vascular and parenchymal basement membranes (BMs) are thickened in diabetes, but alterations in individual BM components in diabetic eyes, especially in diabetic retinopathy (DR), are obscure. To identify abnormalities in the distribution of specific constituents, we analyzed cryostat sections...... of human eyes obtained at autopsy (seven normal, five diabetic without DR, and 13 diabetic with DR) by immunofluorescence with antibodies to 30 BM and extracellular matrix components. In non-DR eyes, no qualitative changes of ocular BM components were seen. In some DR corneas, epithelial BM was stained...... discontinuously for laminin-1, entactin/nidogen, and alpha3-alpha4 Type IV collagen, in contrast to non-DR corneas. Major BM alterations were found in DR retinas compared to normals and non-DR diabetics. The inner limiting membrane (retinal BM) of DR eyes had accumulations of fibronectin (including cellular...

  9. Basement membrane heparan sulfate proteoglycan from the L2 rat yolk sac carcinoma

    DEFF Research Database (Denmark)

    Fenger, M; Wewer, U; Albrechtsen, R

    1984-01-01

    Heparan sulfate proteoglycan from the L2 rat yolk sac carcinoma has been purified and partially characterized. The proteoglycan has an apparent Mr of 750 000, 35% of which represents the core protein. The core protein seems to be homogeneous, whereas the heparan sulfate chains are heterogeneous w...... with an Mr of about 50 000-70 000, with 30% of the glucosamine being N-sulfated. Antibodies raised against the core protein of the heparan sulfate proteoglycan reacted with basement membranes of various rat and human tissue....

  10. Laminin isoforms in endothelial and perivascular basement membranes

    Science.gov (United States)

    Yousif, Lema F.; Di Russo, Jacopo; Sorokin, Lydia

    2013-01-01

    Laminins, one of the major functional components of basement membranes, are found underlying endothelium, and encasing pericytes and smooth muscle cells in the vessel wall. Depending on the type of blood vessel (capillary, venule, postcapillary venule, vein or artery) and their maturation state, both the endothelial and mural cell phenotype vary, with associated changes in laminin isoform expression. Laminins containing the α4 and α5 chains are the major isoforms found in the vessel wall, with the added contribution of laminin α2 in larger vessels. We here summarize current data on the precise localization of these laminin isoforms and their receptors in the different layers of the vessel wall, and their potential contribution to vascular homeostasis. PMID:23263631

  11. The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini.

    Directory of Open Access Journals (Sweden)

    Aljona Gaiko-Shcherbak

    Full Text Available The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function.

  12. Production of monoclonal antibodies to human glomerular basement membrane.

    Directory of Open Access Journals (Sweden)

    Mino,Yasuaki

    1984-10-01

    Full Text Available Using the technique of somatic cell fusion, we produced monoclonal antibodies to collagenase-digested human glomerular basement membrane (GBM. Fourteen monoclonal antibodies which reacted with normal human kidney in indirect immunofluorescence (IIF studies were produced. An analysis of the binding patterns indicated that the antigens recognized could be divided into six broad groups. Monoclonal antibody B3-H10 (Group 1 reacted with only GBM in a fine granular pattern. A5-B12 and B5-C2 (Group 2 reacted with GBM and peritubular capillary in a linear pattern. B2-A12 (Group 3 reacted with only epithelial cells. Al-C9 and A4-E2 (Group 4 showed a mesangial pattern in glomerulus and a lineal pattern in tubular basement membrane (TBM, Bowman's capsule and peritubular capillary. A1-E1, A1-E11, A2-E6, A3-B6, A4-F8 and B5-H2 (Group 5 recognized determinants common to GBM, TBM, Bowman's capsule and/or peritubular capillary. A3-F1 and B5-E10 (Group 6 reacted with TBM and Bowman's capsule. The staining pattern of B3-H10 (Group 1 was characteristic because it was not linear, but finely granular along the GBM. The staining pattern of B2-A12 (Group 3 was also characteristic because only epithelial cells were stained, and processes of epithelial cells were observed as fine fibrils. To the best of our knowledge, these two types of monoclonal antibodies have not been reported previously.

  13. Enhanced assembly of basement membrane matrix by endodermal cells in response to fibronectin substrata

    DEFF Research Database (Denmark)

    Austria, M R; Couchman, J R

    1991-01-01

    Basement membranes are complex extracellular matrices contributing to the regulation of growth, migration and differentiation of many cell types. However, little is known about the mechanisms regulating the deposition and assembly of basement membrane from its constituents. We have investigated t...

  14. Fluid Mechanics of the Vascular Basement Membrane in the Brain

    Science.gov (United States)

    Coloma, Mikhail; Hui, Jonathan; Chiarot, Paul; Huang, Peter; Carare, Roxana; McLeod, Kenneth; Schaffer, David

    2013-11-01

    Beta-amyloid is a normal product of brain metabolic function and is found within the interstitial fluid of the brain. Failure of the clearance of beta-amyloid from the aging brain leads to its accumulation within the walls of arteries and to Alzheimer's disease. The vascular basement membrane (VBM) within the walls of cerebral arteries surrounds the spirally arranged smooth muscle cells and represents an essential pathway for removal of beta-amyloid from the brain. This process fails with the stiffening of arterial walls associated with aging. In this study we hypothesize that the deformation of the VBM associated with arterial pulsations drives the interstitial fluid to drain in the direction opposite of the arterial blood flow. This hypothesis is theoretically investigated by modeling the VBM as a thin, coaxial, fluid-filled porous medium surrounding a periodically deforming cylindrical tube. Flow and boundary conditions required to achieve such a backward clearance are derived through a control volume analysis of mass, momentum, and energy.

  15. The vascular basement membrane as "soil" in brain metastasis.

    Directory of Open Access Journals (Sweden)

    W Shawn Carbonell

    Full Text Available Brain-specific homing and direct interactions with the neural substance are prominent hypotheses for brain metastasis formation and a modern manifestation of Paget's "seed and soil" concept. However, there is little direct evidence for this "neurotropic" growth in vivo. In contrast, many experimental studies have anecdotally noted the propensity of metastatic cells to grow along the exterior of pre-existing vessels of the CNS, a process termed vascular cooption. These observations suggest the "soil" for malignant cells in the CNS may well be vascular, rather than neuronal. We used in vivo experimental models of brain metastasis and analysis of human clinical specimens to test this hypothesis. Indeed, over 95% of early micrometastases examined demonstrated vascular cooption with little evidence for isolated neurotropic growth. This vessel interaction was adhesive in nature implicating the vascular basement membrane (VBM as the active substrate for tumor cell growth in the brain. Accordingly, VBM promoted adhesion and invasion of malignant cells and was sufficient for tumor growth prior to any evidence of angiogenesis. Blockade or loss of the beta1 integrin subunit in tumor cells prevented adhesion to VBM and attenuated metastasis establishment and growth in vivo. Our data establishes a new understanding of CNS metastasis formation and identifies the neurovasculature as the critical partner for such growth. Further, we have elucidated the mechanism of vascular cooption for the first time. These findings may help inform the design of effective molecular therapies for patients with fatal CNS malignancies.

  16. Type IV Collagens and Basement Membrane Diseases: Cell Biology and Pathogenic Mechanisms.

    Science.gov (United States)

    Mao, Mao; Alavi, Marcel V; Labelle-Dumais, Cassandre; Gould, Douglas B

    2015-01-01

    Basement membranes are highly specialized extracellular matrices. Once considered inert scaffolds, basement membranes are now viewed as dynamic and versatile environments that modulate cellular behaviors to regulate tissue development, function, and repair. Increasing evidence suggests that, in addition to providing structural support to neighboring cells, basement membranes serve as reservoirs of growth factors that direct and fine-tune cellular functions. Type IV collagens are a major component of all basement membranes. They evolved along with the earliest multicellular organisms and have been integrated into diverse fundamental biological processes as time and evolution shaped the animal kingdom. The roles of basement membranes in humans are as complex and diverse as their distributions and molecular composition. As a result, basement membrane defects result in multisystem disorders with ambiguous and overlapping boundaries that likely reflect the simultaneous interplay and integration of multiple cellular pathways and processes. Consequently, there will be no single treatment for basement membrane disorders, and therapies are likely to be as varied as the phenotypes. Understanding tissue-specific pathology and the underlying molecular mechanism is the present challenge; personalized medicine will rely upon understanding how a given mutation impacts diverse cellular functions.

  17. cDNA cloning of the basement membrane chondroitin sulfate proteoglycan core protein, bamacan: a five domain structure including coiled-coil motifs

    DEFF Research Database (Denmark)

    Wu, R R; Couchman, J R

    1997-01-01

    . The protein sequence has low overall homology, apart from very small NH2- and COOH-terminal motifs. At the junctions between the distal globular domains and the coiled-coil regions lie glycosylation sites, with up to three N-linked oligosaccharides and probably three chondroitin chains. Three other Ser...

  18. Quantitative Proteome Analysis Reveals Increased Content of Basement Membrane Proteins in Arteries from Patients with Type 2 Diabetes and Lower Levels among Metformin Users

    DEFF Research Database (Denmark)

    Rørdam Preil, Simone; Kristensen, Lars P; Beck, Hans C;

    2015-01-01

    BACKGROUND: -The increased risk of cardiovascular diseases (CVD) in type 2 diabetes has been extensively documented, but the origins of the association remain largely unknown. We sought to determine changes in protein expressions in arterial tissue from patients with type 2 diabetes and moreover...

  19. Anti-glomerular basement membrane disease superimposed on membranous nephropathy: a case report and review of the literature

    Directory of Open Access Journals (Sweden)

    Nivera Noel

    2010-08-01

    Full Text Available Abstract Introduction Anti-glomerular basement membrane disease is a rare autoimmune disorder characterized by pulmonary hemorrhage, crescentic glomerulonephritis and the presence of circulating anti-glomerular basement membrane antibodies. The simultaneous occurrence of both anti-glomerular basement membrane disease and membranous nephropathy is rare. Case presentation A 59-year-old Hispanic man presented with acute onset of nausea and vomiting and was found to have renal insufficiency. Work-up included a kidney biopsy, which revealed anti-glomerular basement membrane disease with underlying membranous nephropathy. He was treated with emergent hemodialysis, intravenous corticosteroids, plasmapheresis, and cyclophosphamide without improvement in his renal function. Conclusion Simultaneous anti-glomerular basement membrane disease and membranous nephropathy is very rare. There have been 16 previous case reports in the English language literature that have been associated with a high mortality and morbidity, and a very high rate of renal failure resulting in hemodialysis. Co-existence of membranous nephropathy and anti-glomerular basement membrane disease may be immune-mediated, although the exact mechanism is not clear.

  20. Remodeling of basement membrane in patients with asthma.

    Science.gov (United States)

    Grigoraş, Adriana; Grigoraş, Constantin Cristian; Giuşcă, Simona Eliza; Căruntu, Irina Draga; Amălinei, Cornelia

    2016-01-01

    The "bronchial remodeling" specific for the asthmatic disease consists in irreversible changes of the bronchial wall, including glandular and smooth muscle fibers hyperplasia and÷or hypertrophy, goblet cells hyperplasia, and thickening of basement membrane (BM). We aimed to analyze the BM thickness in asthma patients, in order to validate the relationship between its changes and the disease severity defined in agreement with the Global Initiative for Asthma (GINA) criteria. The study group has been formed of 38 patients with different degrees of severity of asthma established by spirometry using Forced Expiratory Volume in one second (FEV1), and two patients without asthma symptoms as controls. The specimens harvested by fibrobronchoscopy have been processed by paraffin embedding followed by Hematoxylin-Eosin (HE) and Periodic Acid-Schiff (PAS) staining. For each case, the BM measurement has been realized by a "point-by-point" method. Statistical analysis has been performed using SPSS 17 software, by applying non-parametric correlation tests. The quantitative assessment revealed a progressive increase in BM thickness during the course of the disease, from a mean value of 11.2 μm in stage 1 to that of 15.6 μm in stage 4. Even if this process has been noticed starting with the first stage of asthma, the differences in the BM size were statistically significant only for stages 1 and 3 (p=0.047), stages 1 and 4 (p=0.000), stages 2 and 3 (p=0.000), and stages 3 and 4 (p=0.000). Spearman's test has shown an opposite correlation between the BM thickness and asthma severity defined by FEV1 values (r=-0.86, pasthma and continues in a progressive modality, the BM thickening being correlated with the disease severity. Thus, we support the concept of biological consequences of BM thickening in asthma pathogenesis, a mechanism still incompletely deciphered. PMID:27151696

  1. Pericapillary basement membrane thickening in human skeletal muscles.

    Science.gov (United States)

    Baum, Oliver; Bigler, Marius

    2016-09-01

    The basement membrane (BM) surrounding capillaries in skeletal muscles varies physiologically in thickness according to age, physical fitness, and anatomical site in humans. Furthermore, the pericapillary BM thickness (CBMT) increases pathophysiologically during several common disease states, including peripheral arterial disease and diabetes mellitus. This review on CBM thickening in human skeletal muscles is two pronged. First, it addresses the advantages/disadvantages of grid- and tablet-based measuring and morphometric techniques that are implemented to assess the CBMT on transmission electron micrographs. Second, it deals with the biology of CBM thickening in skeletal muscles, particularly its possible causes, molecular mechanisms, and functional impact. CBM thickening is triggered by several physical factors, including diabetes-associated glycation, hydrostatic pressure, and inflammation. Increased biosynthesis of type IV collagen expression or repetitive cycles in pericyte or endothelial cell degeneration/proliferation appear to be most critical for CBM accumulation. A thickened CBM obviously poses a greater barrier for diffusion, lowers the microvascular elasticity, and impedes transcytosis of inflammatory cells. Our own morphometric data reveal the CBM enlargement to be not accompanied by the pericyte coverage. Owing to an overlap or redundancy in the capillary supply, CBM thickening in skeletal muscles might not be such a devastating occurrence as in organs with endarterial circulation (e.g., kidney and retina). CBM growth in skeletal muscles can be reversed by training or administration of antidiabetic drugs. In conclusion, CBM thickening in skeletal muscles is a microvascular remodeling process by which metabolic, hemodynamic, and inflammatory forces are integrated together and which could play a hitherto underestimated role in etiology/progression of human diseases.

  2. Expression of basement membrane components through morphological changes in the hair growth cycle

    DEFF Research Database (Denmark)

    Couchman, J R; Gibson, W T

    1985-01-01

    The amount and distribution of fibronectin associated with hair follicles was found to vary during the hair growth cycle in the rat. Immunocytochemical staining of follicles in mid-late anagen (the growth stage) revealed the presence of fibronectin in the dermal papilla matrix, in the basement...... membrane separating this from the epithelial cells of the hair bulb, and in the basement membrane and connective tissue sheath which underly the cells of the outer root sheath. Early in catagen, the transitional stage, staining of the dermal papilla matrix disappeared. Fibronectin persisted in the basement...

  3. A Review on the Potential Role of Basement Membrane Laminin in the Pathogenesis of Psoriasis.

    Science.gov (United States)

    McFadden, J P; Kimber, I

    2016-01-01

    We have previously reviewed alterations to basement membrane laminin in psoriasis and how disruption of this layer could lead to at least some of the pathological changes observed. We here postulate that basement membrane laminin is the key antigen in driving psoriasis, inducing a T cell-mediated autoimmune response. For laminin to be considered as the key autoantigen in psoriasis, it would be reasonable to expect the following to be demonstrable: (1) that autoantigens are present in psoriatic inflammation; (2) that basement membrane laminin is perturbed in involved and uninvolved skin, and that some of the pathological changes associated with psoriasis could be predicted as a sequel to this; (3) that disruption of the basement membrane is among the earliest events in the evolution of psoriatic lesions; (4) that as streptococcal pharyngitis is the most clearly defined event to trigger or exacerbate psoriasis, then a T cell-mediated autoimmune response to laminin should be anticipated as a potential sequelae to streptococcal pharyngitis; (5) that T cells in psoriasis can be shown to react to peptides with homology to laminin; (6) that HLACw6, as the most closely related gene associated with psoriasis and which is involved in antigen expression, should be preferentially expressed within lesional psoriasis towards the basement membrane, together with other proximal associated immune activity; and (7) that there is some association between antilaminin pemphigoid, a humorally mediated autoimmune disease to skin basement membrane laminin, and psoriasis. We here review the data relevant to each of these requirements.

  4. Pericytes regulate vascular basement membrane remodeling and govern neutrophil extravasation during inflammation.

    Directory of Open Access Journals (Sweden)

    Shijun Wang

    Full Text Available During inflammation polymorphonuclear neutrophils (PMNs traverse venular walls, composed of the endothelium, pericyte sheath and vascular basement membrane. Compared to PMN transendothelial migration, little is known about how PMNs penetrate the latter barriers. Using mouse models and intravital microscopy, we show that migrating PMNs expand and use the low expression regions (LERs of matrix proteins in the vascular basement membrane (BM for their transmigration. Importantly, we demonstrate that this remodeling of LERs is accompanied by the opening of gaps between pericytes, a response that depends on PMN engagement with pericytes. Exploring how PMNs modulate pericyte behavior, we discovered that direct PMN-pericyte contacts induce relaxation rather than contraction of pericyte cytoskeletons, an unexpected response that is mediated by inhibition of the RhoA/ROCK signaling pathway in pericytes. Taking our in vitro results back into mouse models, we present evidence that pericyte relaxation contributes to the opening of the gaps between pericytes and to the enlargement of the LERs in the vascular BM, facilitating PMN extravasation. Our study demonstrates that pericytes can regulate PMN extravasation by controlling the size of pericyte gaps and thickness of LERs in venular walls. This raises the possibility that pericytes may be targeted in therapies aimed at regulating inflammation.

  5. Vascular basement membranes as pathways for the passage of fluid into and out of the brain.

    Science.gov (United States)

    Morris, Alan W J; Sharp, Matthew MacGregor; Albargothy, Nazira J; Fernandes, Rute; Hawkes, Cheryl A; Verma, Ajay; Weller, Roy O; Carare, Roxana O

    2016-05-01

    In the absence of conventional lymphatics, drainage of interstitial fluid and solutes from the brain parenchyma to cervical lymph nodes is along basement membranes in the walls of cerebral capillaries and tunica media of arteries. Perivascular pathways are also involved in the entry of CSF into the brain by the convective influx/glymphatic system. The objective of this study is to differentiate the cerebral vascular basement membrane pathways by which fluid passes out of the brain from the pathway by which CSF enters the brain. Experiment 1: 0.5 µl of soluble biotinylated or fluorescent Aβ, or 1 µl 15 nm gold nanoparticles was injected into the mouse hippocampus and their distributions determined at 5 min by transmission electron microscopy. Aβ was distributed within the extracellular spaces of the hippocampus and within basement membranes of capillaries and tunica media of arteries. Nanoparticles did not enter capillary basement membranes from the extracellular spaces. Experiment 2: 2 µl of 15 nm nanoparticles were injected into mouse CSF. Within 5min, groups of nanoparticles were present in the pial-glial basement membrane on the outer aspect of cortical arteries between the investing layer of pia mater and the glia limitans. The results of this study and previous research suggest that cerebral vascular basement membranes form the pathways by which fluid passes into and out of the brain but that different basement membrane layers are involved. The significance of these findings for neuroimmunology, Alzheimer's disease, drug delivery to the brain and the concept of the Virchow-Robin space are discussed. PMID:26975356

  6. Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa

    OpenAIRE

    Watt, Stephen A; Dayal, Jasbani H.S; Wright, Sheila; Riddle, Megan; Pourreyron, Celine; McMillan, James R.; Kimble, Roy M; Prisco, Marco; Gartner, Ulrike; Warbrick, Emma; McLean, W H Irwin; Leigh, Irene M.; McGrath, John A.; Salas-Alanis, Julio C; Tolar, Jakub

    2015-01-01

    Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstra...

  7. Changes in the molecular sieve of glomerular basement membrane in rats with aminonucleoside nephrosis.

    Directory of Open Access Journals (Sweden)

    Takaya,Yasumasa

    1980-02-01

    Full Text Available Isolated and purified glomerular basement membranes (GBM of normal and aminonucleoside (PAN nephrosis rats were observed by electron microscopy after negative staining. Although GBM of normal rats appeared as a molecular sieve with uniform pores, GBM of nephrotic rats showed enlargement and elongation of the pores. For an average of fifty pores, the long dimension was 40.4+/-10.7 A and the short dimension 13.8+/-3.6 A in nephrosis whereas the long dimension was 12.3+/-2.5 A and the short dimension 8.4+/-1.0 A in normal rats. Changes in the pores in GBM were thought to result in increased permeability of serum protein and hence proteinuria.

  8. Molecular sieve of the rat glomerular basement membrane: a transmission electron microscopic study of enzyme-treated specimens.

    Directory of Open Access Journals (Sweden)

    Ichiyasu,Akira

    1988-12-01

    Full Text Available Isolated rat glomerular basement membrane was treated with elastase and observed by transmission electron microscopy. The treatment with elastase revealed the fundamental structure of the glomerular basement membrane quite clearly, and enabled the observation of a sieve structure within the glomerular basement membrane. This sieve structure may play a major role in the filtration of blood as well as in the production of urine. Treatment with antibody showed that the sieve was mainly constituted of type IV collagen.

  9. Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane

    Energy Technology Data Exchange (ETDEWEB)

    BARCELLOS-HOFF, M. H; AGGELER, J.; RAM, T. G; BISSELL, M. J

    1989-02-01

    An essential feature of mammary gland differentiation during pregnancy is the formation of alveoli composed of polarized epithelial cells, which, under the influence of lactogenic hormones, secrete vectorially and sequester milk proteins. Previous culture studies have described either organization of cells polarized towards lumina containing little or no demonstrable tissue-specific protein, or establishment of functional secretory cells exhibiting little or no glandular architecture. In this paper, we report that tissue-specific vectorial secretion coincides with the formation of functional alveoli-like structures by primary mammary epithelial cells cultured on a reconstituted basement membrane matrix (derived from Engelbreth-Holm-Swarm murine tumour). Morphogenesis of these unique three-dimensional structures was initiated by cell-directed remodelling of the exogenous matrix leading to reorganization of cells into matrixensheathed aggregates by 24 h after plating. The aggregates subsequently cavitated, so that by day 6 the cells were organized into hollow spheres in which apical cell surfaces faced lumina sealed by tight junctions and basal surfaces were surrounded by a distinct basal lamina. The profiles of proteins secreted into the apical (luminal) and basal (medium) compartments indicated that these alveoli-like structures were capable of an appreciable amount of vectorial secretion. Immunoprecipitation with a broad spectrum milk antiserum showed that more than 80% of caseins were secreted into the lumina, whereas iron-binding proteins (both lactoferrin and transferrin) were present in comparable amounts in each compartment. Thus, these mammary cells established protein targeting pathways directing milk-specific proteins to the luminal compartment. A time course monitoring secretory activity demonstrated that establishment of tissue-specific vectorial secretion and increased total and milk protein secretion coincided with functional alveolar

  10. Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa.

    Directory of Open Access Journals (Sweden)

    Stephen A Watt

    Full Text Available Recessive dystrophic epidermolysis bullosa (RDEB is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3, in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention.

  11. Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa.

    Science.gov (United States)

    Watt, Stephen A; Dayal, Jasbani H S; Wright, Sheila; Riddle, Megan; Pourreyron, Celine; McMillan, James R; Kimble, Roy M; Prisco, Marco; Gartner, Ulrike; Warbrick, Emma; McLean, W H Irwin; Leigh, Irene M; McGrath, John A; Salas-Alanis, Julio C; Tolar, Jakub; South, Andrew P

    2015-01-01

    Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention. PMID:26380979

  12. Development and heterogeneity of antigens in the immature nephron. Reactivity with human antiglomerular basement membrane autoantibodies.

    OpenAIRE

    Jeraj, K.; Fish, A. J.; Yoshioka, K; Michael, A. F.

    1984-01-01

    Indirect immunofluorescence microscopy was performed with 15 human anti-glomerular basement membrane (GBM) antibodies and mouse monoclonal antibodies to Type IV collagen (MBM4) and renal basement membranes (MBM15) on renal tissue from 6 fetuses (gestational age, 15-23 weeks), 8 infants (age, 1-21 days), and 8 children and adults (ages, 3-27 years). Of the 15 human anti-GBM antibodies that react with GBM in adult glomeruli, only 4 identified antigens in the GBM of fetal and infant glomeruli. I...

  13. Rat hair follicle dermal papillae have an extracellular matrix containing basement membrane components

    DEFF Research Database (Denmark)

    Couchman, J R

    1986-01-01

    Dermal papillae are small mesenchymally derived zones at the bases of hair follicles which have an important role in hair morphogenesis in the embryo and control of the hair growth cycle in postnatal mammals. The cells of the papilla are enmeshed in a dense extracellular matrix which undergoes...... extensive changes in concert with the hair cycle. Here it is shown that this matrix in anagen pelage follicles of postnatal rats contains an abundance of basement membrane components rather than dermal components such as interstitial collagens. In particular, type IV collagen, laminin, and basement membrane...

  14. Deposition of nucleosomal antigens (histones and DNA) in the epidermal basement membrane in human lupus nephritis.

    NARCIS (Netherlands)

    Grootscholten, C.; Bruggen, M.C.J. van; Pijl, J.W. van der; Jong, E.M.G.J. de; Ligtenberg, G.; Derksen, R.H.W.M.; Berden, J.H.M.

    2003-01-01

    OBJECTIVE: Antinuclear autoantibodies complexed to nucleosomes can bind to heparan sulfate (HS) in the glomerular basement membrane. This binding is due to the binding of the positively charged histones to the strongly anionic HS. Nucleosomes and histones have been identified in glomerular deposits

  15. Experimental orchitis induced in rats by passive transfer of an antiserum to seminiferous tubule basement membrane.

    Science.gov (United States)

    Lustig, L; Denduchis, B; González, N N; Puig, R P

    1978-09-01

    A multifocal damage of the testis was obtained when rats were injected intravenously or under the tunica albuginea of the testis with a rabbit antiseminiferous tubule basement membrane serum. The damage was characterized by foci of perivascular and peritubular infiltrates of mononuclear round cells, infolding, thickening, and rupture of the seminiferous tubular wall and different degrees of injury of the germinal epithelium such as, cell disorganization, cell sloughing, and atrophy. Delamination and thickening of seminiferous tubule basement membrane and vacuolization of the Sertoli cell cytoplasm was often observed by electron microscopy. A linear deposit of rabbit gamma-globulin was detected by immunohistochemical techniques along the basement membranes of the seminiferous tubules and vessels. Testicular damage was not detected in rats injected with normal rabbit serum, used as control. In the kidneys of rats injected intravenously with the immune serum, a deposit of rabbit gamma-globulin was detected along glomerular basement membrane. Focal areas of mononuclear cell infiltrates, hypercellularity of glomeruli and thickening of glomerular capillary walls and Bowman's capsule were also observed. PMID:367304

  16. Heparan sulfate proteoglycans made by different basement-membrane-producing tumors have immunological and structural similarities

    DEFF Research Database (Denmark)

    Wewer, U M; Albrechtsen, R; Hassell, J R

    1985-01-01

    Using immunological assays, we determined the relationship between the heparan sulfate proteoglycans produced by two different murine basement-membrane-producing tumors, i.e., the mouse Engelbreth-Holm-Swarm (EHS) tumor and the L2 rat yolk-sac tumor. Antibodies prepared against the heparan sulfat...

  17. Anti-DNA autoantibodies initiate experimental lupus nephritis by binding directly to the glomerular basement membrane in mice.

    Science.gov (United States)

    Krishnan, Meera R; Wang, Congmiao; Marion, Tony N

    2012-07-01

    The strongest serological correlate for lupus nephritis is antibody to double-stranded DNA, although the mechanism by which anti-DNA antibodies initiate lupus nephritis is unresolved. Most recent reports indicate that anti-DNA must bind chromatin in the glomerular basement membrane or mesangial matrix to form glomerular deposits. Here we determined whether direct binding of anti-DNA antibody to glomerular basement membrane is critical to initiate glomerular binding of anti-DNA in experimental lupus nephritis. Mice were co-injected with IgG monoclonal antibodies or hybridomas with similar specificity for DNA and chromatin but different IgG subclass and different relative affinity for basement membrane. Only anti-DNA antibodies that bound basement membrane bound to glomeruli, activated complement, and induced proteinuria whether injected alone or co-injected with a non-basement-membrane-binding anti-DNA antibody. Basement membrane-binding anti-DNA antibodies co-localized with heparan sulfate proteoglycan in glomerular basement membrane and mesangial matrix but not with chromatin. Thus, direct binding of anti-DNA antibody to antigens in the glomerular basement membrane or mesangial matrix may be critical to initiate glomerular inflammation. This may accelerate and exacerbate glomerular immune complex formation in human and murine lupus nephritis.

  18. Optically Highlighting Basement Membrane Components in C. elegans

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Elliott Hagedorn & David Sherwood ### Abstract Green fluorescent protein (GFP) and other genetically encoded fluorescent proteins provide a means to study gene expression pattern and protein localization in living tissues. Recently discovered GFP-like fluorophores and engineered variants have further expanded the fluorescent protein toolkit for in vivo imaging. Here we describe a technique using transgenic C. elegans that contain laminin or type IV collagen fused to the g...

  19. Permselectivity Replication of Artificial Glomerular Basement Membranes in Nanoporous Collagen Multilayers

    OpenAIRE

    Pullela, Srinivasa R.; Andres, Christine; Chen, Wei; Xu, Chuanlai; Wang, Libing; Kotov, Nicholas A.

    2011-01-01

    Basement membranes (BMs) play important roles in many biological functions such as tissue regeneration, cancer proliferation, nutrient/drug delivery, breathing, and many others. While there are many theoretical models, adequate experimental analogs of BMs describing basic physicochemical properties of BM, such as diffusion and permselectivity are not available. Taking BMs found in glomerulus of kidneys as an example, adequate reproduction of their permselectivity requires biomimetic membranes...

  20. Ultrastructure of basement membranes in monkey and shark teeth at an early stage of development.

    Science.gov (United States)

    Sawada, Takashi

    2003-12-01

    The basement membrane, which separates the inner enamel epithelium from the dental papilla in the early stages of tooth development, is known to play a significant role in odontogenesis. In this review article, this basement membrane was described in detail based on our recent findings with the use of high-resolution electron microscopy. Tooth germs of a monkey (Macaca fuscata) and a shark (Cephaloscyllium umbratile) were processed for thin-section observations. During the early stage of development, the basement membrane of the inner enamel (dental) epithelium was composed of a lamina lucida, lamina densa, and much wider lamina fibroreticularis. At higher magnification, the lamina densa in both species was made up of a fine network of cords, which are generally the main constituents of the basement membranes. In the monkey tooth, the lamina fibroreticularis was rich in fibrils, which were now characterized as basotubules, 10-nm-wide microfibril-like structures. The space between the basotubules was filled with a cord network that extended from the lamina densa. Dental papilla cell processes were inserted into the lamina fibroreticularis, and their surface was closely associated with numerous parallel basotubules via 1.5- to 3-nm-wide filaments. In the shark tooth during its early stage of development, the basotubules were absent in the lamina fibroreticularis and only narrow extensions, 60-90 nm wide and 1-2 microm long, of the cord network of the lamina densa were present. The dental papilla cells were immobilized by means of the binding of their processes to the extensions. These results indicate that basement membranes in both monkey and shark teeth at early stage of development are specialized for functions as anchoring and firm binding, which are essential for the successful differentiation of the odontoblasts.

  1. Hypoplastic basement membrane of the lens anlage in the inheritable lens aplastic mouse (lap mouse).

    Science.gov (United States)

    Aso, S; Baba, R; Noda, S; Ikuno, S; Fujita, M

    2000-04-01

    Adult homozygous lap mice show various eye abnormalities such as aphakia, retinal disorganization, and dysplasia of the cornea and anterior chamber. In the fetal eye of a homozygous lap mouse, the lens placode appears to develop normally. However, the lens vesicle develops abnormally to form a mass of cells without a cavity, and the mass vanishes soon afterward. Apoptotic cell death is associated with the disappearance of the lens anlage. We examined the basement membranes of the lens anlage of this mutant by immunohistochemical methods under light microscopy using antibodies against basement membrane components of the lens anlage, type IV collagen, fibronectin, laminin, heparan sulfate proteoglycan, and entactin and by transmission electron microscopy. Immunohistochemistry showed the distribution and intensity of antibody binding to the lens anlage to be almost the same for each these antibodies regardless of the stage of gestation or whether the anlagen were from normal BALB/c or lap mice. Thus, positive continuous reactions were observed around the exterior region of the lens anlage from day 10 of gestation for type IV collagen, fibronectin, laminin, heparan sulfate proteoglycan antibodies, and at least from day 11of gestation for entactin antibody. The basement membrane lamina densa of both normal and lap mice was shown by electron microscopy to be discontinuous at days 10 and 10.5 of gestation. However, by day 11 the lamina densa was continuous in the lens anlagen of normal mice but still discontinuous in the lap mice. By day 12 of gestation, the lamina densa had thickened markedly in normal mice, whereas in lap mice it remained discontinuous and its thinness indicated hypoplasia. These results indicate that, while all basement components examined are produced and deposited in the normal region of the lens anlage in the lap mouse, the basement membrane is, for some reason, imperfectly formed. The time at which hypoplasia of the basement membrane was observed

  2. Drugging Membrane Protein Interactions.

    Science.gov (United States)

    Yin, Hang; Flynn, Aaron D

    2016-07-11

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind cells to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally "undruggable" regions of membrane proteins, enabling modulation of protein-protein, protein-lipid, and protein-nucleic acid interactions. In this review, we survey the state of the art of high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  3. Accelerating repaired basement membrane after bevacizumab treatment on alkali-burned mouse cornea

    Directory of Open Access Journals (Sweden)

    Koon-Ja Lee

    2013-04-01

    Full Text Available To understand the corneal regeneration induced by bevacizumab,we investigated the structure changes of stroma andbasement membrane regeneration. A Stick soaked in 0.5 NNaOH onto the mouse cornea and 2.5 mg/ml of bevacizumabwas delivered into an alkali-burned cornea (2 μl by subconjunctivalinjections at 1 hour and 4 days after injury. At 7 daysafter injury, basement membrane regeneration was observedby transmission electron microscope. Uneven and thin epithelialbasement membrane, light density of hemidesmosomes,and edematous collagen fibril bundles are shown in thealkali-burned cornea. Injured epithelial basement membraneand hemidesmosomes and edematous collagen fibril bundlesresulting from alkali-burned mouse cornea was repaired bybevacizumab treatment. This study demonstrates that bevacizumabcan play an important role in wound healing in thecornea by accelerating the reestablishment of basementmembrane integrity that leads to barriers for scar formation.[BMB Reports 2013; 46(4: 195-200

  4. Attachment of cells to basement membrane collagen type IV

    OpenAIRE

    1986-01-01

    Of ten different cell lines examined, three showed distinct attachment and spreading on collagen IV substrates, and neither attachment nor spreading was enhanced by adding soluble laminin or fibronectin. This reaction was not inhibited by cycloheximide or antibodies to laminin, indicating a direct attachment to collagen IV without the need of mediator proteins. Cell-binding sites were localized to the major triple-helical domain of collagen IV and required an intact triple helical conformatio...

  5. The basement membrane constituents in the mouse embryo's tooth. An autoradiographic study

    International Nuclear Information System (INIS)

    Enamel organs isolated from the lower first teeth of 18-days old white mouse embryo by trypsin treatment were used in this study. The organs were cultured during periods of increasing time on a semi-solid medium containing cock serum. In another chase experiments, the organs were cultured on a liquid medium containing proline-3H, leucine-3H, and glucosamine-3H, were studied by autoradiography using both light and electron microscopes. It has been shown that the nature of the culture medium does not apparently interfere with the ability of the enamel to reconstitute the basement membrane. On the other hand, it have been found obvious differences concerning the kinetic of the used isotopes. The results indicate that the turn-over of the basement membrane constituents represents a continuous and homogenous process which continues to take place during, before and after reconstitution. 42 refs. (author)

  6. Basement membrane-specific chondroitin sulfate proteoglycan is abnormally associated with the glomerular capillary basement membrane of diabetic rats

    DEFF Research Database (Denmark)

    McCarthy, K J; Abrahamson, D R; Bynum, K R;

    1994-01-01

    of the pericapillary GBM affects the morphology of the capillary endothelial cells within these areas, directly displacing the cell body from the GBM proper and causing loss of fenestrae. These new data on BM-CSPG distribution reflect abnormal glomerular extracellular matrix protein biosynthesis/turnover in diabetes...

  7. Reticular basement membrane in asthma and COPD: Similar thickness, yet different composition

    OpenAIRE

    Jeroen JW Liesker; Ten Hacken, Nick H.; Mieke Zeinstra-Smith; Rutgers, Steven R; Dirkje S Postma; et al.

    2009-01-01

    Jeroen JW Liesker1, Nick H Ten Hacken1, Mieke Zeinstra-Smith2, Steven R Rutgers1, Dirkje S Postma1, Wim Timens21Department of Pulmonology; 2Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Background: Reticular basement membrane (RBM) thickening has been variably associated with asthma and chronic obstructive pulmonary disease (COPD). Even if RBM thickness is similar in both diseases, its composition might still differ. Objectiv...

  8. Antigens of the basement membranes of the seminiferous tubules induce autoimmunity in Wistar rats.

    Science.gov (United States)

    Lustig, L; Satz, M L; Sztein, M B; Denduchis, B

    1982-05-01

    A preparation enriched in basement membranes from seminiferous tubules was isolated from rat testes (STBM) and injected with complete Freund's adjuvant into Wistar rats. In 60% of animals a mild multifocal orchitis was observed. In damaged areas, perivascular and peritubular mononuclear cell infiltrates and different degrees of cell sloughing of some seminiferous tubules were observed. Electron microscopy revealed focal thickenings and delamination of the basement membrane of the seminiferous tubules as well as vacuolization of Sertoli cell cytoplasm. Using immunofluorescence discontinuous linear deposits of IgG were detected along the seminiferous tubular wall. Moreover, the same pattern of immunofluorescence was observed when the IgG eluted from the testes of the immunized rats was layered on sections of normal rat testis. Circulating antibodies to STBM were detected using passive haemagglutination in approximately 45% of the immunized rats, with titers ranging from 1:20 to 1:80. Leukocyte migration was inhibited when the spleen cells of the immunized rats were incubated with antigens from the basement membrane of seminiferous tubules, whilst a negative reaction was obtained when the soluble fraction of testis homogenate was used. PMID:7050376

  9. Tissue fibrocytes in patients with mild asthma: A possible link to thickness of reticular basement membrane?

    Directory of Open Access Journals (Sweden)

    Bjermer Leif

    2006-03-01

    Full Text Available Abstract Background Myofibroblasts, proposed as being derived from circulating fibrocytes, are considered to be important cells in thickening of the basement membrane in patients with asthma. We have studied the correlation of tissue fibrocyte levels to basement membrane thickness and the presence of fibrocytes in bronchoalveolar lavage fluid (BALF in steroid-naive patients with mild asthma and controls. Methods Patients with mild asthma (n = 9 were recruited and divided into two categories based on whether or not fibroblast-like cells could be established from BALF. Non-asthmatic healthy subjects (n = 5 were used as controls. Colocalization of the fibrocyte markers CD34, CD45RO, procollagen I, and α-smooth muscle actin (α-SMA were identified in bronchial biopsies from patients and controls by confocal microscopy. Kruskall-Wallis method was used to calculate statistical significance and Spearman coefficient of rank correlation was used to assess the degree of association. Results In patients with BALF fibroblasts, a 14-fold increase of tissue cells expressing CD34/CD45RO/α-SMA and a 16-fold increase of tissue cells expressing CD34/procollagen I was observed when compared to controls (p Conclusion These findings indicate a correlation between recruited fibrocytes in tissue and thickness of basement membrane. Fibroblast progenitor cells may therefore be important in airway remodeling in steroid-naive patients with mild asthma.

  10. VP08R from Infectious Spleen and Kidney Necrosis Virus Is a Novel Component of the Virus-Mock Basement Membrane

    OpenAIRE

    Xu, Xiaopeng; Yan, Muting; Wang, Rui; Lin, Ting; Tang, Junliang; Li, Chaozheng; Weng, Shaoping; He, Jian-Guo

    2014-01-01

    Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus, family Iridoviridae, brings great harm to fish farming. In infected tissues, ISKNV infection is characterized by a unique phenomenon, in that the infected cells are attached by lymphatic endothelial cells (LECs), which are speculated to wall off the infected cells from host immune attack. A viral membrane protein, VP23R, binds and recruits the host nidogen-1 protein to construct a basement memb...

  11. C3d fragment of complement interacts with laminin and binds to basement membranes of glomerulus and trophoblast

    OpenAIRE

    1986-01-01

    Two mouse monoclonal antibodies generated against human placental homogenate were found to react specifically with human complement component C3. In immunofluorescence of human tissues, these antibodies gave a bright linear staining outlining the glomerular basement membrane of the adult kidney and the trophoblast basement membrane of placenta. An identical staining pattern was observed with a rabbit C3d antiserum which also prevented binding of the monoclonal antibodies to tissue sections. O...

  12. Proteomic analysis of urinary exosomes from patients of early IgA nephropathy and thin basement membrane nephropathy.

    Science.gov (United States)

    Moon, Pyong-Gon; Lee, Jeong-Eun; You, Sungyong; Kim, Taek-Kyun; Cho, Ji-Hoon; Kim, In-San; Kwon, Tae-Hwan; Kim, Chan-Duck; Park, Sun-Hee; Hwang, Daehee; Kim, Yong-Lim; Baek, Moon-Chang

    2011-06-01

    To identify biomarker candidates associated with early IgA nephropathy (IgAN) and thin basement membrane nephropathy (TBMN), the most common causes presenting isolated hematuria in childhood, a proteomic approach of urinary exosomes from early IgAN and TBMN patients was introduced. The proteomic results from the patients were compared with a normal group to understand the pathophysiological processes associated with these diseases at the protein level. The urinary exosomes, which reflect pathophysiological processes, collected from three groups of young adults (early IgAN, TBMN, and normal) were trypsin-digested using a gel-assisted protocol, and quantified by label-free LC-MS/MS, using an MS(E) mode. A total of 1877 urinary exosome proteins, including cytoplasmic, membrane, and vesicle trafficking proteins, were identified. Among the differentially expressed proteins, four proteins (aminopeptidase N, vasorin precursor, α-1-antitrypsin, and ceruloplasmin) were selected as biomarker candidates to differentiate early IgAN from TBMN. We confirmed the protein levels of the four biomarker candidates by semi-quantitative immunoblot analysis in urinary exosomes independently prepared from other patients, including older adult groups. Further clinical studies are needed to investigate the diagnostic and prognostic value of these urinary markers for early IgAN and TBMN. Taken together, this study showed the possibility of identifying biomarker candidates for human urinary diseases using urinary exosomes and might help to understand the pathophysiology of early IgAN and TBMN at the protein level.

  13. Reticular basement membrane in asthma and COPD: Similar thickness, yet different composition

    Directory of Open Access Journals (Sweden)

    Jeroen JW Liesker

    2009-02-01

    Full Text Available Jeroen JW Liesker1, Nick H Ten Hacken1, Mieke Zeinstra-Smith2, Steven R Rutgers1, Dirkje S Postma1, Wim Timens21Department of Pulmonology; 2Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Background: Reticular basement membrane (RBM thickening has been variably associated with asthma and chronic obstructive pulmonary disease (COPD. Even if RBM thickness is similar in both diseases, its composition might still differ. Objective: To assess whether RBM thickness and composition differ between asthma and COPD. Methods: We investigated 24 allergic asthmatics (forced expiratory volume in one second [FEV1] 92% predicted, and 17 nonallergic COPD patients (FEV1 60% predicted, and for each group a control group of similar age and smoking habits (12 and 10 persons, respectively. Snap-frozen sections of bronchial biopsies were stained with hematoxylin/eosin and for collagen I, III, IV, V, laminin and tenascin. RBM thickening was assessed by digital image analysis. Relative staining intensity of each matrix component was determined.Results: Mean (SD RBM thickness was not significantly different between asthma and COPD 5.5 (1.3 vs 6.0 (1.8 μm, but significantly larger than in their healthy counterparts, ie, 4.7 (0.9 and 4.8 (1.2 μm, respectively. Collagen I and laminin stained significantly stronger in asthma than in COPD. Tenascin stained stronger in asthma than in healthy controls of similar age, and stronger in COPD controls than in asthma controls (p 0.05.Conclusion: RBM thickening occurs both in asthma and COPD. We provide supportive evidence that its composition differs in asthma and COPD. Keywords: reticular basement membrane thickness, reticular basement membrane composition, asthma, biopsy, COPD, remodeling

  14. Immunochemical and ultrastructural assessment of the nature of the pericellular basement membrane of human decidual cells

    DEFF Research Database (Denmark)

    Wewer, U M; Faber, M; Liotta, L A;

    1985-01-01

    Human decidual cells of early and late pregnancy were studied immunochemically and ultrastructurally with respect to the presence and nature of pericellular basement membrane material. The most prominent cell type in decidual tissue of both early and late pregnancy were large, mature epithelioid...... of stromal cells into decidual cells of the pregnant endometrium. Predecidualization of the human endometrium, which is seen in the late secretory phase of the normal menstrual cycle and in some states of hyperplasia, was also shown to be accompanied by the presence of deposits of laminin-positive material...

  15. Nephritogenic Lupus Antibodies Recognize Glomerular Basement Membrane-Associated Chromatin Fragments Released from Apoptotic Intraglomerular Cells

    Science.gov (United States)

    Kalaaji, Manar; Mortensen, Elin; Jørgensen, Leif; Olsen, Randi; Rekvig, Ole Petter

    2006-01-01

    Antibodies to dsDNA represent a classification criterion for systemic lupus erythematosus. Subpopulations of these antibodies are involved in lupus nephritis. No known marker separates nephritogenic from non-nephritogenic anti-dsDNA antibodies. It is not clear whether specificity for glomerular target antigens or intrinsic antibody-affinity for dsDNA or nucleosomes is a critical parameter. Furthermore, it is still controversial whether glomerular target antigen(s) is constituted by nucleosomes or by non-nucleosomal glomerular structures. Previously, we have demonstrated that antibodies eluted from murine nephritic kidneys recognize nucleosomes, but not other glomerular antigens. In this study, we determined the structures that bind nephritogenic autoantibodies in vivo by transmission electron microscopy, immune electron microscopy, and colocalization immune electron microscopy using experimental antibodies to dsDNA, to histones and transcription factors, or to laminin. The data obtained are consistent and point at glomerular basement membrane-associated nucleosomes as target structures for the nephritogenic autoantibodies. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling or caspase-3 assays demonstrate that lupus nephritis is linked to intraglomerular cell apoptosis. The data suggest that nucleosomes are released by apoptosis and associate with glomerulus basement membranes, which may then be targeted by pathogenic anti-nucleosome antibodies. Thus, apoptotic nucleosomes may represent both inducer and target structures for nephritogenic autoantibodies in systemic lupus erythematosus. PMID:16723695

  16. Anti-glomerular basement membrane glomerulonephritis and thrombotic microangiopathy in first degree relatives; a case report

    Directory of Open Access Journals (Sweden)

    Idorn Thomas

    2012-07-01

    Full Text Available Abstract Background Anti-glomerular basement membrane glomerulonephritis and thrombotic microangiopathy are rare diseases with no known coherence. Case Presentation A daughter and her biological mother were diagnosed with pregnancy-induced thrombotic microangiopathy and anti-glomerular basement membrane glomerulonephritis, respectively. Both developed end-stage renal disease. Exploration of a common aetiology included analyses of HLA genotypes, functional and genetic aspects of the complement system, ADAMTS13 activity and screening for autoantibodies. The daughter was heterozygous carrier of the complement factor I G261D mutation, previously described in patients with membranoproliferative glomerulonephritis and atypical haemolytic uremic syndrome. The mother was non-carrier of this mutation. They shared the disease associated complement factor H silent polymorphism Q672Q (79602A>G. Conclusion An unequivocal functional or molecular association between these two family cases was not found suggesting that the patients probably share another, so far undiagnosed and unknown, predisposing factor. It seems highly unlikely that two infrequent immunologic diseases would occur by unrelated pathophysiological mechanisms within first degree relatives.

  17. Mechanical Stretch on Human Skin Equivalents Increases the Epidermal Thickness and Develops the Basement Membrane.

    Directory of Open Access Journals (Sweden)

    Eijiro Tokuyama

    Full Text Available All previous reports concerning the effect of stretch on cultured skin cells dealt with experiments on epidermal keratinocytes or dermal fibroblasts alone. The aim of the present study was to develop a system that allows application of stretch stimuli to human skin equivalents (HSEs, prepared by coculturing of these two types of cells. In addition, this study aimed to analyze the effect of a stretch on keratinization of the epidermis and on the basement membrane. HSEs were prepared in a gutter-like structure created with a porous silicone sheet in a silicone chamber. After 5-day stimulation with stretching, HSEs were analyzed histologically and immunohistologically. Stretch-stimulated HSEs had a thicker epidermal layer and expressed significantly greater levels of laminin 5 and collagen IV/VII in the basal layer compared with HSEs not subjected to stretch stimulation. Transmission electron microscopy revealed that the structure of the basement membrane was more developed in HSEs subjected to stretching. Our model may be relevant for extrapolating the effect of a stretch on the skin in a state similar to an in vivo system. This experimental system may be useful for analysis of the effects of stretch stimuli on skin properties and wound healing and is also expected to be applicable to an in vitro model of a hypertrophic scar in the future.

  18. Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells.

    Science.gov (United States)

    Kalaaji, Manar; Mortensen, Elin; Jørgensen, Leif; Olsen, Randi; Rekvig, Ole Petter

    2006-06-01

    Antibodies to dsDNA represent a classification criterion for systemic lupus erythematosus. Subpopulations of these antibodies are involved in lupus nephritis. No known marker separates nephritogenic from non-nephritogenic anti-dsDNA antibodies. It is not clear whether specificity for glomerular target antigens or intrinsic antibody-affinity for dsDNA or nucleosomes is a critical parameter. Furthermore, it is still controversial whether glomerular target antigen(s) is constituted by nucleosomes or by non-nucleosomal glomerular structures. Previously, we have demonstrated that antibodies eluted from murine nephritic kidneys recognize nucleosomes, but not other glomerular antigens. In this study, we determined the structures that bind nephritogenic autoantibodies in vivo by transmission electron microscopy, immune electron microscopy, and colocalization immune electron microscopy using experimental antibodies to dsDNA, to histones and transcription factors, or to laminin. The data obtained are consistent and point at glomerular basement membrane-associated nucleosomes as target structures for the nephritogenic autoantibodies. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling or caspase-3 assays demonstrate that lupus nephritis is linked to intraglomerular cell apoptosis. The data suggest that nucleosomes are released by apoptosis and associate with glomerulus basement membranes, which may then be targeted by pathogenic anti-nucleosome antibodies. Thus, apoptotic nucleosomes may represent both inducer and target structures for nephritogenic autoantibodies in systemic lupus erythematosus.

  19. Laminin, a noncollagenous component of epithelial basement membranes synthesized by a rat yolk sac tumor

    DEFF Research Database (Denmark)

    Wewer, U; Albrechtsen, R; Ruoslahti, E

    1981-01-01

    Laminin, a glycoprotein antigenically similar or identical to a component of epithelial basement membranes, was identified as a major component of the abundant extracellular matrix synthesized by an experimentally induced rat yolk sac tumor. Immunocytochemical staining revealed laminin in cultured...... polypeptides with molecular weights of approximately 200,000 and 400,000. These comigrated with the polypeptides of mouse laminin isolated previously. The yolk sac tumor tissue grown in vivo contained laminin in the tumor cells and in the extracellular material as evidenced by immunofluorescence and...... membranes in rat tissues in a manner indistinguishable from antilaminin. The presence of laminin in rat yolk sac cells, the presumed origin of our yolk sac tumor, was studied in some detail. Laminin was found to be present in normal cells of the visceral as well as the parietal yolk sac layer and in their...

  20. Defective muscle basement membrane and lack of M-laminin in the dystrophic dy/dy mouse

    DEFF Research Database (Denmark)

    Xu, H; Christmas, P; Wu, X R;

    1994-01-01

    -linked Duchenne and Becker muscular dystrophies. We have examined M-laminin expression in mice with autosomal recessive muscular dystrophy caused by the mutation dy. The heavy chain of M-laminin was undetectable in skeletal muscle, heart muscle, and peripheral nerve by immunofluorescence and immunoblotting......M-laminin is a major member of the laminin family of basement membrane proteins. It is prominently expressed in striated muscle and peripheral nerve. M-laminin is deficient in patients with the autosomal recessive Fukuyama congenital muscular dystrophy but is normal in patients with the sex...... tissue from dy/dy mice, suggesting that M-laminin heavy-chain mRNA may be produced at very low levels or is unstable. Information about the chromosomal localization of the M heavy-chain in human and mouse suggests that a mutation in the M-chain gene causes the muscular dystrophy in dy/dy mice. The dy...

  1. Nephritogenic antigen determinants in epidermal and renal basement membranes of kindreds with Alport-type familial nephritis.

    OpenAIRE

    Kashtan, C; Fish, A. J.; Kleppel, M; Yoshioka, K; Michael, A. F.

    1986-01-01

    We probed epidermal basement membranes (EBM) of acid-urea denatured skin from members of kindreds with Alport-type familial nephritis (FN) for the presence of antigens reactive with Goodpasture sera (GPS) and serum (FNS) from an Alport patient who developed anti-glomerular basement membrane (GBM) nephritis in a renal allograft. By immunoblotting, GPS reacted primarily with the 28,000 molecular weight (mol wt) monomer but also the 24,000 mol wt and 26,000 mol wt monomers of the noncollagenous ...

  2. Basement membrane changes in breast cancer detected by immunohistochemical staining for laminin

    DEFF Research Database (Denmark)

    Albrechtsen, R; Nielsen, M; Wewer, U;

    1981-01-01

    The distribution of the basement membrane glycoprotein laminin was studied by the immunoperoxidase technique in benign and malignant human breast tissue and in axillary lymph nodes from patients with breast cancer. An antiserum prepared against rat laminin was used. The specificity...... with molecular weights of 400,000 and 200,000 of rat laminin in sodium dodecyl sulfate:polyacrylamide gel electrophoresis. The neoplastic cells in malignant breast tissues showed strong cytoplasmic staining for laminin, and a positive reaction was aslo found in lymph node metastases. In some cases in which only...... micrometastases were present, these cells also stained strongly for laminin. In nonmalignant breast tissues, the epithelial cells of the duct were positive for laminin, but the staining was weaker than in the carcinomas. Pretreatment of the fixed tissue sections with trypsin markedly enhanced the staining...

  3. An Overlapping Case of Alport Syndrome and Thin Basement Membrane Disease

    Science.gov (United States)

    Alganabi, Mashriq; Eter, Ahmad

    2016-01-01

    We report a case of a 48-year-old male who presented with hematuria of at least 10 years, and has a daughter with hematuria as well. The patient has a history of degenerative hearing loss, decreased vision and cataract formation, but no diabetes, hypertension or proteinuria. A full serology and urology workup was negative for any abnormality. A kidney biopsy for the patient revealed a diagnosis of Alport syndrome but was unable to rule out thin basement membrane disease. The biopsy was inconclusive in making the diagnosis but the patient’s clinical presentation led to the diagnosis of Alport syndrome. The patient’s 10-year-old daughter also has hematuria with no clear etiology but now can subsequently be anticipatorily managed for Alport syndrome progression. Due to the rarity of the disease, diagnosis is often missed or delayed by primary care providers especially when no associated proteinuria has yet developed. This can lead to confusion and misdiagnosis with thin basement membrane disease, a generally benign hematuria without kidney failure progression. Additionally, biopsy can be inconclusive in these patients, relying on the physician’s history and physical examination findings to diagnose. It is important to appropriately diagnose Alport syndrome not only to manage the patient’s rate of kidney failure progression but also allow for a higher degree of suspicion, screening and intervention in the patient’s family members. Both the inconclusive nature of kidney biopsies and the usefulness of diagnosis for family member screening are often overlooked in medical literature but are explored in this case.

  4. Erythrocyte membrane proteins and membrane skeleton

    Institute of Scientific and Technical Information of China (English)

    LU Yiqin; LIU Junfan

    2007-01-01

    Considerable advances in the research field of erythrocyte membrane were achieved in the recent two decades.New findings in the structure-function correlation and interactions of erythrocyte membrane proteins have attracted extensive attention.Interesting progress was also made in the molecular pathogenesis of erythrocyte membrane disorders.Advances in the composition,function and interaction of erythrocyte membrane proteins,erythrocyte membrane skeleton,and relevant diseases are briefly described and summarized here on the basis of domestic and world literatures.

  5. Basement membrane and interstitial proteoglycans produced by MDCK cells correspond to those expressed in the kidney cortex

    DEFF Research Database (Denmark)

    Erickson, A C; Couchman, J R

    2001-01-01

    Multiple proteoglycans (PGs) are present in all basement membranes (BM) and may contribute to their structure and function, but their effects on cell behavior are not well understood. Their postulated functions include: a structural role in maintaining tissue histoarchitecture, or aid in selectiv...

  6. Intercellular deposits of basement membrane material in active human pituitary adenomas detected by immunostaining for laminin and electron microscopy

    DEFF Research Database (Denmark)

    Holck, S; Wewer, U M; Albrechtsen, R

    1986-01-01

    and one patient with Cushing's syndrome). Concurrently, at the ultrastructural level, bunches of basement membrane-like material intermingled between the adenoma cells were demonstrated in seven of these ten active adenomas. Furthermore, secretory granules were entrapped occasionally in this intercellular...

  7. Electron microscopic study of the myelinated nerve fibres and the perineurial cell basement membrane in the diabetic human peripheral nerves

    International Nuclear Information System (INIS)

    To study the quantitative and ultrastructural changes in myelinated nerve fibers and the basement membranes of the perineurial cells in diabetic nerves. The study was performed at the Department of Anatomy, Faculty of Medicine, King Abdul-Aziz University, Jeddah, Saudi Arabia from 2003 to 2005. Human sural nerves were obtained from 15 lower limbs and 5 diabetic nerve biopsies. The total mean and density of myelinated nerve fibers per fascicle were calculated, with density of microtubules and mitochondria in the axoplasm. The number of the perineurial cell basement membrane layers was counted, and thickness of the basement membrane was measured. Among the 15 diabetic and 5 normal human sural nerves, the average diameters, number and surface area of myelinated nerve fibers and axonal microtubules density were found to be less in diabetic nerves. Mitochondrial density was higher in diabetic axons. Thickness of the perineurial cell basement membrane had a greater mean, but the number of perineurial cell layers was less than that of the diabetic group. The inner cellular layer of the perineurium of the diabetic nerves contained large vacuoles containing electron-dense degenerated myelin. A few specimens showed degenerated myelinated nerve fibers, while others showed recovering ones. Retracted axoplasms were encountered with albumin extravasation. Diabetes caused an increase in perineurial permeability. The diabetic sural nerve showed marked decrease in the myelinated nerve fibres, increase degenerated mitochondria, and decreased microtubules. (author)

  8. Role of 17 beta-estradiol on type IV collagen fibers volumetric density in the basement membrane of bladder wall.

    Science.gov (United States)

    de Fraga, Rogerio; Dambros, Miriam; Miyaoka, Ricardo; Riccetto, Cássio Luís Zanettini; Palma, Paulo César Rodrigues

    2007-10-01

    The authors quantified the type IV collagen fibers volumetric density in the basement membrane of bladder wall of ovariectomized rats with and without estradiol replacement. This study was conducted on 40 Wistar rats (3 months old) randomly divided in 4 groups: group 1, remained intact (control); group 2, submitted to bilateral oophorectomy and daily replacement 4 weeks later of 17 beta-estradiol for 12 weeks; group 3, sham operated and daily replacement 4 weeks later of sesame oil for 12 weeks; and group 4, submitted to bilateral oophorectomy and killed after 12 weeks. It was used in immunohistochemistry evaluation using type IV collagen polyclonal antibody to stain the fibers on paraffin rat bladder sections. The M-42 stereological grid system was used to analyze the fibers. Ovariectomy had an increase effect on the volumetric density of the type IV collagen fibers in the basement membrane of rat bladder wall. Estradiol replacement in castrated animals demonstrated a significative difference in the stereological parameters when compared to the castrated group without hormonal replacement. Surgical castration performed on rats induced an increasing volumetric density of type IV collagen fibers in the basement membrane of rats bladder wall and the estradiol treatment had a significant effect in keeping a low volumetric density of type IV collagen fibers in the basement membrane of rats bladder wall.

  9. Deletion of PPAR-γ in immune cells enhances susceptibility to antiglomerular basement membrane disease

    Directory of Open Access Journals (Sweden)

    Cristen Chafin

    2010-10-01

    Full Text Available Cristen Chafin2, Sarah Muse2, Raquel Hontecillas5, Josep Bassaganya-Riera5, David L Caudell2, Samuel K Shimp III4, M Nichole Rylander4, John Zhang6, Liwu Li3, Christopher M Reilly1,21Virginia College of Osteopathic Medicine, 2Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA; 3Department of Biological Sciences, 4Department of Mechanical Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA; 5Nutritional Immunology and Molecular Medicine Laboratory, Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA; 6Medical University of SC, Charleston, SC, USAAbstract: Activation of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR-γ has been shown to be immunoregulatory in autoimmune diseases by inhibiting production of a number of inflammatory mediators. We investigated whether PPAR-γ gene deletion in hematopoietic cells would alter disease pathogenesis in the antiglomerular basement membrane (anti-GBM mouse model. PPAR-γ+/+ and PPAR-γ-/- mice were immunized with rabbit antimouse GBM antibodies and lipopolysaccharide and evaluated for two weeks. Although both the PPAR-γ+/+ and PPAR-γ-/- mice had IgG deposition in the glomerulus and showed proteinuria two weeks after injection, glomerular and tubulointerstitial disease in PPAR-γ-/- mice were significantly more severe compared with the PPAR-γ+/+ animals. We observed that the PPAR-γ-/- mice had decreased CD4+CD25+ regulatory T cells and an increased CD8+:CD4+ ratio as compared with the PPAR-γ+/+ mice, suggesting that PPAR-γ has a role in the regulation of T cells. Furthermore, plasma interleukin-6 levels were significantly increased in the PPAR-γ-/- mice at two weeks as compared with the PPAR-γ+/+ animals. Taken together, these studies show that

  10. Modelling of proteins in membranes

    DEFF Research Database (Denmark)

    Sperotto, Maria Maddalena; May, S.; Baumgaertner, A.

    2006-01-01

    This review describes some recent theories and simulations of mesoscopic and microscopic models of lipid membranes with embedded or attached proteins. We summarize results supporting our understanding of phenomena for which the activities of proteins in membranes are expected to be significantly...... affected by the lipid environment. Theoretical predictions are pointed out, and compared to experimental findings, if available. Among others, the following phenomena are discussed: interactions of interfacially adsorbed peptides, pore-forming amphipathic peptides, adsorption of charged proteins onto...... oppositely charged lipid membranes, lipid-induced tilting of proteins embedded in lipid bilayers, protein-induced bilayer deformations, protein insertion and assembly, and lipid-controlled functioning of membrane proteins....

  11. Immunohistochemical localization of chondroitin sulfate, chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, entactin, and laminin in basement membranes of postnatal developing and adult rat lungs

    DEFF Research Database (Denmark)

    Sannes, P L; Burch, K K; Khosla, J;

    1993-01-01

    , and laminin. A monoclonal antibody specific for the glycosaminoglycan portion (CS) of CSPG and a monoclonal antibody against the core protein of CSPG were used in an immunoperoxidase sequence to stain extracellular matrix (ECM) components of pulmonary basement membranes (BMs). Anti-CS stained airway BM...... with CSPG, except that entactin showed particular affinity for EL. These results offer a more detailed perspective on previous survey observations of CSPG, HSPG, and entactin in the rat lung, and describe the immunoreactivity of CS for the first time.(ABSTRACT TRUNCATED AT 250 WORDS)...

  12. Vascular Basement Membrane-derived Multifunctional Peptide, a Novel Inhibitor of Angiogenesis and Tumor Growth

    Institute of Scientific and Technical Information of China (English)

    Jian-Guo CAO; Shu-Ping PENG; Li SUN; Hui LI; Li WANG; Han-Wu DENG

    2006-01-01

    Vascular basement membrane-derived multifunctional peptide (VBMDMP) gene (fusion gene of the human immunoglobulin G3 upper hinge region and two tumstatin-derived fragments) obtained by chemical synthesis was cloned into vector pUC 19, and introduced into the expression vector pGEX-4T-1 to construct a prokaryotic expression vector pGEX-4T-1-VBMDMP. Recombinant VBMDMP produced in Escherichia coli has been shown to have significant activity of antitumor growth and antimetastasis in Lewis lung carcinoma transplanted into mouse C57B1/6. In the present study, we have studied the ability of rVBMDMP to inhibit endothelial cell tube formation and proliferation, to induce apoptosis in vitro, and to suppress tumor growth in vivo. The experimental results showed that rVBMDMP potently inhibited proliferation of human endothelial (HUVEC-12) cells and human colon cancer (SW480) cells in vitro, with no inhibition of proliferation in Chinese hamster ovary (CHO-K1) cells. rVBMDMP also significantly inhibited human endothelial cell tube formation and suppressed tumor growth of SW480 cells in a mouse xenograft model. These results suggest that rVBMDMP is a powerful therapeutic agent for suppressing angiogenesis and tumor growth.

  13. Binding of Streptococcus mutans antigens to heart and kidney basement membranes.

    Science.gov (United States)

    Stinson, M W; Barua, P K; Bergey, E J; Nisengard, R J; Neiders, M E; Albini, B

    1984-01-01

    Using indirect immunofluorescence, alkali-extracted components of Streptococcus mutans were found to bind in vitro to capillary walls and sarcolemmal sheaths of monkey cardiac muscle and to glomerular and tubular basement membranes of monkey kidney. Adsorption of S. mutans components to tissue fragments was also detected by indirect radioimmunoassay and immunoblotting on nitrocellulose paper. Antibodies did not bind to untreated, control tissues in these experiments, proving that antigens shared by S. mutans and tissue components were not involved. Rabbit and monkey heart and kidney components bound S. mutans antigens of 24,000, 35,000, and 65,000 Mr. Monkey heart also bound molecules of 90,000 and 120,000 Mr. Rabbits immunized by intravenous injection of disrupted S. mutans cells developed severe nephritis that was characterized by the deposition of immunoglobulins, complement component C3, and S. mutans antigens in the glomeruli. Immunoglobulin G eluted from nephritic kidneys reacted in immunoblots with the 24,000, 35,000, and 65,000 Mr components of S. mutans extract, indicating that the antigens that bound to tissue in vitro also bound in vivo and reacted with antibodies in situ. Antibodies to other S. mutans antigens were not detected in the kidney eluate, although they were present in the serum of the same rabbit. Images PMID:6384042

  14. Anti-glomerular basement membrane glomerulonephritis in an HIV positive patient: case report

    Directory of Open Access Journals (Sweden)

    Eduardo José Bellotto Monteiro

    2006-02-01

    Full Text Available We report on a case of a patient with HIV infection, diagnosed 18 months prior to the development of an anti-glomerular basement membrane (anti-GBM rapidly progressive glomerulonephritis; this is probably the first report of such an association. A 30-year-old white man presented with elevation of serum creatinine (1.3 - 13.5 mg/dL within one month. At admission, the urinalysis showed proteinuria of 7.2 g/L and 8,000,000 erythrocytes/mL. Renal biopsy corresponded to a crescentic diffuse proliferative glomerulonephritis mediated by anti-GBM, and serum testing for anti-GBM antibodies was positive; antinuclear antibodies (ANA and anti-neutrophilic cytoplasmic antibodies (ANCA were also positive. The patient underwent hemodyalisis and was treated with plasmapheresis, cyclophosphamide and prednisone. The association described here is not casual, as crescentic glomerulonephritis is not common in HIV-positive patients, anti-GBM glomerulonephritis is rare and anti-GBM antibodies are frequently observed in HIV-positive subjects when compared to the overall population. Based on the current case and on the elevated frequency of the positivity for such antibodies in this group of patients, it is advisable to be aware of the eventual association between these two conditions and to promote an active search for anti-GBM antibodies and early diagnosis of eventual urinary abnormalities in HIV-positive subjects, considering the severity of anti-GBM glomerulonephritis.

  15. Proteins causing membrane fouling in membrane bioreactors.

    Science.gov (United States)

    Miyoshi, Taro; Nagai, Yuhei; Aizawa, Tomoyasu; Kimura, Katsuki; Watanabe, Yoshimasa

    2015-01-01

    In this study, the details of proteins causing membrane fouling in membrane bioreactors (MBRs) treating real municipal wastewater were investigated. Two separate pilot-scale MBRs were continuously operated under significantly different operating conditions; one MBR was a submerged type whereas the other was a side-stream type. The submerged and side-stream MBRs were operated for 20 and 10 days, respectively. At the end of continuous operation, the foulants were extracted from the fouled membranes. The proteins contained in the extracted foulants were enriched by using the combination of crude concentration with an ultrafiltration membrane and trichloroacetic acid precipitation, and then separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The N-terminal amino acid sequencing analysis of the proteins which formed intensive spots on the 2D-PAGE gels allowed us to partially identify one protein (OmpA family protein originated from genus Brevundimonas or Riemerella anatipestifer) from the foulant obtained from the submerged MBR, and two proteins (OprD and OprF originated from genus Pseudomonas) from that obtained from the side-stream MBR. Despite the significant difference in operating conditions of the two MBRs, all proteins identified in this study belong to β-barrel protein. These findings strongly suggest the importance of β-barrel proteins in developing membrane fouling in MBRs.

  16. Localization of type IV collagen a 1 to a 6 chains in basement membrane during mouse molar germ development.

    Science.gov (United States)

    Nagai, N; Nakano, K; Sado, Y; Naito, I; Gunduz, M; Tsujigiwa, H; Nagatsuka, H; Ninomiya, Y; Siar, C H

    2001-10-01

    The dental basement membrane (BM) putatively mediates epithelial-mesenchymal interactions during tooth morphogenesis and cytodifferentiation. Type IV collagen alpha chains, a major network-forming protein of the dental BM, was studied and results disclosed distinct expression patterns at different stages of mouse molar germ development. At the dental placode and bud stage, the BM of the oral epithelium expressed alpha 1, alpha 2, alpha 5 and alpha 6 chains while the gubernaculum dentis, in addition to the above four chains, also expressed a 4 chain. An asymmetrical expression for alpha 4, alpha 5 and alpha 6 chains was observed at the bud stage. At the early bell stage, the BM associated with the inner enamel epithelium (IEE) of molar germ expressed alpha 1, alpha 2 and alpha 4 chains while the BM of the outer enamel epithelium (OEE) expressed only alpha 1 and a 2 chains. With the onset of dentinogenesis, the collagen a chain profile of the IEE BM gradually disappeared. Howeverfrom the early to late bell stage, the gubernaculum dentis consistently expressed alpha 1, alpha 2, alpha 5 and a 6 chains resembling fetal oral mucosa. These findings suggest that stage- and position-specific distribution of type IV collagen alpha subunits occur during molar germ development and that these changes are essential for molar morphogenesis and cytodifferentiation. PMID:11732842

  17. Reinforcement of epithelial cell adhesion to basement membrane by a bacterial pathogen as a new infectious stratagem.

    Science.gov (United States)

    Kim, Minsoo; Ogawa, Michinaga; Mimuro, Hitomi; Sasakawa, Chihiro

    2010-01-01

    The intestinal epithelium undergoes a rapid turnover in addition to rapid exfoliation in response to bacterial infection, thus acting as an intrinsic defense against microbial intruders. It has long been questioned how mucosal pathogens can circumvent the intestinal defense systems. Our recent discovery of a bacterial ploy used by Shigella provided us with fresh insight. Shigella delivers OspE via the type III secretion system during multiplication within epithelial cells. This effector protein reinforces epithelial adherence to the basement membrane by interacting with integrin-linked kinase (ILK), a unique intracellular Ser/Thr kinase that links the cell-adhesion receptors, integrin, and growth factors to the actin cytoskeleton. The interaction between OspE and ILK increased formation of focal adhesions (FAs) and surface levels of b1-integrin, while suppressing phosphorylation of FAK and paxillin, thus suppressing rapid turnover of FAs, reducing cell motility and promoting cell adhesion to extracellular matrix. The impact of this OspE-ILK interplay was demonstrated both in vitro and in vivo by infecting polarized epithelial cell monolayers and guinea pig colons with Shigella possessing or lacking the ospE gene. The findings thus establish a new class of virulence-associated factors, and provide new insight into the functioning of the intestinal barrier and bacterial strategies for circumventing it. PMID:21178415

  18. Distribution of two basement membrane proteoglycans through hair follicle development and the hair growth cycle in the rat

    DEFF Research Database (Denmark)

    Couchman, J R; King, J L; McCarthy, K J

    1990-01-01

    The distribution of two distinct populations of basement membrane proteoglycans has been monitored through hair growth development in the rat embryo and subsequent hair growth cycle. An antiserum against a small heparan sulfate proteoglycan uniformly stained the dermal-epidermal junction...... of embryonic rats throughout the period of hair follicle formation. On the other hand, monoclonal antibodies recognizing a basement membrane-specific chondroitin sulfate proteoglycan only weakly stained 16-d embryo dermal-epidermal junction, but strong staining was associated with hair follicle buds...... as they developed. Through the hair growth cycle, it was found that the heparan sulfate proteoglycan persisted around the follicles, while the chondroitin sulfate proteoglycan decreased in amount through catagen until it was undetectable at the base and dermal papilla of the telogen follicle. As anagen commenced...

  19. Alport familial nephritis. Absence of 28 kilodalton non-collagenous monomers of type IV collagen in glomerular basement membrane.

    OpenAIRE

    Kleppel, M M; Kashtan, C. E.; Butkowski, R J; Fish, A. J.; Michael, A. F.

    1987-01-01

    Alport-type familial nephritis (FN), a genetic disorder, results in progressive renal insufficiency and sensorineural hearing loss. Immunochemical and biochemical analyses of the non-collagenous (NC1) domain of type IV collagen isolated from the glomerular basement membranes (GBM) of three males with this disease demonstrate absence of the normally occurring 28-kilodalton (kD) NC1 monomers, but persistence of the 26- and 24-kD monomeric subunits derived from alpha 1 and 2 (both type IV) colla...

  20. A Case of Fibrillary Glomerulonephritis Associated with Thrombotic Microangiopathy and Anti-Glomerular Basement Membrane Antibody

    Directory of Open Access Journals (Sweden)

    Akishi Momose

    2015-02-01

    Full Text Available We present the first report of a case of fibrillary glomerulonephritis (FGN associated with thrombotic microangiopathy (TMA and anti-glomerular basement membrane antibody (anti-GBM antibody. A 54-year-old man was admitted to our hospital for high fever and anuria. On the first hospital day, we initiated hemodialysis for renal dysfunction. Laboratory data revealed normocytic-normochromic anemia with schistocytes in the peripheral smear, thrombocytopenia, increased serum lactate dehydrogenase, decreased serum haptoglobin, and negative results for both direct and indirect Coombs tests. Based on these results, we diagnosed TMA. Assays conducted several days later indicated a disintegrin-like and metalloprotease with a thrombospondin motif 13 (ADAMTS13 activity of 31.6%, and ADAMTS13 inhibitors were negative. We started plasma exchange using fresh frozen plasma and steroid pulse therapy. Anti-GBM antibody was found to be positive. Renal biopsy showed FGN. Blood pressure rose on the 46th hospital day, and mild convulsions developed. Based on magnetic resonance imaging of the head, the patient was diagnosed with reversible posterior leukoencephalopathy syndrome. Hypertension persisted despite administration of multiple antihypertensive agents, and the patient experienced a sudden generalized seizure. Computed tomography of the head showed multiple cerebral hemorrhages. However, his blood pressure subsequently decreased and the platelet count increased. TMA remitted following 36 plasma exchange sessions, but renal function was not restored, and maintenance hemodialysis was continued. The patient was discharged on the 119th day of hospitalization. In conclusion, it was shown that TMA, FGN and anti-GBM antibody were closely related.

  1. Ultrastructural morphometry of capillary basement membrane thickness in normal and transgenic diabetic mice.

    Science.gov (United States)

    Carlson, Edward C; Audette, Janice L; Veitenheimer, Nicole J; Risan, Jessica A; Laturnus, Donna I; Epstein, Paul N

    2003-04-01

    Capillary basement membrane (CBM) thickening is an ultrastructural hallmark in diabetic patients and in animal models of diabetes. However, the wide variety of tissues sampled and diverse methods employed have made the interpretation of thickness data difficult. We showed previously that acellular glomerular BMs in OVE26 transgenic diabetic mice were thickened beyond normal age-related thickening, and in the current study we hypothesized that other microvascular BMs likewise would show increased widths relative to age-matched controls. Accordingly, a series of tissues, including skeletal and cardiac muscle, ocular retina and choriod, peripheral nerve, lung, pancreas, and renal glomerulus was collected from 300-350-day-old normal and transgenic mice. Transmission electron micrographs of cross sections through capillary walls were prepared, and CBM thickness (CBMT) was determined by the "orthogonal intercept" method. Morphometric analyses showed highly variable transgene-related BMT increases in the sampled tissues, with glomerular BM showing by far the greatest increase (+87%). Significant thickness increases were also seen in the retina, pulmonary alveolus, and thoracoabdominal diaphragm. BMT increases were not universal; however, most were modestly widened, and those that were thickest in controls generally showed the greatest increase. Although the pathogenesis of diabetes-related increases in CBM is poorly understood, data in the current study showed that in OVE26 transgenic mice increased BMT was a frequent concomitant of hyperglycemia. Accordingly, it seems likely that hyperglycemia-induced microvascular damage may be a contributing factor in diabetic BM disease, and that microvessel cellular and extracellular heterogeneity may limit the extent of CBM thickening in diverse tissues. PMID:12629676

  2. The effect of asthma on the perimeter of the airway basement membrane.

    Science.gov (United States)

    Elliot, John G; Budgeon, Charley A; Harji, Salima; Jones, Robyn L; James, Alan L; Green, Francis H

    2015-11-15

    When comparing the pathology of airways in individuals with and without asthma, the perimeter of the basement membrane (Pbm) is used as a marker of airway size, as it is independent of airway smooth muscle shortening or airway collapse. The extent to which the Pbm is itself altered in asthma has not been quantified. The aim of this study was to compare the Pbm from the same anatomical sites in postmortem lungs from subjects with (n = 55) and without (n = 30) asthma (nonfatal or fatal). Large and small airways were systematically sampled at equidistant "levels" from the apical segment of the left upper lobes and anterior and basal segments of the left lower lobes of lungs fixed in inflation. The length of the Pbm was estimated from cross sections of airway at each relative level. Linear mixed models were used to investigate the relationships between Pbm and sex, age, height, smoking status, airway level, and asthma group. The final model showed significant interactions between Pbm and airway level in small (<3 mm) airways, in subjects having asthma (P < 0.0001), and by sex (P < 0.0001). No significant interactions for Pbm between asthma groups were observed for larger airways (equivalent to a diameter of ∼3 mm and greater) or smoking status. Asthma is not associated with remodeling of the Pbm in large airways. In medium and small airways, the decrease in Pbm in asthma (≤20%) would not account for the published differences in wall area or area of smooth muscle observed in cases of severe asthma.

  3. Long-term outcome of anti-glomerular basement membrane antibody disease treated with immunoadsorption.

    Directory of Open Access Journals (Sweden)

    Peter Biesenbach

    Full Text Available Anti-glomerular basement membrane (GBM antibody disease may lead to acute crescentic glomerulonephritis with poor renal prognosis. Current therapy favours plasma exchange (PE for removal of pathogenic antibodies. Immunoadsorption (IAS is superior to PE regarding efficiency of antibody-removal and safety. Apart from anecdotal data, there is no systemic analysis of the long-term effects of IAS on anti-GBM-disease and antibody kinetics.To examine the long-term effect of high-frequency IAS combined with standard immunosuppression on patient and renal survival in patients with anti-GBM-disease and to quantify antibody removal and kinetics through IAS.Retrospective review of patients treated with IAS for anti-GBM-antibody disease confirmed by biopsy and/or anti-GBM-antibodies.University Hospital of Vienna, Austria.10 patients with anti-GBM-disease treated with IAS.Patient and renal survival, renal histology, anti-GBM-antibodies.Anti-GBM-antibodies were reduced by the first 9 IAS treatments (mean number of 23 to negative levels in all patients. Renal survival was 40% at diagnosis, 70% after the end of IAS, 63% after one year and 50% at the end of observation (mean 84 months, range 9 to 186. Dialysis dependency was successfully reversed in three of six patients. Patient survival was 90% at the end of observation.IAS efficiently eliminates anti-GBM-antibodies suggesting non-inferiority to PE with regard to renal and patient survival. Hence IAS should be considered as a valuable treatment option for anti-GBM-disease, especially in patients presenting with a high percentage of crescents and dialysis dependency due to an unusual high proportion of responders.

  4. Proteins interacting with Membranes: Protein Sorting and Membrane Shaping

    Science.gov (United States)

    Callan-Jones, Andrew

    2015-03-01

    Membrane-bound transport in cells requires generating membrane curvature. In addition, transport is selective, in order to establish spatial gradients of membrane components in the cell. The mechanisms underlying cell membrane shaping by proteins and the influence of curvature on membrane composition are active areas of study in cell biophysics. In vitro approaches using Giant Unilamellar Vesicles (GUVs) are a useful tool to identify the physical mechanisms that drive sorting of membrane components and membrane shape change by proteins. I will present recent work on the curvature sensing and generation of IRSp53, a protein belonging to the BAR family, whose members, sharing a banana-shaped backbone, are involved in endocytosis. Pulling membrane tubes with 10-100 nm radii from GUVs containing encapsulated IRSp53 have, unexpectedly, revealed a non-monotonic dependence of the protein concentration on the tube as a function of curvature. Experiments also show that bound proteins alter the tube mechanics and that protein phase separation along the tube occurs at low tensions. I will present accompanying theoretical work that can explain these findings based on the competition between the protein's intrinsic curvature and the effective rigidity of a membrane-protein patch.

  5. Hydrophobic organization of membrane proteins

    OpenAIRE

    Rees, D C; DeAntonio, L.; Eisenberg, D.

    1989-01-01

    Membrane-exposed residues are more hydrophobic than buried interior residues in the transmembrane regions of the photosynthetic reaction center from Rhodobacter sphaeroides. This hydrophobic organization is opposite to that of water-soluble proteins. The relative polarities of interior and surface residues of membrane and water soluble proteins are not simply reversed, however. The hydrophobicities of interior residues of both membrane and water-soluble proteins are comparable, whereas the bi...

  6. Insufficient Folding of Type IV Collagen and Formation of Abnormal Basement Membrane-like Structure in Embryoid Bodies Derived from Hsp47-Null Embryonic Stem CellsD⃞

    OpenAIRE

    Matsuoka, Yasuhiro; Kubota, Hiroshi; Adachi, Eijiro; Nagai, Naoko; Marutani, Toshihiro; Hosokawa, Nobuko; Nagata, Kazuhiro

    2004-01-01

    Hsp47 is a molecular chaperone that specifically recognizes procollagen in the endoplasmic reticulum. Hsp47-null mouse embryos produce immature type I collagen and form discontinuous basement membranes. We established Hsp47-/- embryonic stem cell lines and examined formation of basement membrane and production of type IV collagen in embryoid bodies, a model for postimplantation egg-cylinder stage embryos. The visceral endodermal cell layers surrounding Hsp47-/- embryoid bodies were often diso...

  7. In vivo laser confocal microscopy findings in patients with map-dot-fingerprint (epithelial basement membrane dystrophy

    Directory of Open Access Journals (Sweden)

    Kobayashi A

    2012-07-01

    Full Text Available Akira Kobayashi, Hideaki Yokogawa, Kazuhisa SugiyamaDepartment of Ophthalmology, Kanazawa University Graduate School of Medical Science, Kanazawa, JapanBackground: The purpose of this study was to investigate pathological changes of the corneal cell layer in patients with map-dot-fingerprint (epithelial basement membrane dystrophy by in vivo laser corneal confocal microscopy.Methods: Two patients were evaluated using a cornea-specific in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph 2 Rostock Cornea Module, HRT 2-RCM. The affected corneal areas of both patients were examined. Image analysis was performed to identify corneal epithelial and stromal deposits correlated with this dystrophy.Results: Variously shaped (linear, multilaminar, curvilinear, ring-shape, geographic highly reflective materials were observed in the “map” area, mainly in the basal epithelial cell layer. In “fingerprint” lesions, multiple linear and curvilinear hyporeflective lines were observed. Additionally, in the affected corneas, infiltration of possible Langerhans cells and other inflammatory cells was observed as highly reflective Langerhans cell-like or dot images. Finally, needle-shaped materials were observed in one patient.Conclusion: HRT 2-RCM laser confocal microscopy is capable of identifying corneal microstructural changes related to map-dot-fingerprint corneal dystrophy in vivo. The technique may be useful in elucidating the pathogenesis and natural course of map-dot-fingerprint corneal dystrophy and other similar basement membrane abnormalities.Keywords: cornea, confocal microscopy, map-dot-fingerprint dystrophy, epithelial basement membrane dystrophy, Heidelberg Retina Tomograph 2 Rostock Cornea Module (HRT 2-RCM

  8. Overexpression of β1-chain-containing laminins in capillary basement membranes of human breast cancer and its metastases

    International Nuclear Information System (INIS)

    Laminins are the major components of vascular and parenchymal basement membranes. We previously documented a switch in the expression of vascular laminins containing the α4 chain from predominantly laminin-9 (α4β2γ1) to predominantly laminin-8 (α4β1γ1) during progression of human brain gliomas to high-grade glioblastoma multiforme. Here, differential expression of laminins was studied in blood vessels and ductal epithelium of the breast. In the present study the expressions of laminin isoforms α1–α5, β1–β3, γ1, and γ2 were examined during progression of breast cancer. Forty-five clinical samples of breast tissues including normal breast, ductal carcinomas in situ, invasive ductal carcinomas, and their metastases to the brain were compared using Western blot analysis and immunohistochemistry for various chains of laminin, in particular laminin-8 and laminin-9. Laminin α4 chain was observed in vascular basement membranes of most studied tissues, with the highest expression in metastases. At the same time, the expression of laminin β2 chain (a constituent of laminin-9) was mostly seen in normal breast and carcinomas in situ but not in invasive carcinomas or metastases. In contrast, laminin β1 chain (a constituent of laminin-8) was typically found in vessel walls of carcinomas and their metastases but not in those of normal breast. The expression of laminin-8 increased in a progression-dependent manner. A similar change was observed from laminin-11 (α5β2γ1) to laminin-10 (α5β1γ1) during breast tumor progression. Additionally, laminin-2 (α2β1γ1) appeared in vascular basement membranes of invasive carcinomas and metastases. Chains of laminin-5 (α3β3γ2) were expressed in the ductal epithelium basement membranes of the breast and diminished with tumor progression. These results suggest that laminin-2, laminin-8, and laminin-10 are important components of tumor microvessels and may associate with breast tumor progression. Angiogenic switch

  9. Molecular dynamics of membrane proteins.

    Energy Technology Data Exchange (ETDEWEB)

    Woolf, Thomas B. (Johns Hopkins University School of Medicine, Baltimore, MD); Crozier, Paul Stewart; Stevens, Mark Jackson

    2004-10-01

    Understanding the dynamics of the membrane protein rhodopsin will have broad implications for other membrane proteins and cellular signaling processes. Rhodopsin (Rho) is a light activated G-protein coupled receptor (GPCR). When activated by ligands, GPCRs bind and activate G-proteins residing within the cell and begin a signaling cascade that results in the cell's response to external stimuli. More than 50% of all current drugs are targeted toward G-proteins. Rho is the prototypical member of the class A GPCR superfamily. Understanding the activation of Rho and its interaction with its Gprotein can therefore lead to a wider understanding of the mechanisms of GPCR activation and G-protein activation. Understanding the dark to light transition of Rho is fully analogous to the general ligand binding and activation problem for GPCRs. This transition is dependent on the lipid environment. The effect of lipids on membrane protein activity in general has had little attention, but evidence is beginning to show a significant role for lipids in membrane protein activity. Using the LAMMPS program and simulation methods benchmarked under the IBIG program, we perform a variety of allatom molecular dynamics simulations of membrane proteins.

  10. Deletion of the basement membrane heparan sulfate proteoglycan type XVIII collagen causes hypertriglyceridemia in mice and humans.

    Directory of Open Access Journals (Sweden)

    Joseph R Bishop

    Full Text Available BACKGROUND: Lipoprotein lipase (Lpl acts on triglyceride-rich lipoproteins in the peripheral circulation, liberating free fatty acids for energy metabolism or storage. This essential enzyme is synthesized in parenchymal cells of adipose tissue, heart, and skeletal muscle and migrates to the luminal side of the vascular endothelium where it acts upon circulating lipoproteins. Prior studies suggested that Lpl is immobilized by way of heparan sulfate proteoglycans on the endothelium, but genetically altering endothelial cell heparan sulfate had no effect on Lpl localization or lipolysis. The objective of this study was to determine if extracellular matrix proteoglycans affect Lpl distribution and triglyceride metabolism. METHODS AND FINDINGS: We examined mutant mice defective in collagen XVIII (Col18, a heparan sulfate proteoglycan present in vascular basement membranes. Loss of Col18 reduces plasma levels of Lpl enzyme and activity, which results in mild fasting hypertriglyceridemia and diet-induced hyperchylomicronemia. Humans with Knobloch Syndrome caused by a null mutation in the vascular form of Col18 also present lower than normal plasma Lpl mass and activity and exhibit fasting hypertriglyceridemia. CONCLUSIONS: This is the first report demonstrating that Lpl presentation on the lumenal side of the endothelium depends on a basement membrane proteoglycan and demonstrates a previously unrecognized phenotype in patients lacking Col18.

  11. An in vitro model of the glomerular capillary wall using electrospun collagen nanofibres in a bioartificial composite basement membrane.

    Directory of Open Access Journals (Sweden)

    Sadie C Slater

    Full Text Available The filtering unit of the kidney, the glomerulus, contains capillaries whose walls function as a biological sieve, the glomerular filtration barrier. This comprises layers of two specialised cells, glomerular endothelial cells (GEnC and podocytes, separated by a basement membrane. Glomerular filtration barrier function, and dysfunction in disease, remains incompletely understood, partly due to difficulties in studying the relevant cell types in vitro. We have addressed this by generation of unique conditionally immortalised human GEnC and podocytes. However, because the glomerular filtration barrier functions as a whole, it is necessary to develop three dimensional co-culture models to maximise the benefit of the availability of these cells. Here we have developed the first two tri-layer models of the glomerular capillary wall. The first is based on tissue culture inserts and provides evidence of cell-cell interaction via soluble mediators. In the second model the synthetic support of the tissue culture insert is replaced with a novel composite bioartificial membrane. This consists of a nanofibre membrane containing collagen I, electrospun directly onto a micro-photoelectroformed fine nickel supporting mesh. GEnC and podocytes grew in monolayers on either side of the insert support or the novel membrane to form a tri-layer model recapitulating the human glomerular capillary in vitro. These models will advance the study of both the physiology of normal glomerular filtration and of its disruption in glomerular disease.

  12. Reciprocal interactions between Beta1-integrin and epidermal growth factor in three-dimensional basement membrane breast cultures: A different perspective in epithelial biology

    Energy Technology Data Exchange (ETDEWEB)

    Wang, F.; Weaver, V.M.; Petersen, O.W.; Larabell, C.A.; Dedhar, S.; Briand, P.; Lupu, R.; Bissell, M.J.

    1998-09-30

    Anchorage and growth factor independence are cardinal features of the transformed phenotype. Although it is logical that the two pathways must be coregulated in normal tissues to maintain homeostasis, this has not been demonstrated directly. We showed previously that down-modulation of {beta}1-integrin signaling reverted the malignant behavior of a human breast tumor cell line (T4-2) derived from phenotypically normal cells (HMT-3522) and led to growth arrest in a threedimensional (3D) basement membrane assay in which the cells formed tissue-like acini (14). Here, we show that there is a bidirectional cross-modulation of {beta}1-integrin and epidermal growth factor receptor (EGFR) signaling via the mitogenactivated protein kinase (MAPK) pathway. The reciprocal modulation does not occur in monolayer (2D) cultures. Antibodymediated inhibition of either of these receptors in the tumor cells, or inhibition of MAPK kinase, induced a concomitant downregulation of both receptors, followed by growth-arrest and restoration of normal breast tissue morphogenesis. Crossmodulation and tissue morphogenesis were associated with attenuation of EGF-induced transient MAPK activation. To specifically test EGFR and {beta}1-integrin interdependency, EGFR was overexpressed in nonmalignant cells, leading to disruption of morphogenesis and a compensatory up-regulation of {beta}1-integrin expression, again only in 3D. Our results indicate that when breast cells are spatially organized as a result of contact with basement membrane, the signaling pathways become coupled and bidirectional. They further explain why breast cells fail to differentiate in monolayer cultures in which these events are mostly uncoupled. Moreover, in a subset of tumor cells in which these pathways are misregulated but functional, the cells could be 'normalized' by manipulating either pathway.

  13. A role for PDGF-C/PDGFRα signaling in the formation of the meningeal basement membranes surrounding the cerebral cortex.

    Science.gov (United States)

    Andrae, Johanna; Gouveia, Leonor; Gallini, Radiosa; He, Liqun; Fredriksson, Linda; Nilsson, Ingrid; Johansson, Bengt R; Eriksson, Ulf; Betsholtz, Christer

    2016-01-01

    Platelet-derived growth factor-C (PDGF-C) is one of three known ligands for the tyrosine kinase receptor PDGFRα. Analysis ofPdgfcnull mice has demonstrated roles for PDGF-C in palate closure and the formation of cerebral ventricles, but redundancy with other PDGFRα ligands might obscure additional functions. In search of further developmental roles for PDGF-C, we generated mice that were double mutants forPdgfc(-/-)andPdgfra(GFP/+) These mice display a range of severe phenotypes including spina bifida, lung emphysema, abnormal meninges and neuronal over-migration in the cerebral cortex. We focused our analysis on the central nervous system (CNS), where PDGF-C was identified as a critical factor for the formation of meninges and assembly of the glia limitans basement membrane. We also present expression data onPdgfa,PdgfcandPdgfrain the cerebral cortex and microarray data on cerebral meninges. PMID:26988758

  14. Creatinine clearance, urinary excretion of glomerular basement membrane antigens and renal histology in congenital nephrotic syndrome of Finnish type.

    Science.gov (United States)

    Huttunen, N P

    1977-04-01

    The endogenous creatinine clearance and urinary excretion rate of glomerular basement membrane (GBM) antigens were followed from 2 to 19 months in fifteen patients with congenital nephrotic syndrome (CNF). The quantitative examination of renal morphology was made on fourteen of these patients. Creatinine clearance increased during the first few months of life and thereafter gradually decreased. The urinary excretion rate of GBM antigens rose during the course of the disease. The creatinine clearance did not correlate significantly with glomerular fibrosis but it did correlate with tubular atrophy and interstitial fibrosis. The urinary excretion of GBM antigens correlated significantly with glomerular and interstitial fibrosis and with tubular atrophy. It is concluded that there is a clear progress in the disease and the renal histological changes probably are caused by accumulation of GBM material in glomeruli.

  15. Histones have high affinity for the glomerular basement membrane. Relevance for immune complex formation in lupus nephritis

    Energy Technology Data Exchange (ETDEWEB)

    Schmiedeke, T.M.; Stoeckl, F.W.W.; Weber, R.; Sugisaki, Y.; Batsford, S.R.; Vogt, A.

    1989-06-01

    An effort has been made to integrate insights on charge-based interactions in immune complex glomerulonephritis with nuclear antigen involvement in lupus nephritis. Attention was focussed on the histones, a group of highly cationic nuclear constituents, which could be expected to bind to fixed anionic sites present in the glomerular basement membrane (GBM). We demonstrated that all histone subfractions, prepared according to Johns, have a high affinity for GBM and the basement membrane of peritubular capillaries. Tissue uptake of /sup 125/I-labeled histones was measured by injecting 200 micrograms of each fraction into the left kidney via the aorta and measuring organ uptake after 15 min. In glomeruli isolated from the left kidneys, the following quantities of histones were found: f1, 13 micrograms; f2a (f2al + f2a2), 17 micrograms; f2b, 17 micrograms; and f3, 32 micrograms. Kinetic studies of glomerular binding showed that f1 disappeared much more rapidly than f2a. The high affinity of histones (pI between 10.5 and 11.0; mol wt 10,000-22,000) for the GBM correlates well with their ability to form aggregates (mol wt greater than 100,000) for comparison lysozyme (pI 11, mol wt 14,000), which does not aggregate spontaneously bound poorly (0.4 micrograms in isolated glomeruli). The quantity of histones and lysozyme found in the isolated glomeruli paralleled their in vitro affinity for a Heparin-Sepharose column (gradient elution studies). This gel matrix contains the sulfated, highly anionic polysaccharide heparin, which is similar to the negatively charged heparan sulfate present in the GBM. Lysozyme eluted with 0.15 M NaCl, f1 with 1 M NaCl, and f2a, f2b, and f3 could not be fully desorbed even with 2 M NaCl; 6 M guanidine-HCl was necessary.

  16. Reconstitution of Membrane Proteins into Model Membranes: Seeking Better Ways to Retain Protein Activities

    OpenAIRE

    Trevor Lithgow; Lisa Martin; Hsin-Hui Shen

    2013-01-01

    The function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. The activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. The reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. However, the activity of mem...

  17. The effect of protein-protein and protein-membrane interactions on membrane fouling in ultrafiltration

    NARCIS (Netherlands)

    Huisman, I.H.; Prádanos, P.; Hernández, A.

    2000-01-01

    It was studied how protein-protein and protein-membrane interactions influence the filtration performance during the ultrafiltration of protein solutions over polymeric membranes. This was done by measuring flux, streaming potential, and protein transmission during filtration of bovine serum albumin

  18. ANTI-NUCLEOSOME ANTIBODIES COMPLEXED TO NUCLEOSOMAL ANTIGENS SHOW ANTI-DNA REACTIVITY AND BIND TO RAT GLOMERULAR-BASEMENT-MEMBRANE IN-VIVO

    NARCIS (Netherlands)

    KRAMERS, C; HYLKEMA, MN; VANBRUGGEN, MCJ; VANDELAGEMAAT, R; DIJKMAN, HBPM; ASSMANN, KJM; SMEENK, RJT; BERDEN, JHM; Hylkema, Machteld

    1994-01-01

    Histones can mediate the binding of DNA and anti-DNA to the glomerular basement membrane (GBM). Zn ELISA histone/DNA/anti-DNA complexes are able to bind to heparan sulfate (HS), an intrinsic constituent of the GBM. We questioned whether histone containing immune complexes are able to bind to the GBM

  19. Elastase, but not proteinase 3 (PR3), induces proteinuria associated with loss of glomerular basement membrane heparan sulphate after in vivo renal perfusion in rats

    NARCIS (Netherlands)

    Heeringa, P; VanDenBorn, J; Brouwer, E; Dolman, KM; Klok, PA; Huitema, MG; Limburg, PC; Bakker, MAH; Berden, JHM; Daha, MR; Kallenberg, CGM

    1996-01-01

    Elastase, but not PR3, induces proteinuria associated with loss of glomerular basement membrane (GEM) heparan sulphate after in vivo renal perfusion in rats. PR3 and elastase are cationic neutral serine proteinases present in the azurophilic granules of polymorphonuclear leucocytes. Release of these

  20. Targeted Expression of Stromelysin-1 in Mammary Gland Provides Evidence for a Role of Proteinases in Branching Morphogenesis and the Requirement for an Intact Basement Membrane for Tissue-specific Gene Expression

    Energy Technology Data Exchange (ETDEWEB)

    Sympson, Carolyn J; Talhouk, Rabih S; Alexander, Caroline M; Chin, Jennie R; Cliff, Shirley M; Bissell, Mina J; Werb, Zena

    1994-05-01

    The extracellular matrix (ECM) is an important regulator of the differentiated phenotype of mammary epithelial cells in culture. Despite the fact that ECM-degrading enzymes have been implicated in morphogenesis and tissue remodeling, there is little evidence for a direct role for such regulation in vivo. We generated transgenic mice that express autoactivated isoforms of the matrix metalloproteinase stromelysin-1, under the control of the whey acidic protein gene promoter, to examine the effect of inappropriate expression of this enzyme. Stromelysin-1 is implicated as the primary player in the loss of basement membrane and loss of function in the mammary gland during involution. The transgene was expressed at low levels in mammary glands of virgin female mice, leading to an unexpected phenotype: The primary ducts had supernumerary branches and showed precocious development of alveoli that expressed beta-casein at levels similar to that of an early- to mid-pregnant gland. Lactating glands showed high levels of transgene expression, with accumulation at the basement membrane, and a decrease in laminin and collagen IV, resulting in a loss of basement membrane integrity; this was accompanied by a dramatic alteration of alveolar morphology, with decreased size and shrunken lumina containing little beta-casein. During pregnancy, expression of endogenous whey acidic protein and beta-casein was reduced in transgenic glands, confirming the observed dependence of milk protein transcription of ECM in mammary epithelial cells in culture. These data provide direct evidence that stromelysin-1 activity can be morphogenic for mammary epithelial cells, inducing hyperproliferation and differentiation in virgin animals, and that its lytic activity can, indeed, disrupt membrane integrity and reduce mammary-specific function. We conclude that the balance of ECM-degrading enzymes with their inhibitors, and the associated regulation of ECM structure, is crucial for tissue-specific gene

  1. A direct contact between astrocyte and vitreous body is possible in the rabbit eye due to discontinuities in the basement membrane of the retinal inner limiting membrane

    Directory of Open Access Journals (Sweden)

    A. Haddad

    2003-02-01

    Full Text Available Different from most mammalian species, the optic nerve of the rabbit eye is initially formed inside the retina where myelination of the axons of the ganglion cells starts and vascularization occurs. Astrocytes are confined to these regions. The aforementioned nerve fibers known as medullated nerve fibers form two bundles that may be identified with the naked eye. The blood vessels run on the inner surface of these nerve fiber bundles (epivascularization and, accordingly, the accompanying astrocytes lie mostly facing the vitreous body from which they are separated only by the inner limiting membrane of the retina. The arrangement of the astrocytes around blood vessels leads to the formation of structures known as glial tufts. Fragments (N = 3 or whole pieces (N = 3 of the medullated nerve fiber region of three-month-old male rabbits (Orictolagus cuniculus were fixed in glutaraldehyde followed by osmium tetroxide, and their thin sections were examined with a transmission electron microscope. Randomly located discontinuities (up to a few micrometers long of the basement membrane of the inner limiting membrane of the retina were observed in the glial tufts. As a consequence, a direct contact between the astrocyte plasma membrane and vitreous elements was demonstrated, making possible functional interactions such as macromolecular exchanges between this glial cell type and the components of the vitreous body.

  2. Heparanase expression,degradation of basement membrane and low degree of infiltration by immunocytes correlate with invasion and progression of human gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Zun-Jiang Xie; Ying Liu; Li-Min Jia; Ye-Chun He

    2008-01-01

    AIM: To disclose the mechanisms that accelerate or limit tumor invasion and metastasis in gastric cancer patients.METHODS: The heparanase expression,continuity of basement,degree of infiltration by dendritic cells and lymphocytes in gastric cancer tissues from 33 the early and late stage patients were examined by immunohistochemistry,in situ hybridization and transmission electron microscopy.RESULTS: Heparanase mRNA expression in the late stage patients with gastric cancer was stronger than that in the early stage gastric cancer patients.In the early stage gastric cancer tissues,basement membrane (BlVl) appeared intact,whereas in the late stage,discontinuous BM was often present.The density of $100 protein positive tumor infiltrating dendritic cells (TIDC) in the early stage gastric cancer tissues was higher than that in the late stage.The infiltrating degree of tumor infiltrating lymphocytes (TIL) in the early stage patients whose tumor tissues contained a high density of TIDC was significantly higher than that in the late stage gastric cancer tissues patients with a low density of TIDC.There were few cancer cells penetrated through the continuous BM of cancer nests in the early stage gastric cancers,but many cancer cells were found outside of the defective BM of cancer nests in the late stage.CONCLUSION: Our results suggest that strong heparanase expression is related with the degradation of BM which allows or accelerates tumor invasion and metastasis.However,high density of TIDC and degree of infiltration by TIL are associated with tumor progression in human gastric cancers.

  3. Multiscale Simulation of Protein Mediated Membrane Remodeling

    OpenAIRE

    Ayton, Gary S.; Voth, Gregory A.

    2009-01-01

    Proteins interacting with membranes can result in substantial membrane deformations and curvatures. This effect is known in its broadest terms as membrane remodeling. This review article will survey current multiscale simulation methodologies that have been employed to examine protein-mediated membrane remodeling.

  4. Analysis of Protein-Membrane Interactions

    DEFF Research Database (Denmark)

    Kemmer, Gerdi Christine

    Cellular membranes are complex structures, consisting of hundreds of different lipids and proteins. These membranes act as barriers between distinct environments, constituting hot spots for many essential functions of the cell, including signaling, energy conversion, and transport. These functions...... are implemented by soluble proteins reversibly binding to, as well as by integral membrane proteins embedded in, cellular membranes. The activity and interaction of these proteins is furthermore modulated by the lipids of the membrane. Here, liposomes were used as model membrane systems to investigate...... interactions between proteins and lipids. First, interactions of soluble proteins with membranes and specific lipids were studied, using two proteins: Annexin V and Tma1. The protein was first subjected to a lipid/protein overlay assay to identify candidate interaction partners in a fast and efficient way...

  5. Novel Tripod Amphiphiles for Membrane Protein Analysis

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Kruse, Andrew C; Gotfryd, Kamil;

    2013-01-01

    Integral membrane proteins play central roles in controlling the flow of information and molecules across membranes. Our understanding of membrane protein structures and functions, however, is seriously limited, mainly due to difficulties in handling and analysing these proteins in aqueous solution...

  6. Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

    Science.gov (United States)

    Laible, Philip D; Hanson, Deborah K

    2013-06-04

    The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

  7. Efficient differentiation of embryonic stem cells into hepatic cells in vitro using a feeder-free basement membrane substratum.

    Directory of Open Access Journals (Sweden)

    Nobuaki Shiraki

    Full Text Available The endoderm-inducing effect of the mesoderm-derived supportive cell line M15 on embryonic stem (ES cells is partly mediated through the extracellular matrix, of which laminin α5 is a crucial component. Mouse ES or induced pluripotent stem cells cultured on a synthesized basement membrane (sBM substratum, using an HEK293 cell line (rLN10-293 cell stably expressing laminin-511, could differentiate into definitive endoderm and subsequently into pancreatic lineages. In this study, we investigated the differentiation on sBM of mouse and human ES cells into hepatic lineages. The results indicated that the BM components played an important role in supporting the regional-specific differentiation of ES cells into hepatic endoderm. We show here that knockdown of integrin β1 (Itgb1 in ES cells reduced their differentiation into hepatic lineages and that this is mediated through Akt signaling activation. Moreover, under optimal conditions, human ES cells differentiated to express mature hepatocyte markers and secreted high levels of albumin. This novel procedure for inducing hepatic differentiation will be useful for elucidating the molecular mechanisms controlling lineage-specific fates during gut regionalization. It could also represent an attractive approach to providing a surrogate cell source, not only for regenerative medicine, but also for pharmaceutical and toxicologic studies.

  8. Basement membrane and vascular remodelling in smokers and chronic obstructive pulmonary disease: a cross-sectional study

    Directory of Open Access Journals (Sweden)

    Muller H Konrad

    2010-07-01

    Full Text Available Abstract Background Little is known about airway remodelling in bronchial biopsies (BB in smokers and chronic obstructive pulmonary disease (COPD. We conducted an initial pilot study comparing BB from COPD patients with nonsmoking controls. This pilot study suggested the presence of reticular basement membrane (Rbm fragmentation and altered vessel distribution in COPD. Methods To determine whether Rbm fragmentation and altered vessel distribution in BB were specific for COPD we designed a cross-sectional study and stained BB from 19 current smokers and 14 ex-smokers with mild to moderate COPD and compared these to 15 current smokers with normal lung function and 17 healthy and nonsmoking subjects. Results Thickness of the Rbm was not significantly different between groups; although in COPD this parameter was quite variable. The Rbm showed fragmentation and splitting in both current smoking groups and ex-smoker COPD compared with healthy nonsmokers (p Conclusions Airway remodelling in smokers and mild to moderate COPD is associated with fragmentation of the Rbm and altered distribution of vessels in the airway wall. Rbm fragmentation was also present to as great an extent in ex-smokers with COPD. These characteristics may have potential physiological consequences.

  9. Complement and Humoral Adaptive Immunity in the Human Choroid Plexus: Roles for Stromal Concretions, Basement Membranes, and Epithelium.

    Science.gov (United States)

    Moore, G R Wayne; Laule, Cornelia; Leung, Esther; Pavlova, Vladimira; Morgan, B Paul; Esiri, Margaret M

    2016-05-01

    The choroid plexus (CP) provides a barrier to entry of toxic molecules from the blood into the brain and transports vital molecules into the cerebrospinal fluid. While a great deal is known about CP physiology, relatively little is known about its immunology. Here, we show immunohistochemical data that help define the role of the CP in innate and adaptive humoral immunity. The results show that complement, in the form of C1q, C3d, C9, or C9neo, is preferentially deposited in stromal concretions. In contrast, immunoglobulin (Ig) G (IgG) and IgA are more often found in CP epithelial cells, and IgM is found in either locale. C4d, IgD, and IgE are rarely, if ever, seen in the CP. In multiple sclerosis CP, basement membrane C9 or stromal IgA patterns were common but were not specific for the disease. These findings indicate that the CP may orchestrate the clearance of complement, particularly by deposition in its concretions, IgA and IgG preferentially via its epithelium, and IgM by either mechanism.

  10. Skin Basement Membrane: The Foundation of Epidermal Integrity—BM Functions and Diverse Roles of Bridging Molecules Nidogen and Perlecan

    Directory of Open Access Journals (Sweden)

    Dirk Breitkreutz

    2013-01-01

    Full Text Available The epidermis functions in skin as first defense line or barrier against environmental impacts, resting on extracellular matrix (ECM of the dermis underneath. Both compartments are connected by the basement membrane (BM, composed of a set of distinct glycoproteins and proteoglycans. Herein we are reviewing molecular aspects of BM structure, composition, and function regarding not only (i the dermoepidermal interface but also (ii the resident microvasculature, primarily focusing on the per se nonscaffold forming components perlecan and nidogen-1 and nidogen-2. Depletion or functional deficiencies of any BM component are lethal at some stage of development or around birth, though BM defects vary between organs and tissues. Lethality problems were overcome by developmental stage- and skin-specific gene targeting or by cell grafting and organotypic (3D cocultures of normal or defective cells, which allows recapitulating BM formation de novo. Thus, evidence is accumulating that BM assembly and turnover rely on mechanical properties and composition of the adjacent ECM and the dynamics of molecular assembly, including further “minor” local components, nidogens largely functioning as catalysts or molecular adaptors and perlecan as bridging stabilizer. Collectively, orchestration of BM assembly, remodeling, and the role of individual players herein are determined by the developmental, tissue-specific, or functional context.

  11. Membrane tension and peripheral protein density mediate membrane shape transitions

    Science.gov (United States)

    Shi, Zheng; Baumgart, Tobias

    2015-01-01

    Endocytosis is a ubiquitous eukaryotic membrane budding, vesiculation and internalization process fulfilling numerous roles including compensation of membrane area increase after bursts of exocytosis. The mechanism of the coupling between these two processes to enable homeostasis is not well understood. Recently, an ultrafast endocytosis (UFE) pathway was revealed with a speed significantly exceeding classical clathrin-mediated endocytosis (CME). Membrane tension reduction is a potential mechanism by which endocytosis can be rapidly activated at remote sites. Here, we provide experimental evidence for a mechanism whereby membrane tension reduction initiates membrane budding and tubulation mediated by endocytic proteins, such as endophilin A1. We find that shape instabilities occur at well-defined membrane tensions and surface densities of endophilin. From our data, we obtain a membrane shape stability diagram that shows remarkable consistency with a quantitative model. This model applies to all laterally diffusive curvature-coupling proteins and therefore a wide range of endocytic proteins.

  12. DIFFERENT APPROACHES TO CRYSTALLIZATION OF MEMBRANE PROTEINS

    Directory of Open Access Journals (Sweden)

    Prakash G. Doiphode

    2012-01-01

    Full Text Available Crystallography is more like an art than science. Crystallizing membrane proteins are a big challenge; membrane proteins are present in the cell membrane and serve as cell support. The most important feature of membrane protein is that it contains both hydrophobic and hydrophilic regions on its surface. They are generally much more difficult to study than soluble proteins. The problem becomes more difficult when trying to obtain crystals to determine the high resolution structures of membrane proteins. We want to utilize this opportunity to briefly examine various approaches for crystallization of membrane proteins. The important factors for determining the success of crystallization experiments for membrane proteins lies in the purification, preparation of membrane samples, the environment in which the crystals are grown and the technique used to grow the crystals. All the X-ray structures of membrane protein are grown from preparations of detergents by different methods developed to crystallize. In this review different techniques for the crystallization of membrane proteins are being described. The cubic phase method also known as in meso method is discussed along with other methods to understand about the crystallization of membrane proteins, its general applicability, salt, detergent and screening effects on crystallization. Low volumes as nano-liter of samples can be used for crystallization. The effects of different detergents on the crystallization of membrane protein, as well as the use of surfactants like polyoxyethylene. Approach based on the detergent complexation to prove the ability of cyclodextrins to remove detergent from ternary mixtures in order to get 2D crystals. Crystallization of membrane proteins using non-ionic surfactants as well as Lipidic sponge phase and with swollen lipidic mesophases is discussed to better understand the crystallization of membrane proteins.

  13. Proteins and Peptides in Biomimetic Polymeric Membranes

    DEFF Research Database (Denmark)

    Perez, Alfredo Gonzalez

    2013-01-01

    This chapter discusses recent advances and the main advantages of block copolymers for functional membrane protein reconstitution in biomimetic polymeric membranes. A rational approach to the reconstitution of membrane proteins in a functional form can be addressed by a more holistic view by usin...

  14. Assessment of proteolytic degradation of the basement membrane: a fragment of type IV collagen as a biochemical marker for liver fibrosis

    OpenAIRE

    Veidal Sanne S; Karsdal Morten A; Nawrocki Arkadiusz; Larsen Martin R; Dai Yueqin; Zheng Qinlong; Hägglund Per; Vainer Ben; Skjøt-Arkil Helene; Leeming Diana J

    2011-01-01

    Abstract Background Collagen deposition and an altered matrix metalloproteinase (MMP) expression profile are hallmarks of fibrosis. Type IV collagen is the most abundant structural basement membrane component of tissue, which increases 14-fold during fibrogenesis in the liver. Proteolytic degradation of collagens by proteases produces small fragments, so-called neoepitopes, which are released systemically. Technologies investigating MMP-generated fragments of collagens may provide more useful...

  15. Effect of membrane curvature on lateral distribution of membrane proteins

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    membrane tubes out of Giant Unilamellar lipid Vesicles (GUVs). The tube diameter can be tuned by aspirating the GUV into a micropipette for controlling the membrane tension. By using fluorescently labled proteins we have shown that sorting of proteins like e.g. FBAR onto tubes is significantly increased...

  16. First Identification of a Triple Corneal Dystrophy Association: Keratoconus, Epithelial Basement Membrane Corneal Dystrophy and Fuchs' Endothelial Corneal Dystrophy

    Directory of Open Access Journals (Sweden)

    Cosimo Mazzotta

    2014-09-01

    Full Text Available Purpose: To report the observation of a triple corneal dystrophy association consisting of keratoconus (KC, epithelial basement membrane corneal dystrophy (EBMCD and Fuchs' endothelial corneal dystrophy (FECD. Methods: A 55-year-old male patient was referred to our cornea service for blurred vision and recurrent foreign body sensation. He reported bilateral recurrent corneal erosions with diurnal visual fluctuations. He underwent corneal biomicroscopy, Scheimpflug tomography, in vivo HRT confocal laser scanning microscopy and genetic testing for TGFBI and ZEB1 mutations using direct DNA sequencing. Results: Biomicroscopic examination revealed the presence of subepithelial central and paracentral corneal opacities. The endothelium showed a bilateral flecked appearance, and the posterior corneal curvature suggested a possible concomitant ectatic disorder. Corneal tomography confirmed the presence of a stage II KC in both eyes. In vivo confocal laser scanning microscopy revealed a concomitant bilateral EBMCD with hyperreflective deposits in basal epithelial cells, subbasal Bowman's layer microfolds and ridges with truncated subbasal nerves as pseudodendritic elements. Stromal analysis revealed honeycomb edematous areas, and the endothelium showed a strawberry surface configuration typical of FECD. The genetic analysis resulted negative for TGFBI mutations and positive for a heterozygous mutation in exon 7 of the gene ZEB1. Conclusion: This is the first case reported in the literature in which KC, EBMCD and FECD are present in the same patient and associated with ZEB1 gene mutation. The triple association was previously established by means of morphological analysis of the cornea using corneal Scheimpflug tomography and in vivo HRT II confocal laser scanning microscopy.

  17. Characterization and mechanisms of photoageing-related changes in skin. Damages of basement membrane and dermal structures.

    Science.gov (United States)

    Amano, Satoshi

    2016-08-01

    Sun-exposed skin is characterized by superficial changes such as wrinkles, sagging and pigmentary changes, and also many internal changes in the structure and function of epidermis, basement membrane (BM) and dermis. These changes (so-called photoageing) are predominantly induced by the ultraviolet (UV) component of sunlight. Epidermis of UV-irradiated skin produced several enzymes such as matrix metalloproteinases (MMPs), urinary plasminogen activator (uPA)/plasmin and heparanase, which degrade dermal collagen fibres and elastic fibres in the dermis, and components of epidermal BM. The BM at the dermal-epidermal junction (DEJ) controls dermal-epidermal signalling and plays an important role in the maintenance of a healthy epidermis and dermis. BM is repetitively damaged in sun-exposed skin compared with unexposed skin, leading to epidermal and dermal deterioration and accelerated skin ageing. UV exposure also induces an increase in vascular endothelial growth factor (VEGF), an angiogenic factor, while thrombospondin-1 (TSP-1), an anti-angiogenic factor, is decreased; these changes induce angiogenesis in papillary dermis with increased migration of elastase-positive leucocytes, leading to dermal elastic fibre damage. Elastic fibres, such as oxytalan fibres in papillary dermis, are associated with not only skin resilience, but also skin surface texture, and elastic fibre formation by fibroblasts is facilitated by increased expression of fibulin-5. Thus, induction of fibulin-5 expression is a damage-repair mechanism, and fibulin-5 is an early marker of photoaged skin. UV-induced skin damage is cumulative and leads to premature ageing of skin. However, appropriate daily skincare may ameliorate photoageing by inhibiting processes causing damage and enhancing repair processes. PMID:27539897

  18. Subepithelial basement membrane thickness in patients with normal colonic mucosal appearance in colonoscopy:Results from southern Turkey

    Institute of Scientific and Technical Information of China (English)

    Fazilet Kayaselcuk; Ender Serin; Yǖksel Gumurdulu; Birol Ozer; Ilhan Tuncer; Sedat Boyacioglu

    2004-01-01

    AIM: Our aims were to determine the normal limits of subepithelial basement membrane (SEBM) thickness in order to more accurately diagnose collagenous colitis in the population from southern Turkey and to investigate into links between SEBM thickness and age, and sex.METHODS: The study included 100 patients (mean age 50.0±13.3 years; male, 34; female, 66) with miscellaneous gastrointestinal symptoms, and normal colonic mucosal appearance in colonoscopic evaluation. Biopsies were taken from five different regions of the colon. SEBM was measured with a calibrated eyepiece on specimens prepared with specific stains for collagen. Intensity of inflammatory cells was graded semiquantitatively. Differences in SEBM thickness among the different colon regions, and relationships between SEBM thickness and age, sex, and density of inflammatory cells were statistically evaluated.RESULTS: The cecum and rectum showed the largest amounts of infiltrate. None of the specimens showed histologic findings of collagenous colitis. The SEBM thicknesses measured for each case ranged from 3-20 μm. The biggest thickness was observed in rectal mucosa (median value: 10 μm).Cecum and ascending colon showed similar SEBM thickness (median value: 5 μm). SEBM thickness was not correlated with patient age or sex, but was positively correlated with the intensity of inflammatory cells in each colon segment.CONCLUSION: In this patient group from southern Turkey,SEBM was thickest in the rectum. Our results indicate that,in this population, SEBM thickness is not correlated with age or sex, but is positively correlated with severity of inflammation. The findings also support the concept that measuring SEBM thickness at one segment in the colon is inadequate and may be misleading.

  19. Kinetics and Thermodynamics of Membrane Protein Folding

    Directory of Open Access Journals (Sweden)

    Ernesto A. Roman

    2014-03-01

    Full Text Available Understanding protein folding has been one of the great challenges in biochemistry and molecular biophysics. Over the past 50 years, many thermodynamic and kinetic studies have been performed addressing the stability of globular proteins. In comparison, advances in the membrane protein folding field lag far behind. Although membrane proteins constitute about a third of the proteins encoded in known genomes, stability studies on membrane proteins have been impaired due to experimental limitations. Furthermore, no systematic experimental strategies are available for folding these biomolecules in vitro. Common denaturing agents such as chaotropes usually do not work on helical membrane proteins, and ionic detergents have been successful denaturants only in few cases. Refolding a membrane protein seems to be a craftsman work, which is relatively straightforward for transmembrane β-barrel proteins but challenging for α-helical membrane proteins. Additional complexities emerge in multidomain membrane proteins, data interpretation being one of the most critical. In this review, we will describe some recent efforts in understanding the folding mechanism of membrane proteins that have been reversibly refolded allowing both thermodynamic and kinetic analysis. This information will be discussed in the context of current paradigms in the protein folding field.

  20. Effects of protein crowding on membrane systems.

    Science.gov (United States)

    Guigas, Gernot; Weiss, Matthias

    2016-10-01

    Cellular membranes are typically decorated with a plethora of embedded and adsorbed macromolecules, e.g. proteins, that participate in numerous vital processes. With typical surface densities of 30,000 proteins per μm(2) cellular membranes are indeed crowded places that leave only few nanometers of private space for individual proteins. Here, we review recent advances in our understanding of protein crowding in membrane systems. We first give a brief overview on state-of-the-art approaches in experiment and simulation that are frequently used to study crowded membranes. After that, we review how crowding can affect diffusive transport of proteins and lipids in membrane systems. Next, we discuss lipid and protein sorting in crowded membrane systems, including effects like protein cluster formation, phase segregation, and lipid droplet formation. Subsequently, we highlight recent progress in uncovering crowding-induced conformational changes of membranes, e.g. membrane budding and vesicle formation. Finally, we give a short outlook on potential future developments in the field of crowded membrane systems. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:26724385

  1. Tandem Facial Amphiphiles for Membrane Protein Stabilization

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Gotfryd, Kamil; Pacyna, Jennifer;

    2010-01-01

    We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles....

  2. Active Nuclear Import of Membrane Proteins Revisited

    NARCIS (Netherlands)

    Laba, Justyna K; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the m

  3. Activity assay of membrane transport proteins

    Institute of Scientific and Technical Information of China (English)

    Hao Xie

    2008-01-01

    Membrane transport proteins are integral membrane proteins and considered as potential drug targets. Activity assay of transport proteins is essential for developing drugs to target these proteins. Major issues related to activity assessment of transport proteins include availability of transporters,transport activity of transporters, and interactions between ligands and transporters. Researchers need to consider the physiological status of proteins (bound in lipid membranes or purified), availability and specificity of substrates, and the purpose of the activity assay (screening, identifying, or comparing substrates and inhibitors) before choosing appropriate assay strategies and techniques. Transport proteins bound in vesicular membranes can be assayed for transporting substrate across membranes by means of uptake assay or entrance counterflow assay. Alternatively, transport proteins can be assayed for interactions with ligands by using techniques such as isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, or surface plasmon resonance. Other methods and techniques such as fluorometry, scintillation proximity assay, electrophysiological assay, or stopped-flow assay could also be used for activity assay of transport proteins. In this paper the major strategies and techniques for activity assessment of membrane transport proteins are reviewed.

  4. Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition

    Energy Technology Data Exchange (ETDEWEB)

    Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Villadsen, Rene; Rank, Fritz; Bissell, Mina J.; Petersen, Ole William

    2001-10-04

    The signals that determine the correct polarity of breast epithelial structures in vivo are not understood. We have shown previously that luminal epithelial cells can be polarized when cultured within a reconstituted basement membrane gel. We reasoned that such cues in vivo may be given by myoepithelial cells. Accordingly, we used an assay where luminal epithelial cells are incorrectly polarized to test this hypothesis. We show that culturing human primary luminal epithelial cells within collagen-I gels leads to formation of structures with no lumina and with reverse polarity as judged by dual stainings for sialomucin, epithelial specific antigen or occludin. No basement membrane is deposited, and {beta}4-integrin staining is negative. Addition of purified human myoepithelial cells isolated from normal glands corrects the inverse polarity, and leads to formation of double-layered acini with central lumina. Among the laminins present in the human breast basement membrane (laminin-1, -5 and -10/11), laminin-1 was unique in its ability to substitute for myoepithelial cells in polarity reversal. Myoepithelial cells were purified also from four different breast cancer sources including a biphasic cell line. Three out of four samples either totally lacked the ability to interact with luminal epithelial cells, or conveyed only correction of polarity in a fraction of acini. This behavior was directly related to the ability of the tumor myoepithelial cells to produce {alpha}-1 chain of laminin. In vivo, breast carcinomas were either negative for laminin-1 (7/12 biopsies) or showed a focal, fragmented deposition of a less intensely stained basement membrane (5/12 biopsies). Dual staining with myoepithelial markers revealed that tumorassociated myoepithelial cells were either negative or weakly positive for expression of laminin-1, establishing a strong correlation between loss of laminin-1 and breast cancer. We conclude that the double-layered breast acinus may be

  5. MMP Mediated Degradation of Type IV Collagen Alpha 1 and Alpha 3 Chains Reflects Basement Membrane Remodeling in Experimental and Clinical Fibrosis - Validation of Two Novel Biomarker Assays

    DEFF Research Database (Denmark)

    Sand, Jannie Marie; Larsen, Lise Skakkebæk; Hogaboam, Cory;

    2013-01-01

    Fibrosis is characterized by excessive tissue remodeling resulting from altered expression of various growth factors, cytokines and proteases. We hypothesized that matrix metalloproteinase (MMP) mediated degradation of type IV collagen, a main component of the basement membrane, will release...... peptide fragments (neo-epitopes) into the circulation. Here we present the development of two competitive enzyme-linked immunosorbent assays (ELISAs) for assessing the levels of specific fragments of type IV collagen α1 (C4M12a1) and α3 (C4M12a3) chains in serum as indicators of fibrosis....

  6. Functional dynamics of cell surface membrane proteins

    Science.gov (United States)

    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

  7. Glasslike Membrane Protein Diffusion in a Crowded Membrane.

    Science.gov (United States)

    Munguira, Ignacio; Casuso, Ignacio; Takahashi, Hirohide; Rico, Felix; Miyagi, Atsushi; Chami, Mohamed; Scheuring, Simon

    2016-02-23

    Many functions of the plasma membrane depend critically on its structure and dynamics. Observation of anomalous diffusion in vivo and in vitro using fluorescence microscopy and single particle tracking has advanced our concept of the membrane from a homogeneous fluid bilayer with freely diffusing proteins to a highly organized crowded and clustered mosaic of lipids and proteins. Unfortunately, anomalous diffusion could not be related to local molecular details given the lack of direct and unlabeled molecular observation capabilities. Here, we use high-speed atomic force microscopy and a novel analysis methodology to analyze the pore forming protein lysenin in a highly crowded environment and document coexistence of several diffusion regimes within one membrane. We show the formation of local glassy phases, where proteins are trapped in neighbor-formed cages for time scales up to 10 s, which had not been previously experimentally reported for biological membranes. Furthermore, around solid-like patches and immobile molecules a slower glass phase is detected leading to protein trapping and creating a perimeter of decreased membrane diffusion. PMID:26859708

  8. Polyene antibiotic that inhibits membrane transport proteins.

    Science.gov (United States)

    te Welscher, Yvonne Maria; van Leeuwen, Martin Richard; de Kruijff, Ben; Dijksterhuis, Jan; Breukink, Eefjan

    2012-07-10

    The limited therapeutic arsenal and the increase in reports of fungal resistance to multiple antifungal agents have made fungal infections a major therapeutic challenge. The polyene antibiotics are the only group of antifungal antibiotics that directly target the plasma membrane via a specific interaction with the main fungal sterol, ergosterol, often resulting in membrane permeabilization. In contrast to other polyene antibiotics that form pores in the membrane, the mode of action of natamycin has remained obscure but is not related to membrane permeabilization. Here, we demonstrate that natamycin inhibits growth of yeasts and fungi via the immediate inhibition of amino acid and glucose transport across the plasma membrane. This is attributable to ergosterol-specific and reversible inhibition of membrane transport proteins. It is proposed that ergosterol-dependent inhibition of membrane proteins is a general mode of action of all the polyene antibiotics, of which some have been shown additionally to permeabilize the plasma membrane. Our results imply that sterol-protein interactions are fundamentally important for protein function even for those proteins that are not known to reside in sterol-rich domains. PMID:22733749

  9. Statistical thermodynamics of membrane bending mediated protein-protein attraction

    OpenAIRE

    Chou, Tom; Kim, Ken S.; Oster, George

    1999-01-01

    Integral membrane proteins deform the surrounding bilayer creating long-ranged forces that influence distant proteins. These forces can be attractive or repulsive, depending on the proteins' shape, height, contact angle with the bilayer, as well as the local membrane curvature. Although interaction energies are not pairwise additive, for sufficiently low protein density, thermodynamic properties depend only upon pair interactions. Here, we compute pair interaction potentials and entropic cont...

  10. Flagellar membrane proteins in kinetoplastid parasites.

    Science.gov (United States)

    Landfear, Scott M; Tran, Khoa D; Sanchez, Marco A

    2015-09-01

    All kinetoplastid parasites, including protozoa such as Leishmania species, Trypanosoma brucei, and Trypanosoma cruzi that cause devastating diseases in humans and animals, are flagellated throughout their life cycles. Although flagella were originally thought of primarily as motility organelles, flagellar functions in other critical processes, especially in sensing and signal transduction, have become more fully appreciated in the recent past. The flagellar membrane is a highly specialized subdomain of the surface membrane, and flagellar membrane proteins are likely to be critical components for all the biologically important roles of flagella. In this review, we summarize recent discoveries relevant to flagellar membrane proteins in these parasites, including the identification of such proteins, investigation of their biological functions, and mechanisms of selective trafficking to the flagellar membrane. Prospects for future investigations and current unsolved problems are highlighted.

  11. Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp

    Directory of Open Access Journals (Sweden)

    R. Puccia

    1999-05-01

    Full Text Available We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37oC, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB, suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme.

  12. The central role of vascular extracellular matrix and basement membrane remodeling in metabolic syndrome and type 2 diabetes: the matrix preloaded

    Directory of Open Access Journals (Sweden)

    Tyagi Suresh C

    2005-06-01

    Full Text Available Abstract The vascular endothelial basement membrane and extra cellular matrix is a compilation of different macromolecules organized by physical entanglements, opposing ionic charges, chemical covalent bonding, and cross-linking into a biomechanically active polymer. These matrices provide a gel-like form and scaffolding structure with regional tensile strength provided by collagens, elasticity by elastins, adhesiveness by structural glycoproteins, compressibility by proteoglycans – hyaluronans, and communicability by a family of integrins, which exchanges information between cells and between cells and the extracellular matrix of vascular tissues. Each component of the extracellular matrix and specifically the capillary basement membrane possesses unique structural properties and interactions with one another, which determine the separate and combined roles in the multiple diabetic complications or diabetic opathies. Metabolic syndrome, prediabetes, type 2 diabetes mellitus, and their parallel companion (atheroscleropathy are associated with multiple metabolic toxicities and chronic injurious stimuli. The adaptable quality of a matrix or form genetically preloaded with the necessary information to communicate and respond to an ever-changing environment, which supports the interstitium, capillary and arterial vessel wall is individually examined.

  13. A Systematic Assessment of Mature MBP in Membrane Protein Production: Overexpression, Membrane targeting and Purification

    OpenAIRE

    Hu, Jian; Qin, Huajun; Gao, Fei Philip; Cross, Timothy A

    2011-01-01

    Obtaining enough membrane protein in native or native-like status is still a challenge in membrane protein structure biology. Maltose binding protein (MBP) has been widely used as a fusion partner in improving membrane protein production. In the present work, a systematic assessment on the application of mature MBP (mMBP) for membrane protein overexpression and purification was performed on 42 membrane proteins, most of which showed no or poor expression level in membrane fraction fused with ...

  14. Detergents in Membrane Protein Purification and Crystallisation.

    Science.gov (United States)

    Anandan, Anandhi; Vrielink, Alice

    2016-01-01

    Detergents play a significant role in structural and functional characterisation of integral membrane proteins (IMPs). IMPs reside in the biological membranes and exhibit a great variation in their structural and physical properties. For in vitro biophysical studies, structural and functional analyses, IMPs need to be extracted from the membrane lipid bilayer environment in which they are found and purified to homogeneity while maintaining a folded and functionally active state. Detergents are capable of successfully solubilising and extracting the IMPs from the membrane bilayers. A number of detergents with varying structure and physicochemical properties are commercially available and can be applied for this purpose. Nevertheless, it is important to choose a detergent that is not only able to extract the membrane protein but also provide an optimal environment while retaining the correct structural and physical properties of the protein molecule. Choosing the best detergent for this task can be made possible by understanding the physical and chemical properties of the different detergents and their interaction with the IMPs. In addition, understanding the mechanism of membrane solubilisation and protein extraction along with crystallisation requirements, if crystallographic studies are going to be undertaken, can help in choosing the best detergent for the purpose. This chapter aims to present the fundamental properties of detergents and highlight information relevant to IMP crystallisation. The first section of the chapter reviews the physicochemical properties of detergents and parameters essential for predicting their behaviour in solution. The second section covers the interaction of detergents with the biologic membranes and proteins followed by their role in membrane protein crystallisation. The last section will briefly cover the types of detergent and their properties focusing on custom designed detergents for membrane protein studies. PMID:27553232

  15. Detergent-Free Membrane Protein Purification.

    Science.gov (United States)

    Rothnie, Alice J

    2016-01-01

    Membrane proteins are localized within a lipid bilayer; in order to purify them for functional and structural studies the first step must involve solubilizing or extracting the protein from these lipids. To date this has been achieved using detergents which disrupt the bilayer and bind to the protein in the transmembrane region. However finding conditions for optimal extraction, without destabilizing protein structure, is time consuming and expensive. Here we present a recently-developed method using a styrene-maleic acid (SMA) co-polymer instead of detergents. The SMA co-polymer extracts membrane proteins in a small disc of lipid bilayer which can be used for affinity chromatography purification, thus enabling the purification of membrane proteins while maintaining their native lipid bilayer environment. PMID:27485341

  16. Efficient preparation and analysis of membrane and membrane protein systems.

    Science.gov (United States)

    Javanainen, Matti; Martinez-Seara, Hector

    2016-10-01

    Molecular dynamics (MD) simulations have become a highly important technique to consider lipid membrane systems, and quite often they provide considerable added value to laboratory experiments. Rapid development of both software and hardware has enabled the increase of time and size scales reachable by MD simulations to match those attainable by several accurate experimental techniques. However, until recently, the quality and maturity of software tools available for building membrane models for simulations as well as analyzing the results of these simulations have seriously lagged behind. Here, we discuss the recent developments of such tools from the end-users' point of view. In particular, we review the software that can be employed to build lipid bilayers and other related structures with or without embedded membrane proteins to be employed in MD simulations. Additionally, we provide a brief critical insight into force fields and MD packages commonly used for membrane and membrane protein simulations. Finally, we list analysis tools that can be used to study the properties of membrane and membrane protein systems. In all these points we comment on the respective compatibility of the covered tools. We also share our opinion on the current state of the available software. We briefly discuss the most commonly employed tools and platforms on which new software can be built. We conclude the review by providing a few ideas and guidelines on how the development of tools can be further boosted to catch up with the rapid pace at which the field of membrane simulation progresses. This includes improving the compatibility between software tools and promoting the openness of the codes on which these applications rely. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:26947184

  17. NMR of Membrane Proteins: Beyond Crystals.

    Science.gov (United States)

    Rajesh, Sundaresan; Overduin, Michael; Bonev, Boyan B

    2016-01-01

    Membrane proteins are essential for the flow of signals, nutrients and energy between cells and between compartments of the cell. Their mechanisms can only be fully understood once the precise structures, dynamics and interactions involved are defined at atomic resolution. Through advances in solution and solid state NMR spectroscopy, this information is now available, as demonstrated by recent studies of stable peripheral and transmembrane proteins. Here we highlight recent cases of G-protein coupled receptors, outer membrane proteins, such as VDAC, phosphoinositide sensors, such as the FAPP-1 pleckstrin homology domain, and enzymes including the metalloproteinase MMP-12. The studies highlighted have resulted in the determination of the 3D structures, dynamical properties and interaction surfaces for membrane-associated proteins using advanced isotope labelling strategies, solubilisation systems and NMR experiments designed for very high field magnets. Solid state NMR offers further insights into the structure and multimeric assembly of membrane proteins in lipid bilayers, as well as into interactions with ligands and targets. Remaining challenges for wider application of NMR to membrane structural biology include the need for overexpression and purification systems for the production of isotope-labelled proteins with fragile folds, and the availability of only a few expensive perdeuterated detergents.Step changes that may transform the field include polymers, such as styrene maleic acid, which obviate the need for detergent altogether, and allow direct high yield purification from cells or membranes. Broader demand for NMR may be facilitated by MODA software, which instantly predicts membrane interactive residues that can subsequently be validated by NMR. In addition, recent developments in dynamic nuclear polarization NMR instrumentation offer a remarkable sensitivity enhancement from low molarity samples and cell surfaces. These advances illustrate the current

  18. NMR of Membrane Proteins: Beyond Crystals.

    Science.gov (United States)

    Rajesh, Sundaresan; Overduin, Michael; Bonev, Boyan B

    2016-01-01

    Membrane proteins are essential for the flow of signals, nutrients and energy between cells and between compartments of the cell. Their mechanisms can only be fully understood once the precise structures, dynamics and interactions involved are defined at atomic resolution. Through advances in solution and solid state NMR spectroscopy, this information is now available, as demonstrated by recent studies of stable peripheral and transmembrane proteins. Here we highlight recent cases of G-protein coupled receptors, outer membrane proteins, such as VDAC, phosphoinositide sensors, such as the FAPP-1 pleckstrin homology domain, and enzymes including the metalloproteinase MMP-12. The studies highlighted have resulted in the determination of the 3D structures, dynamical properties and interaction surfaces for membrane-associated proteins using advanced isotope labelling strategies, solubilisation systems and NMR experiments designed for very high field magnets. Solid state NMR offers further insights into the structure and multimeric assembly of membrane proteins in lipid bilayers, as well as into interactions with ligands and targets. Remaining challenges for wider application of NMR to membrane structural biology include the need for overexpression and purification systems for the production of isotope-labelled proteins with fragile folds, and the availability of only a few expensive perdeuterated detergents.Step changes that may transform the field include polymers, such as styrene maleic acid, which obviate the need for detergent altogether, and allow direct high yield purification from cells or membranes. Broader demand for NMR may be facilitated by MODA software, which instantly predicts membrane interactive residues that can subsequently be validated by NMR. In addition, recent developments in dynamic nuclear polarization NMR instrumentation offer a remarkable sensitivity enhancement from low molarity samples and cell surfaces. These advances illustrate the current

  19. Helix-packing motifs in membrane proteins.

    Science.gov (United States)

    Walters, R F S; DeGrado, W F

    2006-09-12

    The fold of a helical membrane protein is largely determined by interactions between membrane-imbedded helices. To elucidate recurring helix-helix interaction motifs, we dissected the crystallographic structures of membrane proteins into a library of interacting helical pairs. The pairs were clustered according to their three-dimensional similarity (rmsd universe of common transmembrane helix-pairing motifs is relatively simple. The largest cluster, which comprises 29% of the library members, consists of an antiparallel motif with left-handed packing angles, and it is frequently stabilized by packing of small side chains occurring every seven residues in the sequence. Right-handed parallel and antiparallel structures show a similar tendency to segregate small residues to the helix-helix interface but spaced at four-residue intervals. Position-specific sequence propensities were derived for the most populated motifs. These structural and sequential motifs should be quite useful for the design and structural prediction of membrane proteins.

  20. Collective Molecular Dynamics in Proteins and Membranes

    CERN Document Server

    Rheinstadter, Maikel C

    2008-01-01

    The understanding of dynamics and functioning of biological membranes and in particular of membrane embedded proteins is one of the most fundamental problems and challenges in modern biology and biophysics. In particular the impact of membrane composition and properties and of structure and dynamics of the surrounding hydration water on protein function is an upcoming hot topic, which can be addressed by modern experimental and computational techniques. Correlated molecular motions might play a crucial role for the understanding of, for instance, transport processes and elastic properties, and might be relevant for protein function. Experimentally that involves determining dispersion relations for the different molecular components, i.e., the length scale dependent excitation frequencies and relaxation rates. Only very few experimental techniques can access dynamical properties in biological materials on the nanometer scale, and resolve dynamics of lipid molecules, hydration water molecules and proteins and t...

  1. Crystallization of Membrane Proteins by Vapor Diffusion

    Science.gov (United States)

    Delmar, Jared A.; Bolla, Jani Reddy; Su, Chih-Chia; Yu, Edward W.

    2016-01-01

    X-ray crystallography remains the most robust method to determine protein structure at the atomic level. However, the bottlenecks of protein expression and purification often discourage further study. In this chapter, we address the most common problems encountered at these stages. Based on our experiences in expressing and purifying antimicrobial efflux proteins, we explain how a pure and homogenous protein sample can be successfully crystallized by the vapor diffusion method. We present our current protocols and methodologies for this technique. Case studies show step-by-step how we have overcome problems related to expression and diffraction, eventually producing high quality membrane protein crystals for structural determinations. It is our hope that a rational approach can be made of the often anecdotal process of membrane protein crystallization. PMID:25950974

  2. Glycosylation of human glomerular basement membrane collagen: increased content of hexose in ketoamine linkage and unaltered hydroxylysine-O-glycosides in patients with diabetes.

    Science.gov (United States)

    Uitto, J; Perejda, A J; Grant, G A; Rowold, E A; Kilo, C; Williamson, J R

    1982-01-01

    To study the glycosylation of glomerular basement membrane collagen (GBMC) in diabetes, kidneys were obtained at autopsy from 5 patients with insulin-requiring diabetes of long duration and diabetic complications, and from 5 control subjects. Glomeruli were prepared by sieving and collagen was isolated by limited pepsin proteolysis followed by salt precipitations. Amino acid analyses of the collagen preparations, after acid hydrolysis, indicated a composition consistent with that of type IV collagen. No differences in the relative contents of various amino acids, and in particular, 3-hydroxyproline, 4-hydroxyproline and hydroxylysine, were noted between diabetic and control samples. Non-enzymatic glucosylation was assessed by measuring hexose in ketoamine linkage with thiobarbituric acid after conversion to 5-hydroxymethylfurfural. In 4 of the 5 patients studied, glucosylation values exceeded the mean +2 S.D. of the controls; in the fifth subject glucosylation was in the high normal range. No correlation between the severity of diabetes and hexose content of GBMC was noted, however. In further studies, enzymatic glycosylation of GBMC was assayed after alkaline hydrolysis by separation of glucosylgalactosyl-O-hydroxylysine, galactosyl-O-hydroxylysine, and unsubstituted hydroxylysine in an amino acid analyzer. No differences in the relative contents of hydroxylysine-O-glycosides were evident between diabetic and control GBMC. The results suggest that non-enzymatic glucosylation, but not glycosylation catalyzed by collagen glucosyl and galactosyl transferases, is increased in diabetes. The increased carbohydrate content of collagen may lead to decreased turnover and/or excessive accumulations of basement membrane collagen thus contributing to the vascular complications of diabetes. PMID:6218960

  3. Transmembrane protein sorting driven by membrane curvature

    Science.gov (United States)

    Strahl, H.; Ronneau, S.; González, B. Solana; Klutsch, D.; Schaffner-Barbero, C.; Hamoen, L. W.

    2015-11-01

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization.

  4. Model-building codes for membrane proteins.

    Energy Technology Data Exchange (ETDEWEB)

    Shirley, David Noyes; Hunt, Thomas W.; Brown, W. Michael; Schoeniger, Joseph S. (Sandia National Laboratories, Livermore, CA); Slepoy, Alexander; Sale, Kenneth L. (Sandia National Laboratories, Livermore, CA); Young, Malin M. (Sandia National Laboratories, Livermore, CA); Faulon, Jean-Loup Michel; Gray, Genetha Anne (Sandia National Laboratories, Livermore, CA)

    2005-01-01

    We have developed a novel approach to modeling the transmembrane spanning helical bundles of integral membrane proteins using only a sparse set of distance constraints, such as those derived from MS3-D, dipolar-EPR and FRET experiments. Algorithms have been written for searching the conformational space of membrane protein folds matching the set of distance constraints, which provides initial structures for local conformational searches. Local conformation search is achieved by optimizing these candidates against a custom penalty function that incorporates both measures derived from statistical analysis of solved membrane protein structures and distance constraints obtained from experiments. This results in refined helical bundles to which the interhelical loops and amino acid side-chains are added. Using a set of only 27 distance constraints extracted from the literature, our methods successfully recover the structure of dark-adapted rhodopsin to within 3.2 {angstrom} of the crystal structure.

  5. Major intrinsic proteins in biomimetic membranes.

    Science.gov (United States)

    Nielsen, Claus Hélix

    2010-01-01

    Biological membranes define the structural and functional boundaries in living cells and their organelles. The integrity of the cell depends on its ability to separate inside from outside and yet at the same time allow massive transport of matter in and out the cell. Nature has elegantly met this challenge by developing membranes in the form of lipid bilayers in which specialized transport proteins are incorporated. This raises the question: is it possible to mimic biological membranes and create a membrane based sensor and/or separation device? In the development of a biomimetic sensor/separation technology, a unique class of membrane transport proteins is especially interesting-the major intrinsic proteins (MIPs). Generally, MIPs conduct water molecules and selected solutes in and out of the cell while preventing the passage of other solutes, a property critical for the conservation of the cells internal pH and salt concentration. Also known as water channels or aquaporins they are highly efficient membrane pore proteins some of which are capable of transporting water at very high rates up to 10(9) molecules per second. Some MIPs transport other small, uncharged solutes, such as glycerol and other permeants such as carbon dioxide, nitric oxide, ammonia, hydrogen peroxide and the metalloids antimonite, arsenite, silicic and boric acid depending on the effective restriction mechanism of the protein. The flux properties of MIPs thus lead to the question ifMIPs can be used in separation devices or as sensor devices based on, e.g., the selective permeation of metalloids. In principle a MIP based membrane sensor/separation device requires the supporting biomimetic matrix to be virtually impermeable to anything but water or the solute in question. In practice, however, a biomimetic support matrix will generally have finite permeabilities to both electrolytes and non-electrolytes. The feasibility of a biomimetic MIP device thus depends on the relative transport

  6. Revolutionizing membrane protein overexpression in bacteria

    NARCIS (Netherlands)

    Schlegel, Susan; Klepsch, Mirjam; Gialama, Dimitra; Wickstrom, David; Slotboom, Dirk Jan; de Gier, Jan-Willem; Wickström, David

    2010-01-01

    The bacterium Escherichia coli is the most widely used expression host for overexpression trials of membrane proteins. Usually, different strains, culture conditions and expression regimes are screened for to identify the optimal overexpression strategy. However, yields are often not satisfactory, e

  7. Membrane Protein Solubilization and Composition of Protein Detergent Complexes.

    Science.gov (United States)

    Duquesne, Katia; Prima, Valérie; Sturgis, James N

    2016-01-01

    Membrane proteins are typically expressed in heterologous systems with a view to in vitro characterization. A critical step in the preparation of membrane proteins after expression in any system is the solubilization of the protein in aqueous solution, typically using detergents and lipids, to obtain the protein in a form suitable for purification, structural or functional analysis. This process is particularly difficult as the objective is to prepare the protein in an unnatural environment, a protein detergent complex, separating it from its natural lipid partners while causing the minimum destabilization or modification of the structure. Although the process is difficult, and relatively hard to master, an increasing number of membrane proteins have been successfully isolated after expression in a wide variety of systems. In this chapter we give a general protocol for preparing protein detergent complexes that is aimed at guiding the reader through the different critical steps. In the second part of the chapter we illustrate how to analyze the composition of protein detergent complexes; this analysis is important as it has been found that compositional variation often causes irreproducible results. PMID:27485340

  8. A framework for protein and membrane interactions

    Directory of Open Access Journals (Sweden)

    Giorgio Bacci

    2009-11-01

    Full Text Available We introduce the BioBeta Framework, a meta-model for both protein-level and membrane-level interactions of living cells. This formalism aims to provide a formal setting where to encode, compare and merge models at different abstraction levels; in particular, higher-level (e.g. membrane activities can be given a formal biological justification in terms of low-level (i.e., protein interactions. A BioBeta specification provides a protein signature together a set of protein reactions, in the spirit of the kappa-calculus. Moreover, the specification describes when a protein configuration triggers one of the only two membrane interaction allowed, that is "pinch" and "fuse". In this paper we define the syntax and semantics of BioBeta, analyse its properties, give it an interpretation as biobigraphical reactive systems, and discuss its expressivity by comparing with kappa-calculus and modelling significant examples. Notably, BioBeta has been designed after a bigraphical metamodel for the same purposes. Hence, each instance of the calculus corresponds to a bigraphical reactive system, and vice versa (almost. Therefore, we can inherith the rich theory of bigraphs, such as the automatic construction of labelled transition systems and behavioural congruences.

  9. A framework for protein and membrane interactions

    CERN Document Server

    Bacci, Giorgio; Miculan, Marino; 10.4204/EPTCS.11.2

    2009-01-01

    We introduce the BioBeta Framework, a meta-model for both protein-level and membrane-level interactions of living cells. This formalism aims to provide a formal setting where to encode, compare and merge models at different abstraction levels; in particular, higher-level (e.g. membrane) activities can be given a formal biological justification in terms of low-level (i.e., protein) interactions. A BioBeta specification provides a protein signature together a set of protein reactions, in the spirit of the kappa-calculus. Moreover, the specification describes when a protein configuration triggers one of the only two membrane interaction allowed, that is "pinch" and "fuse". In this paper we define the syntax and semantics of BioBeta, analyse its properties, give it an interpretation as biobigraphical reactive systems, and discuss its expressivity by comparing with kappa-calculus and modelling significant examples. Notably, BioBeta has been designed after a bigraphical metamodel for the same purposes. Hence, each ...

  10. Correlated Diffusion of Membrane Proteins and Their Effect on Membrane Viscosity

    OpenAIRE

    Oppenheimer, Naomi; Diamant, Haim

    2009-01-01

    We extend the Saffman theory of membrane hydrodynamics to account for the correlated motion of membrane proteins, along with the effect of protein concentration on that correlation and on the response of the membrane to stresses. Expressions for the coupling diffusion coefficients of protein pairs and their concentration dependence are derived in the limit of small protein size relative to the inter-protein separation. The additional role of membrane viscosity as determining the characteristi...

  11. Subdiffusion of proteins and oligomers on membranes

    Science.gov (United States)

    Lepzelter, David; Zaman, Muhammad

    2012-11-01

    Diffusion of proteins on lipid membranes plays a central role in cell signaling processes. From a mathematical perspective, most membrane diffusion processes are explained by the Saffman-Delbrück theory. However, recent studies have suggested a major limitation in the theoretical framework, the lack of complexity in the modeled lipid membrane. Lipid domains (sometimes termed membrane rafts) are known to slow protein diffusion, but there have been no quantitative theoretical examinations of how much diffusion is slowed in a general case. We provide an overall theoretical framework for confined-domain ("corralled") diffusion. Further, there have been multiple apparent contradictions of the basic conclusions of Saffman and Delbrück, each involving cases in which a single protein or an oligomer has multiple transmembrane regions passing through a lipid phase barrier. We present a set of corrections to the Saffman-Delbrück theory to account for these experimental observations. Our corrections are able to provide a quantitative explanation of numerous cellular signaling processes that have been considered beyond the scope of the Saffman-Delbrück theory, and may be extendable to other forms of subdiffusion.

  12. Identification of extracellularly phosphorylated membrane proteins.

    Science.gov (United States)

    Burghoff, Sandra; Willberg, Wibke; Schrader, Jürgen

    2015-10-01

    Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling. PMID:26152529

  13. Protein permeation through an electrically tunable membrane

    Science.gov (United States)

    Jou, Ining A.; Melnikov, Dmitriy V.; Gracheva, Maria E.

    2016-05-01

    Protein filtration is important in many fields of science and technology such as medicine, biology, chemistry, and engineering. Recently, protein separation and filtering with nanoporous membranes has attracted interest due to the possibility of fast separation and high throughput volume. This, however, requires understanding of the protein’s dynamics inside and in the vicinity of the nanopore. In this work, we utilize a Brownian dynamics approach to study the motion of the model protein insulin in the membrane–electrolyte electrostatic potential. We compare the results of the atomic model of the protein with the results of a coarse-grained and a single-bead model, and find that the coarse-grained representation of protein strikes the best balance between the accuracy of the results and the computational effort required. Contrary to common belief, we find that to adequately describe the protein, a single-bead model cannot be utilized without a significant effort to tabulate the simulation parameters. Similar to results for nanoparticle dynamics, our findings also indicate that the electric field and the electro-osmotic flow due to the applied membrane and electrolyte biases affect the capture and translocation of the biomolecule by either attracting or repelling it to or from the nanopore. Our computational model can also be applied to other types of proteins and separation conditions.

  14. Exploiting Microbeams for Membrane Protein Structure Determination.

    Science.gov (United States)

    Warren, Anna J; Axford, Danny; Paterson, Neil G; Owen, Robin L

    2016-01-01

    A reproducible, and sample independent means of predictably obtaining large, well-ordered crystals has proven elusive in macromolecular crystallography. In the structure determination pipeline, crystallisation often proves to be a rate-limiting step, and the process of obtaining even small or badly ordered crystals can prove time-consuming and laborious. This is particularly true in the field of membrane protein crystallography and this is reflected in the limited number of unique membrane protein structures deposited in the protein data bank (less than 650 by June 2016 - http://blanco.biomol.uci.edu/mpstruc ). Over recent years the requirement for, and time and cost associated with obtaining, large crystals has been partially alleviated through the development of beamline instrumentation allowing data collection, and structure solution, from ever-smaller crystals. Advances in several areas have led to a step change in what might be considered achievable during a synchrotron trip over the last decade. This chapter will briefly review the current status of the field, the tools available to ease data collection and processing, and give some examples of exploitation of these for membrane protein microfocus macromolecular crystallography. PMID:27553238

  15. Membrane topology and insertion of membrane proteins : Search for topogenic signals

    NARCIS (Netherlands)

    Geest, Marleen van; Lolkema, Juke S.

    2000-01-01

    Integral membrane proteins are found in all cellular membranes and carry out many of the functions that are essential to life. The membrane-embedded domains of integral membrane proteins are structurally quite simple, allowing the use of various prediction methods and biochemical methods to obtain s

  16. Membrane Compartmentalization Reducing the Mobility of Lipids and Proteins within a Model Plasma Membrane.

    Science.gov (United States)

    Koldsø, Heidi; Reddy, Tyler; Fowler, Philip W; Duncan, Anna L; Sansom, Mark S P

    2016-09-01

    The cytoskeleton underlying cell membranes may influence the dynamic organization of proteins and lipids within the bilayer by immobilizing certain transmembrane (TM) proteins and forming corrals within the membrane. Here, we present coarse-grained resolution simulations of a biologically realistic membrane model of asymmetrically organized lipids and TM proteins. We determine the effects of a model of cytoskeletal immobilization of selected membrane proteins using long time scale coarse-grained molecular dynamics simulations. By introducing compartments with varying degrees of restraints within the membrane models, we are able to reveal how compartmentalization caused by cytoskeletal immobilization leads to reduced and anomalous diffusional mobility of both proteins and lipids. This in turn results in a reduced rate of protein dimerization within the membrane and of hopping of membrane proteins between compartments. These simulations provide a molecular realization of hierarchical models often invoked to explain single-molecule imaging studies of membrane proteins.

  17. Organization and Dynamics of Receptor Proteins in a Plasma Membrane.

    Science.gov (United States)

    Koldsø, Heidi; Sansom, Mark S P

    2015-11-25

    The interactions of membrane proteins are influenced by their lipid environment, with key lipid species able to regulate membrane protein function. Advances in high-resolution microscopy can reveal the organization and dynamics of proteins and lipids within living cells at resolutions membranes of in vivo-like complexity. We explore the dynamics of proteins and lipids in crowded and complex plasma membrane models, thereby closing the gap in length and complexity between computations and experiments. Our simulations provide insights into the mutual interplay between lipids and proteins in determining mesoscale (20-100 nm) fluctuations of the bilayer, and in enabling oligomerization and clustering of membrane proteins.

  18. Stochastic single-molecule dynamics of synaptic membrane protein domains

    CERN Document Server

    Kahraman, Osman; Haselwandter, Christoph A

    2016-01-01

    Motivated by single-molecule experiments on synaptic membrane protein domains, we use a stochastic lattice model to study protein reaction and diffusion processes in crowded membranes. We find that the stochastic reaction-diffusion dynamics of synaptic proteins provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the single-molecule trajectories observed for synaptic proteins, and spatially inhomogeneous protein lifetimes at the cell membrane. Our results suggest that central aspects of the single-molecule and collective dynamics observed for membrane protein domains can be understood in terms of stochastic reaction-diffusion processes at the cell membrane.

  19. Lipidic phase membrane protein serial femtosecond crystallography

    OpenAIRE

    Johansson, LC; Arnlund, D.; White, TA; Katona, G.; DePonte, DP; Weierstall, U.; Doak, RB; Shoeman, RL; Lomb, L; Malmerberg, E.; Davidsson, J; Nass, K.; Liang, MN; Andreasson, J.; Dell'Aquila, A.

    2012-01-01

    X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.

  20. Theoretical analysis of protein organization in lipid membranes.

    Science.gov (United States)

    Gil, T; Ipsen, J H; Mouritsen, O G; Sabra, M C; Sperotto, M M; Zuckermann, M J

    1998-11-10

    The fundamental physical principles of the lateral organization of trans-membrane proteins and peptides as well as peripheral membrane proteins and enzymes are considered from the point of view of the lipid-bilayer membrane, its structure, dynamics, and cooperative phenomena. Based on a variety of theoretical considerations and model calculations, the nature of lipid-protein interactions is considered both for a single protein and an assembly of proteins that can lead to aggregation and protein crystallization in the plane of the membrane. Phenomena discussed include lipid sorting and selectivity at protein surfaces, protein-lipid phase equilibria, lipid-mediated protein-protein interactions, wetting and capillary condensation as means of protein organization, mechanisms of two-dimensional protein crystallization, as well as non-equilibrium organization of active proteins in membranes. The theoretical findings are compared with a variety of experimental data. PMID:9804966

  1. Osteonectin/SPARC/BM-40 in human decidua and carcinoma, tissues characterized by de novo formation of basement membrane

    DEFF Research Database (Denmark)

    Wewer, U M; Albrechtsen, R; Fisher, L W;

    1988-01-01

    decidua tissue in organ culture demonstrated significant osteonectin biosynthesis. Further, Northern analysis and in situ hybridization showed that the osteonectin mRNA level in human decidua is very high. Immunohistochemically, the large mature decidual cells exhibited faint cytoplasmic immunoreactivity...... immunoreactivity was found in poorly differentiated carcinomas. Stromal cells in the peritumoral tissue and some vessels were immunoreactive. Using in situ hybridization, it appeared that both the tumor cells and the stromal cells contained osteonectin transcripts. In normal steady state human nonosseus tissues......The identification and cDNA cloning of bone-enriched osteonectin and parietal endoderm-enriched SPARC indicated that these proteins share greater than 90% homology. The present study reports substantial expression of this Mr 43,000 protein in human decidua and carcinoma. Metabolic labeling of human...

  2. A two-dimensional model of the colonic crypt accounting for the role of the basement membrane and pericryptal fibroblast sheath.

    Directory of Open Access Journals (Sweden)

    Sara-Jane Dunn

    Full Text Available The role of the basement membrane is vital in maintaining the integrity and structure of an epithelial layer, acting as both a mechanical support and forming the physical interface between epithelial cells and the surrounding connective tissue. The function of this membrane is explored here in the context of the epithelial monolayer that lines the colonic crypt, test-tube shaped invaginations that punctuate the lining of the intestine and coordinate a regular turnover of cells to replenish the epithelial layer every few days. To investigate the consequence of genetic mutations that perturb the system dynamics and can lead to colorectal cancer, it must be possible to track the emerging tissue level changes that arise in the crypt. To that end, a theoretical crypt model with a realistic, deformable geometry is required. A new discrete crypt model is presented, which focuses on the interaction between cell- and tissue-level behaviour, while incorporating key subcellular components. The model contains a novel description of the role of the surrounding tissue and musculature, based upon experimental observations of the tissue structure of the crypt, which are also reported. A two-dimensional (2D cross-sectional geometry is considered, and the shape of the crypt is allowed to evolve and deform. Simulation results reveal how the shape of the crypt may contribute mechanically to the asymmetric division events typically associated with the stem cells at the base. The model predicts that epithelial cell migration may arise due to feedback between cell loss at the crypt collar and density-dependent cell division, an hypothesis which can be investigated in a wet lab. This work forms the basis for investigation of the deformation of the crypt structure that can occur due to proliferation of cells exhibiting mutant phenotypes, experiments that would not be possible in vivo or in vitro.

  3. Type VII collagen associated with the basement membrane of amniotic epithelium forms giant anchoring rivets which penetrate a massive lamina reticularis.

    Science.gov (United States)

    Ockleford, C D; McCracken, S A; Rimmington, L A; Hubbard, A R D; Bright, N A; Cockcroft, N; Jefferson, T B; Waldron, E; d'Lacey, C

    2013-09-01

    In human amnion a simple cuboidal epithelium and underlying fibroblast layer are separated by an almost acellular compact layer rich in collagen types I and III. This (>10 μm) layer, which may be a thick lamina reticularis, apparently presents an unusual set of conditions. Integration of the multilaminous tissue across it is apparently achieved by waisted structures which we have observed with the light microscope in frozen, paraffin-wax and semi-thin resin sections. We have also captured transmission and scanning electron micrographs of the structures. These structures which cross the compact layer we call "rivets". The composition of these "rivets" has been examined immunocytochemically and in three dimensions using the confocal laser scanning epi-fluorescence microscope. The rivets contain type VII collagen and an α6 integrin. They associate with type IV collagen containing structures (basement membrane lamina densa and spongy coils) and a special population of fibroblasts which may generate, maintain or anchor rivets to the underlying mesenchymal layer. Although type VII collagen is well known to anchor basal lamina to underlying mesodermal collagen fibres these "rivets" are an order of magnitude larger than any previously described type VII collagen containing anchoring structures. Intriguing possible functions of these features include nodes for growth of fibrous collagen sheets and sites of possible enzymatic degradation during regulated amnion weakening approaching term. If these sites are confirmed to be involved in amnion degradation and growth they may represent important targets for therapeutic agents that are designed to delay preterm premature rupture of the membranes a major cause of fetal morbidity and mortality.

  4. Reconstitution of the membrane protein OmpF into biomimetic block copolymer–phospholipid hybrid membranes

    Science.gov (United States)

    Bieligmeyer, Matthias; Artukovic, Franjo; Hirth, Thomas; Schiestel, Thomas

    2016-01-01

    Summary Structure and function of many transmembrane proteins are affected by their environment. In this respect, reconstitution of a membrane protein into a biomimetic polymer membrane can alter its function. To overcome this problem we used membranes formed by poly(1,4-isoprene-block-ethylene oxide) block copolymers blended with 1,2-diphytanoyl-sn-glycero-3-phosphocholine. By reconstituting the outer membrane protein OmpF from Escherichia coli into these membranes, we demonstrate functionality of this protein in biomimetic lipopolymer membranes, independent of the molecular weight of the block copolymers. At low voltages, the channel conductance of OmpF in 1 M KCl was around 2.3 nS. In line with these experiments, integration of OmpF was also revealed by impedance spectroscopy. Our results indicate that blending synthetic polymer membranes with phospholipids allows for the reconstitution of transmembrane proteins under preservation of protein function, independent of the membrane thickness. PMID:27547605

  5. Reconstitution of the membrane protein OmpF into biomimetic block copolymer–phospholipid hybrid membranes

    Science.gov (United States)

    Bieligmeyer, Matthias; Artukovic, Franjo; Hirth, Thomas; Schiestel, Thomas

    2016-01-01

    Summary Structure and function of many transmembrane proteins are affected by their environment. In this respect, reconstitution of a membrane protein into a biomimetic polymer membrane can alter its function. To overcome this problem we used membranes formed by poly(1,4-isoprene-block-ethylene oxide) block copolymers blended with 1,2-diphytanoyl-sn-glycero-3-phosphocholine. By reconstituting the outer membrane protein OmpF from Escherichia coli into these membranes, we demonstrate functionality of this protein in biomimetic lipopolymer membranes, independent of the molecular weight of the block copolymers. At low voltages, the channel conductance of OmpF in 1 M KCl was around 2.3 nS. In line with these experiments, integration of OmpF was also revealed by impedance spectroscopy. Our results indicate that blending synthetic polymer membranes with phospholipids allows for the reconstitution of transmembrane proteins under preservation of protein function, independent of the membrane thickness.

  6. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

    Science.gov (United States)

    Milovanovic, Dragomir; Honigmann, Alf; Koike, Seiichi; Göttfert, Fabian; Pähler, Gesa; Junius, Meike; Müllar, Stefan; Diederichsen, Ulf; Janshoff, Andreas; Grubmüller, Helmut; Risselada, Herre J.; Eggeling, Christian; Hell, Stefan W.; van den Bogaart, Geert; Jahn, Reinhard

    2015-01-01

    The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein-protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes.

  7. Ultrastructural immunocytochemical localization of chondroitin sulfate proteoglycan in Bruch's membrane of the rat

    DEFF Research Database (Denmark)

    Lin, W L; Essner, E; McCarthy, K J;

    1992-01-01

    Two monoclonal antibodies (Mab 4D5 and 2D6) raised against the core protein of a basement membrane chondroitin sulfate proteoglycan from Reichert's membrane of the rat, were used for ultrastructural immunoperoxidase localization of this protein in Bruch's membrane of the rat. Immunoreactivity for...... both antibodies was found in the basal lamina (basement membrane) of the choriocapillary endothelium and retinal pigment epithelium, in collagen fibers in the collagenous zones, and surrounding the elastic layer....

  8. Bilayer-thickness-mediated interactions between integral membrane proteins

    CERN Document Server

    Kahraman, Osman; Klug, William S; Haselwandter, Christoph A

    2016-01-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology al...

  9. Proteopolymersomes: in vitro production of a membrane protein in polymersome membranes.

    Science.gov (United States)

    Nallani, Madhavan; Andreasson-Ochsner, Mirjam; Tan, Cherng-Wen Darren; Sinner, Eva-Kathrin; Wisantoso, Yudi; Geifman-Shochat, Susana; Hunziker, Walter

    2011-12-01

    Polymersomes are stable self-assembled architectures which mimic cell membranes. For characterization, membrane proteins can be incorporated into such bio-mimetic membranes by reconstitution methods, leading to so-called proteopolymersomes. In this work, we demonstrate the direct incorporation of a membrane protein into polymersome membranes by a cell-free expression system. Firstly, we demonstrate pore formation in the preformed polymersome membrane using α-hemolysin. Secondly, we use claudin-2, a protein involved in cell-cell interactions, to demonstrate the in vitro expression of a membrane protein into these polymersomes. Surface plasmon resonance (Biacore) binding studies with the claudin-2 proteopolymersomes and claudin-2 specific antibodies are performed to show the presence of the in vitro expressed protein in polymersome membranes.

  10. Biogenesis of inner membrane proteins in Escherichia coli.

    Science.gov (United States)

    Luirink, Joen; Yu, Zhong; Wagner, Samuel; de Gier, Jan-Willem

    2012-06-01

    The inner membrane proteome of the model organism Escherichia coli is composed of inner membrane proteins, lipoproteins and peripherally attached soluble proteins. Our knowledge of the biogenesis of inner membrane proteins is rapidly increasing. This is in particular true for the early steps of biogenesis - protein targeting to and insertion into the membrane. However, our knowledge of inner membrane protein folding and quality control is still fragmentary. Furthering our knowledge in these areas will bring us closer to understand the biogenesis of individual inner membrane proteins in the context of the biogenesis of the inner membrane proteome of Escherichia coli as a whole. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

  11. Research progress on Helicobacter pyloriouter membrane protein

    Institute of Scientific and Technical Information of China (English)

    Shi-He Shao; Hua Wang; Shun-Gen Chai; Li-Mei Liu

    2005-01-01

    Helicobacter pylori (H pylori), one of the most common bacterial pathogens on human beings, colonizes the gastric mucosa. In its 95 paralogous gene families, there is a large outer membrane protein (OMP) family. It includes 32 members. These OMP are important for the diagnosis, protective immunity, pathogenicity of H pylori and so on. They are significantly associated with high H pylori density,the damage of gastric mucosa, high mucosal IL-8 levels and severe neutrophil infiltration. We introduce their research progress on pathogenicity.

  12. Poliomyelitis in MuLV-infected ICR-SCID mice after injection of basement membrane matrix contaminated with lactate dehydrogenase-elevating virus.

    Science.gov (United States)

    Carlson Scholz, Jodi A; Garg, Rohit; Compton, Susan R; Allore, Heather G; Zeiss, Caroline J; Uchio, Edward M

    2011-10-01

    The arterivirus lactate dehydrogenase-elevating virus (LDV) causes life-long viremia in mice. Although LDV infection generally does not cause disease, infected mice that are homozygous for the Fv1(n) allele are prone to develop poliomyelitis when immunosuppressed, a condition known as age-dependent poliomyelitis. The development of age-dependent poliomyelitis requires coinfection with endogenous murine leukemia virus. Even though LDV is a common contaminant of transplantable tumors, clinical signs of poliomyelitis after inadvertent exposure to LDV have not been described in recent literature. In addition, LDV-induced poliomyelitis has not been reported in SCID or ICR mice. Here we describe the occurrence of poliomyelitis in ICR-SCID mice resulting from injection of LDV-contaminated basement membrane matrix. After exposure to LDV, a subset of mice presented with clinical signs including paresis, which was associated with atrophy of the hindlimb musculature, and tachypnea; in addition, some mice died suddenly with or without premonitory signs. Mice presenting within the first 6 mo after infection had regions of spongiosis, neuronal necrosis and astrocytosis of the ventral spinal cord, and less commonly, brainstem. Axonal degeneration of ventral roots prevailed in more chronically infected mice. LDV was identified by RT-PCR in 12 of 15 mice with typical neuropathology; positive antiLDV immunolabeling was identified in all PCR-positive animals (n = 7) tested. Three of 8 mice with neuropathology but no clinical signs were LDV negative by RT-PCR. RT-PCR yielded murine leukemia virus in spinal cords of all mice tested, regardless of clinical presentation or neuropathology.

  13. Nonautonomous Roles of MAB-5/Hox and the Secreted Basement Membrane Molecule SPON-1/F-Spondin in Caenorhabditis elegans Neuronal Migration.

    Science.gov (United States)

    Josephson, Matthew P; Miltner, Adam M; Lundquist, Erik A

    2016-08-01

    Nervous system development and circuit formation requires neurons to migrate from their birthplaces to specific destinations.Migrating neurons detect extracellular cues that provide guidance information. In Caenorhabditis elegans, the Q right (QR) and Q left (QL) neuroblast descendants migrate long distances in opposite directions. The Hox gene lin-39 cell autonomously promotes anterior QR descendant migration, and mab-5/Hox cell autonomously promotes posterior QL descendant migration. Here we describe a nonautonomous role of mab-5 in regulating both QR and QL descendant migrations, a role masked by redundancy with lin-39 A third Hox gene, egl-5/Abdominal-B, also likely nonautonomously regulates Q descendant migrations. In the lin-39 mab-5 egl-5 triple mutant, little if any QR and QL descendant migration occurs. In addition to well-described roles of lin-39 and mab-5 in the Q descendants, our results suggest that lin-39, mab-5, and egl-5 might also pattern the posterior region of the animal for Q descendant migration. Previous studies showed that the spon-1 gene might be a target of MAB-5 in Q descendant migration. spon-1 encodes a secreted basement membrane molecule similar to vertebrate F-spondin. Here we show that spon-1 acts nonautonomously to control Q descendant migration, and might function as a permissive rather than instructive signal for cell migration. We find that increased levels of MAB-5 in body wall muscle (BWM) can drive the spon-1 promoter adjacent to the Q cells, and loss of spon-1 suppresses mab-5 gain of function. Thus, MAB-5 might nonautonomously control Q descendant migrations by patterning the posterior region of the animal to which Q cells respond. spon-1 expression from BWMs might be part of the posterior patterning necessary for directed Q descendant migration.

  14. Zein synthesis and processing on zein protein body membranes. [Maize proteins

    Energy Technology Data Exchange (ETDEWEB)

    Burr, F A

    1978-01-01

    The storage protein of maize, zein, is translated from messenger RNA on ribosomes bound to the outer membrane of the zein protein bodies. No other proteins appear to be made on this membrane. Before zein is transported through the protein body membrane it undergoes at least two post-translational modifications, which are discussed.

  15. Study of polytopic membrane protein topological organization as a function of membrane lipid composition.

    Science.gov (United States)

    Bogdanov, Mikhail; Heacock, Philip N; Dowhan, William

    2010-01-01

    A protocol is described using lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by the substituted-cysteine accessibility method as applied to transmembrane domains (SCAM). SCAM is adapted to follow changes in membrane protein topology as a function of changes in membrane lipid composition. The strategy described can be adapted to any membrane system. PMID:20419405

  16. NMR-based screening of membrane protein ligands

    NARCIS (Netherlands)

    Yanamala, Naveena; Dutta, Arpana; Beck, Barbara; Van Fleet, Bart; Hay, Kelly; Yazbak, Ahmad; Ishima, Rieko; Doemling, Alexander; Klein-Seetharaman, Judith

    2010-01-01

    Membrane proteins pose problems for the application of NMR-based ligand-screening methods because of the need to maintain the proteins in a membrane mimetic environment such as detergent micelles: they add to the molecular weight of the protein, increase the viscosity of the solution, interact with

  17. A novel lipoprotein nanoparticle system for membrane proteins

    Science.gov (United States)

    Frauenfeld, Jens; Löving, Robin; Armache, Jean-Paul; Sonnen, Andreas; Guettou, Fatma; Moberg, Per; Zhu, Lin; Jegerschöld, Caroline; Flayhan, Ali; Briggs, John A.G.; Garoff, Henrik; Löw, Christian; Cheng, Yifan; Nordlund, Pär

    2016-01-01

    Membrane proteins are of outstanding importance in biology, drug discovery and vaccination. A common limiting factor in research and applications involving membrane proteins is the ability to solubilize and stabilize membrane proteins. Although detergents represent the major means for solubilizing membrane proteins, they are often associated with protein instability and poor applicability in structural and biophysical studies. Here, we present a novel lipoprotein nanoparticle system that allows for the reconstitution of membrane proteins into a lipid environment that is stabilized by a scaffold of Saposin proteins. We showcase the applicability of the method on two purified membrane protein complexes as well as the direct solubilization and nanoparticle-incorporation of a viral membrane protein complex from the virus membrane. We also demonstrate that this lipid nanoparticle methodology facilitates high-resolution structural studies of membrane proteins in a lipid environment by single-particle electron cryo-microscopy (cryo-EM) and allows for the stabilization of the HIV-envelope glycoprotein in a functional state. PMID:26950744

  18. Membrane-mediated interaction between strongly anisotropic protein scaffolds.

    Directory of Open Access Journals (Sweden)

    Yonatan Schweitzer

    2015-02-01

    Full Text Available Specialized proteins serve as scaffolds sculpting strongly curved membranes of intracellular organelles. Effective membrane shaping requires segregation of these proteins into domains and is, therefore, critically dependent on the protein-protein interaction. Interactions mediated by membrane elastic deformations have been extensively analyzed within approximations of large inter-protein distances, small extents of the protein-mediated membrane bending and small deviations of the protein shapes from isotropic spherical segments. At the same time, important classes of the realistic membrane-shaping proteins have strongly elongated shapes with large and highly anisotropic curvature. Here we investigated, computationally, the membrane mediated interaction between proteins or protein oligomers representing membrane scaffolds with strongly anisotropic curvature, and addressed, quantitatively, a specific case of the scaffold geometrical parameters characterizing BAR domains, which are crucial for membrane shaping in endocytosis. In addition to the previously analyzed contributions to the interaction, we considered a repulsive force stemming from the entropy of the scaffold orientation. We computed this interaction to be of the same order of magnitude as the well-known attractive force related to the entropy of membrane undulations. We demonstrated the scaffold shape anisotropy to cause a mutual aligning of the scaffolds and to generate a strong attractive interaction bringing the scaffolds close to each other to equilibrium distances much smaller than the scaffold size. We computed the energy of interaction between scaffolds of a realistic geometry to constitute tens of kBT, which guarantees a robust segregation of the scaffolds into domains.

  19. An Integrated Framework Advancing Membrane Protein Modeling and Design.

    Directory of Open Access Journals (Sweden)

    Rebecca F Alford

    2015-09-01

    Full Text Available Membrane proteins are critical functional molecules in the human body, constituting more than 30% of open reading frames in the human genome. Unfortunately, a myriad of difficulties in overexpression and reconstitution into membrane mimetics severely limit our ability to determine their structures. Computational tools are therefore instrumental to membrane protein structure prediction, consequently increasing our understanding of membrane protein function and their role in disease. Here, we describe a general framework facilitating membrane protein modeling and design that combines the scientific principles for membrane protein modeling with the flexible software architecture of Rosetta3. This new framework, called RosettaMP, provides a general membrane representation that interfaces with scoring, conformational sampling, and mutation routines that can be easily combined to create new protocols. To demonstrate the capabilities of this implementation, we developed four proof-of-concept applications for (1 prediction of free energy changes upon mutation; (2 high-resolution structural refinement; (3 protein-protein docking; and (4 assembly of symmetric protein complexes, all in the membrane environment. Preliminary data show that these algorithms can produce meaningful scores and structures. The data also suggest needed improvements to both sampling routines and score functions. Importantly, the applications collectively demonstrate the potential of combining the flexible nature of RosettaMP with the power of Rosetta algorithms to facilitate membrane protein modeling and design.

  20. Membrane interacting regions of Dengue virus NS2A protein.

    Science.gov (United States)

    Nemésio, Henrique; Villalaín, José

    2014-08-28

    The Dengue virus (DENV) NS2A protein, essential for viral replication, is a poorly characterized membrane protein. NS2A displays both protein/protein and membrane/protein interactions, yet neither its functions in the viral cycle nor its active regions are known with certainty. To highlight the different membrane-active regions of NS2A, we characterized the effects of peptides derived from a peptide library encompassing this protein's full length on different membranes by measuring their membrane leakage induction and modulation of lipid phase behavior. Following this initial screening, one region, peptide dens25, had interesting effects on membranes; therefore, we sought to thoroughly characterize this region's interaction with membranes. This peptide presents an interfacial/hydrophobic pattern characteristic of a membrane-proximal segment. We show that dens25 strongly interacts with membranes that contain a large proportion of lipid molecules with a formal negative charge, and that this effect has a major electrostatic contribution. Considering its membrane modulating capabilities, this region might be involved in membrane rearrangements and thus be important for the viral cycle.

  1. A topological and conformational stability alphabet for multipass membrane proteins.

    Science.gov (United States)

    Feng, Xiang; Barth, Patrick

    2016-03-01

    Multipass membrane proteins perform critical signal transduction and transport across membranes. How transmembrane helix (TMH) sequences encode the topology and conformational flexibility regulating these functions remains poorly understood. Here we describe a comprehensive analysis of the sequence-structure relationships at multiple interacting TMHs from all membrane proteins with structures in the Protein Data Bank (PDB). We found that membrane proteins can be deconstructed in interacting TMH trimer units, which mostly fold into six distinct structural classes of topologies and conformations. Each class is enriched in recurrent sequence motifs from functionally unrelated proteins, revealing unforeseen consensus and evolutionary conserved networks of stabilizing interhelical contacts. Interacting TMHs' topology and local protein conformational flexibility were remarkably well predicted in a blinded fashion from the identified binding-hotspot motifs. Our results reveal universal sequence-structure principles governing the complex anatomy and plasticity of multipass membrane proteins that may guide de novo structure prediction, design, and studies of folding and dynamics. PMID:26780406

  2. Detergent-Specific Membrane Protein Crystallization Screens

    Science.gov (United States)

    Wiener, Michael

    2007-01-01

    A suite of reagents has been developed for three-dimensional crystallization of integral membranes present in solution as protein-detergent complexes (PDCs). The compositions of these reagents have been determined in part by proximity to the phase boundaries (lower consolute boundaries) of the detergents present in the PDCs. The acquisition of some of the requisite phase-boundary data and the preliminary design of several of the detergent- specific screens was supported by a NASA contract. At the time of expiration of the contract, a partial set of preliminary screens had been developed. This work has since been extended under non-NASA sponsorship, leading to near completion of a set of 20 to 30 different and unique detergent- specific 96-condition screens.

  3. On the analysis of membrane protein circular dichroism spectra

    OpenAIRE

    Sreerama, Narasimha; Woody, Robert W.

    2004-01-01

    Analysis of circular dichroism spectra of proteins provides information about protein secondary structure. Analytical methods developed for such an analysis use structures and spectra of a set of reference proteins. The reference protein sets currently in use include soluble proteins with a wide range of secondary structures, and perform quite well in analyzing CD spectra of soluble proteins. The utility of soluble protein reference sets in analyzing membrane protein CD spectra, however, has ...

  4. Bilayer-thickness-mediated interactions between integral membrane proteins.

    Science.gov (United States)

    Kahraman, Osman; Koch, Peter D; Klug, William S; Haselwandter, Christoph A

    2016-04-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology allows accurate prediction of thickness-mediated protein interactions for arbitrary protein symmetries at arbitrary protein separations and relative orientations. We provide exact analytic solutions for cylindrical integral membrane proteins with constant and varying hydrophobic thickness, and develop perturbative analytic solutions for noncylindrical protein shapes. We complement these analytic solutions, and assess their accuracy, by developing both finite element and finite difference numerical solution schemes. We provide error estimates of our numerical solution schemes and systematically assess their convergence properties. Taken together, the work presented here puts into place an analytic and numerical framework which allows calculation of bilayer-mediated elastic interactions between integral membrane proteins for the complicated protein shapes suggested by structural biology and at the small protein separations most relevant for the crowded membrane

  5. Bilayer-thickness-mediated interactions between integral membrane proteins

    Science.gov (United States)

    Kahraman, Osman; Koch, Peter D.; Klug, William S.; Haselwandter, Christoph A.

    2016-04-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology allows accurate prediction of thickness-mediated protein interactions for arbitrary protein symmetries at arbitrary protein separations and relative orientations. We provide exact analytic solutions for cylindrical integral membrane proteins with constant and varying hydrophobic thickness, and develop perturbative analytic solutions for noncylindrical protein shapes. We complement these analytic solutions, and assess their accuracy, by developing both finite element and finite difference numerical solution schemes. We provide error estimates of our numerical solution schemes and systematically assess their convergence properties. Taken together, the work presented here puts into place an analytic and numerical framework which allows calculation of bilayer-mediated elastic interactions between integral membrane proteins for the complicated protein shapes suggested by structural biology and at the small protein separations most relevant for the crowded membrane

  6. Discriminating lysosomal membrane protein types using dynamic neural network.

    Science.gov (United States)

    Tripathi, Vijay; Gupta, Dwijendra Kumar

    2014-01-01

    This work presents a dynamic artificial neural network methodology, which classifies the proteins into their classes from their sequences alone: the lysosomal membrane protein classes and the various other membranes protein classes. In this paper, neural networks-based lysosomal-associated membrane protein type prediction system is proposed. Different protein sequence representations are fused to extract the features of a protein sequence, which includes seven feature sets; amino acid (AA) composition, sequence length, hydrophobic group, electronic group, sum of hydrophobicity, R-group, and dipeptide composition. To reduce the dimensionality of the large feature vector, we applied the principal component analysis. The probabilistic neural network, generalized regression neural network, and Elman regression neural network (RNN) are used as classifiers and compared with layer recurrent network (LRN), a dynamic network. The dynamic networks have memory, i.e. its output depends not only on the input but the previous outputs also. Thus, the accuracy of LRN classifier among all other artificial neural networks comes out to be the highest. The overall accuracy of jackknife cross-validation is 93.2% for the data-set. These predicted results suggest that the method can be effectively applied to discriminate lysosomal associated membrane proteins from other membrane proteins (Type-I, Outer membrane proteins, GPI-Anchored) and Globular proteins, and it also indicates that the protein sequence representation can better reflect the core feature of membrane proteins than the classical AA composition.

  7. Divergent mechanisms underlie Smad4-mediated positive regulation of the three genes encoding the basement membrane component laminin-332 (laminin-5

    Directory of Open Access Journals (Sweden)

    Hahn Stephan A

    2008-07-01

    Full Text Available Abstract Background Functional inactivation of the tumor suppressor Smad4 in colorectal and pancreatic carcinogenesis occurs coincident with the transition to invasive growth. Breaking the basement membrane (BM barrier, a prerequisite for invasive growth, can be due to tumor induced proteolytic tissue remodeling or to reduced synthesis of BM molecules by incipient tumor cells. Laminin-332 (laminin-5, a heterotrimeric BM component composed of α3-, β3- and γ2-chains, has recently been identified as a target structure of Smad4 and represents the first example for expression control of an essential BM component by a tumor and invasion suppressor. Biochemically Smad4 is a transmitter of signals of the TGFβ superfamily of cytokines. We have reported previously, that Smad4 functions as a positive transcriptional regulator of constitutive and of TGFβ-induced transcription of all three genes encoding Laminin-332, LAMA3, LAMB3 and LAMC2. Methods Promoter-reporter constructs harboring 4 kb upstream regions, each of the three genes encoding Laminin-322 as well as deletion and mutations constructs were established. Promoter activities and TGFβ induction were assayed through transient transfections in Smad4-negative human cancer cells and their stable Smad4-positive derivatives. Functionally relevant binding sites were subsequently confirmed through chromatin immunoprecipitation. Results Herein, we report that Smad4 mediates transcriptional regulation through three different mechanisms, namely through Smad4 binding to a functional SBE site exclusively in the LAMA3 promoter, Smad4 binding to AP1 (and Sp1 sites presumably via interaction with AP1 family components and lastly a Smad4 impact on transcription of AP1 factors. Whereas Smad4 is essential for positive regulation of all three genes, the molecular mechanisms are significantly divergent between the LAMA3 promoter as compared to the LAMB3 and LAMC2 promoters. Conclusion We hypothesize that this

  8. Size-dependent protein segregation at membrane interfaces

    Science.gov (United States)

    Schmid, Eva M.; Bakalar, Matthew H.; Choudhuri, Kaushik; Weichsel, Julian; Ann, Hyoung Sook; Geissler, Phillip L.; Dustin, Michael L.; Fletcher, Daniel A.

    2016-07-01

    Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane proteins whose organization is critical for intracellular signalling. To isolate the role of membrane protein size in pattern formation, we reconstituted model membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between membrane proteins can drastically alter their organization at membrane interfaces, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally driven membrane height fluctuations that transiently limit access to the interface. This sensitive and highly effective means of physically segregating proteins has implications for cell-cell contacts such as T-cell immunological synapses (for example, CD45 exclusion) and epithelial cell junctions (for example, E-cadherin enrichment), as well as for protein sorting at intracellular contact points between membrane-bound organelles.

  9. Membrane interaction of retroviral Gag proteins

    Directory of Open Access Journals (Sweden)

    Robert Alfred Dick

    2014-04-01

    Full Text Available Assembly of an infectious retroviral particle relies on multimerization of the Gag polyprotein at the inner leaflet of the plasma membrane. The three domains of Gag common to all retroviruses-- MA, CA, and NC-- provide the signals for membrane binding, assembly, and viral RNA packaging, respectively. These signals do not function independently of one another. For example, Gag multimerization enhances membrane binding and is more efficient when NC is interacting with RNA. MA binding to the plasma membrane is governed by several principles, including electrostatics, recognition of specific lipid head groups, hydrophobic interactions, and membrane order. HIV-1 uses many of these principles while Rous sarcoma virus (RSV appears to use fewer. This review describes the principles that govern Gag interactions with membranes, focusing on RSV and HIV-1 Gag. The review also defines lipid and membrane behavior, and discusses the complexities in determining how lipid and membrane behavior impact Gag membrane binding.

  10. Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

    Science.gov (United States)

    Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

    2016-03-29

    Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions

  11. Scaffolding proteins in membrane trafficking : the role of ELKS

    NARCIS (Netherlands)

    Yu, K.L.

    2015-01-01

    Intracellular membrane trafficking is an essential cellular process that involves cooperation of many factors such as scaffolding proteins, GTPases and SNAREs. These proteins work together to ensure proper delivery of different membrane-enclosed cargoes to specific cellular destinations. In this the

  12. Glucose-neopentyl glycol (GNG) amphiphiles for membrane protein study

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Rana, Rohini R; Gotfryd, Kamil;

    2013-01-01

    The development of a new class of surfactants for membrane protein manipulation, "GNG amphiphiles", is reported. These amphiphiles display promising behavior for membrane proteins, as demonstrated recently by the high resolution structure of a sodium-pumping pyrophosphatase reported by Kellosalo et...

  13. Optimization of membrane protein overexpression and purification using GFP fusions

    NARCIS (Netherlands)

    Drew, David; Lerch, Mirjam; Kunji, Edmund; Slotboom, Dirk-Jan; de Gier, Jan-Willem

    2006-01-01

    Optimizing conditions for the overexpression and purification of membrane proteins for functional and structural studies is usually a Laborious and time-consuming process. This process can be accelerated using membrane protein-GFP fusions(1-3), which allows direct monitoring and visualization of mem

  14. Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes

    Directory of Open Access Journals (Sweden)

    Bunai Christine L

    2009-02-01

    Full Text Available Abstract Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr, an alkaline solution (180 mM Na2CO3, pH 11.3 and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14. Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries; (ii peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries; (iii cytoplasmic or ribosomal membrane-contaminating proteins (80 entries. Thirty-one proteins were experimentally associated with the outer membrane (OM. Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM proteins with two or more predicted transmembrane domains (TMDs were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families.

  15. Membrane's Eleven: heavy-atom derivatives of membrane-protein crystals

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Sørensen, Thomas Lykke-Møller; Nissen, Poul

    2006-01-01

    A database has been assembled of heavy-atom derivatives used in the structure determination of membrane proteins. The database can serve as a guide to the design of experiments in the search for heavy-atom derivatives of new membrane-protein crystals. The database pinpoints organomercurials...

  16. PECAM-1 (CD31) Homophilic Interaction Up-Regulates α6β1 on Transmigrated Neutrophils In Vivo and Plays a Functional Role in the Ability of α6 Integrins to Mediate Leukocyte Migration through the Perivascular Basement Membrane

    OpenAIRE

    Dangerfield, John; Larbi, Karen Y.; Huang, Miao-Tzu; Dewar, Ann; Nourshargh, Sussan

    2002-01-01

    Platelet-endothelial cell adhesion molecule (PECAM)-1 has been implicated in leukocyte migration through the perivascular basement membrane (PBM) though the mechanisms involved are unclear. The present results demonstrate that the ability of α6 integrins to mediate neutrophil migration through the PBM is PECAM-1 dependent, a response associated with PECAM-1–mediated increased expression of α6β1 on transmigrating neutrophils in vivo. An anti-α6 integrins mAb (GoH3) inhibited (78%, P < 0.001) n...

  17. Formation of functional cell membrane domains: the interplay of lipid- and protein-mediated interactions.

    OpenAIRE

    Harder, Thomas

    2003-01-01

    Numerous cell membrane associated processes, including signal transduction, membrane sorting, protein processing and virus trafficking take place in membrane subdomains. Protein-protein interactions provide the frameworks necessary to generate biologically functional membrane domains. For example, coat proteins define membrane areas destined for sorting processes, viral proteins self-assemble to generate a budding virus, and adapter molecules organize multimolecular signalling assemblies, whi...

  18. Misfolding of Amyloidogenic Proteins and Their Interactions with Membranes

    Science.gov (United States)

    Relini, Annalisa; Marano, Nadia; Gliozzi, Alessandra

    2013-01-01

    In this paper, we discuss amyloidogenic proteins, their misfolding, resulting structures, and interactions with membranes, which lead to membrane damage and subsequent cell death. Many of these proteins are implicated in serious illnesses such as Alzheimer’s disease and Parkinson’s disease. Misfolding of amyloidogenic proteins leads to the formation of polymorphic oligomers and fibrils. Oligomeric aggregates are widely thought to be the toxic species, however, fibrils also play a role in membrane damage. We focus on the structure of these aggregates and their interactions with model membranes. Study of interactions of amlyoidogenic proteins with model and natural membranes has shown the importance of the lipid bilayer in protein misfolding and aggregation and has led to the development of several models for membrane permeabilization by the resulting amyloid aggregates. We discuss several of these models: formation of structured pores by misfolded amyloidogenic proteins, extraction of lipids, interactions with receptors in biological membranes, and membrane destabilization by amyloid aggregates perhaps analogous to that caused by antimicrobial peptides. PMID:24970204

  19. Misfolding of Amyloidogenic Proteins and Their Interactions with Membranes

    Directory of Open Access Journals (Sweden)

    Annalisa Relini

    2013-12-01

    Full Text Available In this paper, we discuss amyloidogenic proteins, their misfolding, resulting structures, and interactions with membranes, which lead to membrane damage and subsequent cell death. Many of these proteins are implicated in serious illnesses such as Alzheimer’s disease and Parkinson’s disease. Misfolding of amyloidogenic proteins leads to the formation of polymorphic oligomers and fibrils. Oligomeric aggregates are widely thought to be the toxic species, however, fibrils also play a role in membrane damage. We focus on the structure of these aggregates and their interactions with model membranes. Study of interactions of amlyoidogenic proteins with model and natural membranes has shown the importance of the lipid bilayer in protein misfolding and aggregation and has led to the development of several models for membrane permeabilization by the resulting amyloid aggregates. We discuss several of these models: formation of structured pores by misfolded amyloidogenic proteins, extraction of lipids, interactions with receptors in biological membranes, and membrane destabilization by amyloid aggregates perhaps analogous to that caused by antimicrobial peptides.

  20. The Origin and Early Evolution of Membrane Proteins

    Science.gov (United States)

    Pohorille, Andrew; Schweighofer, Karl; Wilson, Michael A.

    2005-01-01

    Membrane proteins mediate functions that are essential to all cells. These functions include transport of ions, nutrients and waste products across cell walls, capture of energy and its transduction into the form usable in chemical reactions, transmission of environmental signals to the interior of the cell, cellular growth and cell volume regulation. In the absence of membrane proteins, ancestors of cell (protocells), would have had only very limited capabilities to communicate with their environment. Thus, it is not surprising that membrane proteins are quite common even in simplest prokaryotic cells. Considering that contemporary membrane channels are large and complex, both structurally and functionally, a question arises how their presumably much simpler ancestors could have emerged, perform functions and diversify in early protobiological evolution. Remarkably, despite their overall complexity, structural motifs in membrane proteins are quite simple, with a-helices being most common. This suggests that these proteins might have evolved from simple building blocks. To explain how these blocks could have organized into functional structures, we performed large-scale, accurate computer simulations of folding peptides at a water-membrane interface, their insertion into the membrane, self-assembly into higher-order structures and function. The results of these simulations, combined with analysis of structural and functional experimental data led to the first integrated view of the origin and early evolution of membrane proteins.

  1. Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography.

    Science.gov (United States)

    Weierstall, Uwe; James, Daniel; Wang, Chong; White, Thomas A; Wang, Dingjie; Liu, Wei; Spence, John C H; Bruce Doak, R; Nelson, Garrett; Fromme, Petra; Fromme, Raimund; Grotjohann, Ingo; Kupitz, Christopher; Zatsepin, Nadia A; Liu, Haiguang; Basu, Shibom; Wacker, Daniel; Han, Gye Won; Katritch, Vsevolod; Boutet, Sébastien; Messerschmidt, Marc; Williams, Garth J; Koglin, Jason E; Marvin Seibert, M; Klinker, Markus; Gati, Cornelius; Shoeman, Robert L; Barty, Anton; Chapman, Henry N; Kirian, Richard A; Beyerlein, Kenneth R; Stevens, Raymond C; Li, Dianfan; Shah, Syed T A; Howe, Nicole; Caffrey, Martin; Cherezov, Vadim

    2014-01-01

    Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously renewed source of material for serial femtosecond crystallography. Data collected from sub-10-μm-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor.

  2. Amyloid Aggregation and Membrane Disruption by Amyloid Proteins

    Science.gov (United States)

    Ramamoorthy, Ayyalusamy

    2013-03-01

    Amyloidogenesis has been the focus of intense basic and clinical research, as an increasing number of amyloidogenic proteins have been linked to common and incurable degenerative diseases including Alzheimer's, type II diabetes, and Parkinson's. Recent studies suggest that the cell toxicity is mainly due to intermediates generated during the assembly process of amyloid fibers, which have been proposed to attack cells in a variety of ways. Disruption of cell membranes is believed to be one of the key components of amyloid toxicity. However, the mechanism by which this occurs is not fully understood. Our research in this area is focused on the investigation of the early events in the aggregation and membrane disruption of amyloid proteins, Islet amyloid polypeptide protein (IAPP, also known as amylin) and amyloid-beta peptide, on the molecular level. Structural insights into the mechanisms of membrane disruption by these amyloid proteins and the role of membrane components on the membrane disruption will be presented.

  3. Quenching of fluorescence in membrane protein by hypocrellin B

    Institute of Scientific and Technical Information of China (English)

    乐加昌; 庞素珍

    1997-01-01

    The hypocrellin B (HB) was used as a fluorescence quencher to study the basic physical characteris-tics of HB in membrane systems, including the diffusion speed of quencher from aqueous phase into membrane phase, the partition coefficient (P) of quencher between membrane and water, and the fluorescence quenching constant of protein (Ksv; Kq). The experimental results show that the quenching of fluorescence in membrane protein by HB can be determined by the principle of dynamic quenching. The experimental process of fluorescence quenching was ob-served in detail by using the ESR technique. The signal of HB" was found to arise from an electron transfer from ex-cited trytophan to HB.

  4. Statistical thermodynamics of membrane bending-mediated protein-protein attractions.

    OpenAIRE

    Chou, T; Kim, K. S.; Oster, G

    2001-01-01

    Highly wedge-shaped integral membrane proteins, or membrane-adsorbed proteins can induce long-ranged deformations. The strain in the surrounding bilayer creates relatively long-ranged forces that contribute to interactions with nearby proteins. In contrast, to direct short-ranged interactions such as van der Waal's, hydrophobic, or electrostatic interactions, both local membrane Gaussian curvature and protein ellipticity can induce forces acting at distances of up to a few times their typical...

  5. Experimental study of application of anti-glomerular basement membrane antibodies neutralizing monoclonal antibody on anti-glomerular basement membrane nephritis rats%应用抗肾小球基底膜抗体的中和性单克隆抗体治疗抗肾小球基底膜肾炎大鼠的实验研究

    Institute of Scientific and Technical Information of China (English)

    肖静; 刘章锁; 聂志勇; 王雅楠; 赵国强

    2010-01-01

    目的 应用抗肾小球基底膜(GBM)抗体的中和性单克隆抗体注射抗GBM肾炎大鼠,观察各种生化指标及肾脏病理学的变化.方法 将Wistar大鼠随机分为5组,每组9只:(1)肾炎模型组:经尾静脉注入人抗GBM抗体;(2)正常对照Ⅰ组:经尾静脉注入非抗体性的健康人IgG;(3)对照Ⅱ组:经尾静脉注入抗GBM抗体的中和性单克降抗体;(4)干预Ⅰ组:经尾静脉注入人抗GBM抗体,第7天后再经尾静脉注入抗GBM抗体的中和性单克隆抗体(1.5ml/100 g);(5)干预Ⅱ组:经尾静脉注入人抗GBM抗体,第14天后再经尾静脉注入抗GBM抗体的中和性单克隆抗体.分别在实验后第7、14、21天观察大鼠24 h尿蛋白量、BUN、Scr和肾组织病理学的变化.结果 第21天干预Ⅰ组尿蛋白量为(16.62±5.53)g/d、BUN为(11.53±2.26)mmol/L、Scr为(102.46±16.86)μmol/L,均显著低于肾炎模型组(P<0.05);干预Ⅱ组较肾炎模型组也有所降低,但差异无统计学意义(P>0.05).干预Ⅰ组和干预Ⅱ组肾脏细胞增生、新月体的形成及免疫复合物的沉积均少于肾炎模型组,但干预Ⅰ组更为明显.对照Ⅰ组和对照Ⅱ组之间无明显变化.结论 早期应用抗GBM抗体的中和性单克隆抗体能够有效改善抗GBM肾炎大鼠的肾脏病变.%Objective To observe the effect of neutralizing monoclonal antibodies to antiglomerular basement membrane (GBM) antibody on anti-GBM nephritis rats. Methods Wistar rats were randomly divided into five groups: control group Ⅰ was a negative control and was injected with healthy human IgG via the caudal vein. Control group Ⅱ was injected with neutralizing monoclonal antibodies to anti-GBM antibody only. Anti- GBM nephritis group was injected with human anti-GBM antibody via the caudal vein only. Intervention group Ⅰ was injected with human anti-GBM antibody via the caudal vein and then with neutralizing monoclonal antibodies to anti-GBM antibody at day 7. Intervention group Ⅱ was

  6. 3D pressure field in lipid membranes and membrane-protein complexes

    DEFF Research Database (Denmark)

    Ollila, O H Samuli; Risselada, H Jelger; Louhivuori, Martti;

    2009-01-01

    a protein-membrane complex. We show that the 3D pressure field is distinctly different for curved and planar bilayers, the pressure field depends strongly on the phase of the membrane, and that an integral protein modulates the tension and elastic properties of the membrane.......We calculate full 3D pressure fields for inhomogeneous nanoscale systems using molecular dynamics simulation data. The fields represent systems with increasing level of complexity, ranging from semivesicles and vesicles to membranes characterized by coexistence of two phases, including also...

  7. Studying Membrane Protein Structure and Function Using Nanodiscs

    DEFF Research Database (Denmark)

    Huda, Pie

    The structure and dynamic of membrane proteins can provide valuable information about general functions, diseases and effects of various drugs. Studying membrane proteins are a challenge as an amphiphilic environment is necessary to stabilise the protein in a functionally and structurally relevant...... form. This is most typically achieved through the use of detergent based reconstitution systems. However, time and again such systems fail to provide a suitable environment causing aggregation and inactivation. Nanodiscs are self-assembled lipoproteins containing two membrane scaffold proteins...... for understanding the key mechanisms during reconstitution of membrane proteins in these lipoproteins. In this project the self-assembly of nanodiscs has been structurally characterized with small angle X-ray scattering (SAXS) in a time resolved fashion. This brought knowledge about the structural development...

  8. Membrane Protein Production in the Yeast, S. cerevisiae.

    Science.gov (United States)

    Cartwright, Stephanie P; Mikaliunaite, Lina; Bill, Roslyn M

    2016-01-01

    The first crystal structures of recombinant mammalian membrane proteins were solved in 2005 using protein that had been produced in yeast cells. One of these, the rabbit Ca(2+)-ATPase SERCA1a, was synthesized in Saccharomyces cerevisiae. All host systems have their specific advantages and disadvantages, but yeast has remained a consistently popular choice in the eukaryotic membrane protein field because it is quick, easy and cheap to culture, whilst being able to post-translationally process eukaryotic membrane proteins. Very recent structures of recombinant membrane proteins produced in S. cerevisiae include those of the Arabidopsis thaliana NRT1.1 nitrate transporter and the fungal plant pathogen lipid scramblase, TMEM16. This chapter provides an overview of the methodological approaches underpinning these successes. PMID:27485327

  9. Self-assembling peptides form nanodiscs that stabilize membrane proteins

    DEFF Research Database (Denmark)

    Midtgaard, Søren Roi; Pedersen, Martin Cramer; Kirkensgaard, Jacob Judas Kain;

    2014-01-01

    -assemble in combination with phospholipids to form discoidal shaped particles that can stabilize membrane proteins. In the present study, we have investigated an ApoA1 mimetic peptide with respect to its solution structure when in complex with phospholipids. This was achieved using a powerful combination of small-angle X...... show that, like the ApoA1 and derived nanodiscs, these peptide discs can accommodate and stabilize a membrane protein. Finally, we exploit their dynamic properties and show that the 18A discs may be used for transferring membrane proteins and associated phospholipids directly and gently...

  10. Structural Aspects of Bacterial Outer Membrane Protein Assembly.

    Science.gov (United States)

    Calmettes, Charles; Judd, Andrew; Moraes, Trevor F

    2015-01-01

    The outer membrane of Gram-negative bacteria is predominantly populated by β-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The β-barrel assembly machinery (Bam) complex is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane. PMID:26621472

  11. Integral membrane protein structure determination using pseudocontact shifts

    International Nuclear Information System (INIS)

    Obtaining enough experimental restraints can be a limiting factor in the NMR structure determination of larger proteins. This is particularly the case for large assemblies such as membrane proteins that have been solubilized in a membrane-mimicking environment. Whilst in such cases extensive deuteration strategies are regularly utilised with the aim to improve the spectral quality, these schemes often limit the number of NOEs obtainable, making complementary strategies highly beneficial for successful structure elucidation. Recently, lanthanide-induced pseudocontact shifts (PCSs) have been established as a structural tool for globular proteins. Here, we demonstrate that a PCS-based approach can be successfully applied for the structure determination of integral membrane proteins. Using the 7TM α-helical microbial receptor pSRII, we show that PCS-derived restraints from lanthanide binding tags attached to four different positions of the protein facilitate the backbone structure determination when combined with a limited set of NOEs. In contrast, the same set of NOEs fails to determine the correct 3D fold. The latter situation is frequently encountered in polytopical α-helical membrane proteins and a PCS approach is thus suitable even for this particularly challenging class of membrane proteins. The ease of measuring PCSs makes this an attractive route for structure determination of large membrane proteins in general

  12. Integral membrane protein structure determination using pseudocontact shifts

    Energy Technology Data Exchange (ETDEWEB)

    Crick, Duncan J.; Wang, Jue X. [University of Cambridge, Department of Biochemistry (United Kingdom); Graham, Bim; Swarbrick, James D. [Monash University, Monash Institute of Pharmaceutical Sciences (Australia); Mott, Helen R.; Nietlispach, Daniel, E-mail: dn206@cam.ac.uk [University of Cambridge, Department of Biochemistry (United Kingdom)

    2015-04-15

    Obtaining enough experimental restraints can be a limiting factor in the NMR structure determination of larger proteins. This is particularly the case for large assemblies such as membrane proteins that have been solubilized in a membrane-mimicking environment. Whilst in such cases extensive deuteration strategies are regularly utilised with the aim to improve the spectral quality, these schemes often limit the number of NOEs obtainable, making complementary strategies highly beneficial for successful structure elucidation. Recently, lanthanide-induced pseudocontact shifts (PCSs) have been established as a structural tool for globular proteins. Here, we demonstrate that a PCS-based approach can be successfully applied for the structure determination of integral membrane proteins. Using the 7TM α-helical microbial receptor pSRII, we show that PCS-derived restraints from lanthanide binding tags attached to four different positions of the protein facilitate the backbone structure determination when combined with a limited set of NOEs. In contrast, the same set of NOEs fails to determine the correct 3D fold. The latter situation is frequently encountered in polytopical α-helical membrane proteins and a PCS approach is thus suitable even for this particularly challenging class of membrane proteins. The ease of measuring PCSs makes this an attractive route for structure determination of large membrane proteins in general.

  13. Electron crystallography for structural and functional studies of membrane proteins.

    Science.gov (United States)

    Fujiyoshi, Yoshinori

    2011-01-01

    Membrane proteins are important research targets for basic biological sciences and drug design, but studies of their structure and function are considered difficult to perform. Studies of membrane structures have been greatly facilitated by technological and instrumental advancements in electron microscopy together with methodological advancements in biology. Electron crystallography is especially useful in studying the structure and function of membrane proteins. Electron crystallography is now an established method of analyzing the structures of membrane proteins in lipid bilayers, which resembles their natural biological environment. To better understand the neural system function from a structural point of view, we developed the cryo-electron microscope with a helium-cooled specimen stage, which allows for analysis of the structures of membrane proteins at a resolution higher than 3 Å. This review introduces recent instrumental advances in cryo-electron microscopy and presents some examples of structure analyses of membrane proteins, such as bacteriorhodopsin, water channels and gap junction channels. This review has two objectives: first, to provide a personal historical background to describe how we came to develop the cryo-electron microscope and second, to discuss some of the technology required for the structural analysis of membrane proteins based on cryo-electron microscopy.

  14. Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins

    NARCIS (Netherlands)

    Kralt, Annemarie; Jagalur, Noorjahan B.; van den Boom, Vincent; Lokareddy, Ravi K.; Steen, Anton; Cingolani, Gino; Fornerod, Maarten; Veenhoff, Liesbeth M.

    2015-01-01

    Endoplasmic reticulum-synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a

  15. Seismic basement in Poland

    Science.gov (United States)

    Grad, Marek; Polkowski, Marcin

    2016-06-01

    The area of contact between Precambrian and Phanerozoic Europe in Poland has complicated structure of sedimentary cover and basement. The thinnest sedimentary cover in the Mazury-Belarus anteclize is only 0.3-1 km thick, increases to 7-8 km along the East European Craton margin, and 9-12 km in the Trans-European Suture Zone (TESZ). The Variscan domain is characterized by a 1- to 2-km-thick sedimentary cover, while the Carpathians are characterized by very thick sediments, up to c. 20 km. The map of the basement depth is created by combining data from geological boreholes with a set of regional seismic refraction profiles. These maps do not provide data about the basement depth in the central part of the TESZ and in the Carpathians. Therefore, the data set is supplemented by 32 models from deep seismic sounding profiles and a map of a high-resistivity (low-conductivity) layer from magnetotelluric soundings, identified as a basement. All of these data provide knowledge about the basement depth and of P-wave seismic velocities of the crystalline and consolidated type of basement for the whole area of Poland. Finally, the differentiation of the basement depth and velocity is discussed with respect to geophysical fields and the tectonic division of the area.

  16. Analysis of protein interactions at native chloroplast membranes by ellipsometry.

    Directory of Open Access Journals (Sweden)

    Verena Kriechbaumer

    Full Text Available Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE. We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins.

  17. Overexpression of membrane proteins from higher eukaryotes in yeasts.

    Science.gov (United States)

    Emmerstorfer, Anita; Wriessnegger, Tamara; Hirz, Melanie; Pichler, Harald

    2014-09-01

    Heterologous expression and characterisation of the membrane proteins of higher eukaryotes is of paramount interest in fundamental and applied research. Due to the rather simple and well-established methods for their genetic modification and cultivation, yeast cells are attractive host systems for recombinant protein production. This review provides an overview on the remarkable progress, and discusses pitfalls, in applying various yeast host strains for high-level expression of eukaryotic membrane proteins. In contrast to the cell lines of higher eukaryotes, yeasts permit efficient library screening methods. Modified yeasts are used as high-throughput screening tools for heterologous membrane protein functions or as benchmark for analysing drug-target relationships, e.g., by using yeasts as sensors. Furthermore, yeasts are powerful hosts for revealing interactions stabilising and/or activating membrane proteins. We also discuss the stress responses of yeasts upon heterologous expression of membrane proteins. Through co-expression of chaperones and/or optimising yeast cultivation and expression strategies, yield-optimised hosts have been created for membrane protein crystallography or efficient whole-cell production of fine chemicals. PMID:25070595

  18. Peroxisome Fission is Associated with Reorganization of Specific Membrane Proteins

    NARCIS (Netherlands)

    Krygowska, Malgorzata; Veenhuis, Marten; Klei, Ida J. van der; Nagotu, Shirisha

    2011-01-01

    Membrane remodeling is an important aspect in organelle biogenesis. We show that different peroxisome membrane proteins that play a role in organelle biogenesis and proliferation (Pex8, Pex10, Pex14, Pex25 and Pex11) are subject to spatiotemporal behavior during organelle development. Using fluoresc

  19. On the mechanism of transport of Inner Nuclear Membrane Proteins

    NARCIS (Netherlands)

    Laba, Justyna Katarzyna

    2016-01-01

    The nucleus is usually the biggest, round-shaped organelle in the cell, which contains numerous proteins and nucleic acids and protects the DNA. Nuclear components are contained within the boarders of Nuclear Envelope (NE), a double membrane system, formed by the fusion of Outer Nuclear Membrane (OM

  20. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

    Energy Technology Data Exchange (ETDEWEB)

    Baker, Lindsay A. [University of Oxford, Oxford Particle Imaging Centre, The Wellcome Trust Centre for Human Genetics, Division of Structural Biology, Nuffield Department of Medicine (United Kingdom); Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc, E-mail: m.baldus@uu.nl [Utrecht University, NMR Spectroscopy, Department of Chemistry, Faculty of Science, Bijvoet Center for Biomolecular Research (Netherlands)

    2015-06-15

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR.

  1. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

    International Nuclear Information System (INIS)

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR

  2. Architecture and Function of Mechanosensitive Membrane Protein Lattices

    CERN Document Server

    Kahraman, Osman; Klug, William S; Haselwandter, Christoph A

    2016-01-01

    Experiments have revealed that membrane proteins can form two-dimensional clusters with regular translational and orientational protein arrangements, which may allow cells to modulate protein function. However, the physical mechanisms yielding supramolecular organization and collective function of membrane proteins remain largely unknown. Here we show that bilayer-mediated elastic interactions between membrane proteins can yield regular and distinctive lattice architectures of protein clusters, and may provide a link between lattice architecture and lattice function. Using the mechanosensitive channel of large conductance (MscL) as a model system, we obtain relations between the shape of MscL and the supramolecular architecture of MscL lattices. We predict that the tetrameric and pentameric MscL symmetries observed in previous structural studies yield distinct lattice architectures of MscL clusters and that, in turn, these distinct MscL lattice architectures yield distinct lattice activation barriers. Our res...

  3. Structuring detergents for extracting and stabilizing functional membrane proteins.

    Directory of Open Access Journals (Sweden)

    Rima Matar-Merheb

    Full Text Available BACKGROUND: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. METHODOLOGY/PRINCIPAL FINDINGS: Anionic calix[4]arene based detergents (C4Cn, n=1-12 were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein, a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM. They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux much more efficiently than SDS (sodium dodecyl sulphate, FC12 (Foscholine 12 or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. CONCLUSION/SIGNIFICANCE: These compounds seem promising to extract in a functional state

  4. Proteome array identification of bioactive soluble proteins/peptides in matrigel; relevance to stem cell responses

    Science.gov (United States)

    Matrigel and similar commercial products are extracts of the Engelbreth-Holm-Swarm sarcoma that provide a basement-membrane-like attachment factor or gel that is used to grow cells on or in. To ascertain further what proteins may be present in Matrigel, besides its major basement-membrane constitue...

  5. A new window into the molecular physiology of membrane proteins

    OpenAIRE

    Landreh, Michael; Robinson, Carol V.

    2014-01-01

    Integral membrane proteins comprise ∼25% of the human proteome. Yet, our understanding of their molecular physiology is still in its infancy. This can be attributed to two factors: the experimental challenges that arise from the difficult chemical nature of membrane proteins, and the unclear relationship between their activity and their native environment. New approaches are therefore required to address these challenges. Recent developments in mass spectrometry have shown that it is possible...

  6. Domain formation in membranes caused by lipid wetting of protein

    OpenAIRE

    Akimov, Sergey A.; Frolov, Vladimir A. J.; Kuzmin, Peter I.; Zimmerberg, Joshua; Chizmadzhev, Yuri A.; Cohen, Fredric S.

    2008-01-01

    Formation of rafts and other domains in cell membranes is considered as wetting of proteins by lipids. The membrane is modeled as a continuous elastic medium. Thermodynamic functions of the lipid films that wet proteins are calculated using a mean-field theory of liquid crystals as adapted to biomembranes. This approach yields the conditions necessary for a macroscopic wetting film to form; its thickness could also be determined. It is shown that films of macroscopic thicknesses form around l...

  7. Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast.

    Science.gov (United States)

    Grossmann, Guido; Opekarová, Miroslava; Malinsky, Jan; Weig-Meckl, Ina; Tanner, Widmar

    2007-01-10

    The plasma membrane potential is mainly considered as the driving force for ion and nutrient translocation. Using the yeast Saccharomyces cerevisiae as a model organism, we have discovered a novel role of the membrane potential in the organization of the plasma membrane. Within the yeast plasma membrane, two non-overlapping sub-compartments can be visualized. The first one, represented by a network-like structure, is occupied by the proton ATPase, Pma1, and the second one, forming 300-nm patches, houses a number of proton symporters (Can1, Fur4, Tat2 and HUP1) and Sur7, a component of the recently described eisosomes. Evidence is presented that sterols, the main lipid constituent of the plasma membrane, also accumulate within the patchy compartment. It is documented that this compartmentation is highly dependent on the energization of the membrane. Plasma membrane depolarization causes reversible dispersion of the H(+)-symporters, not however of the Sur7 protein. Mitochondrial mutants, affected in plasma membrane energization, show a significantly lower degree of membrane protein segregation. In accordance with these observations, depolarized membranes also considerably change their physical properties (detergent sensitivity).

  8. Amphiphilic biopolymers (amphibiopols) as new surfactants for membrane protein solubilization

    Science.gov (United States)

    Duval-Terrié, Caroline; Cosette, Pascal; Molle, Gérard; Muller, Guy; Dé, Emmanuelle

    2003-01-01

    The aim of this study was to develop new surfactants for membrane protein solubilization, from a natural, biodegradable polymer: the polysaccharide pullulan. A set of amphiphilic pullulans (HMCMPs), differing in hydrophobic modification ratio, charge ratio, and the nature of the hydrophobic chains introduced, were synthesized and tested in solubilization experiments with outer membranes of Pseudomonas fluorescens. The membrane proteins were precipitated, and then resolubilized with various HMCMPs. The decyl alkyl chain (C10) was the hydrophobic graft that gave the highest level of solubilization. Decyl alkyl chain-bearing HMCMPs were also able to extract integral membrane proteins from their lipid environment. The best results were obtained with an amphiphilic pullulan bearing 18% decyl groups (18C10). Circular dichroism spectroscopy and membrane reconstitution experiments were used to test the structural and functional integrity of 18C10-solubilized proteins (OmpF from Escherichia coli and bacteriorhodopsin from Halobacterium halobium). Whatever their structure type (α or β), 18C10 did not alter either the structure or the function of the proteins analyzed. Thus, HMCMPs appear to constitute a promising new class of polymeric surfactants for membrane protein studies. PMID:12649425

  9. Use of reverse micelles in membrane protein structural biology

    Energy Technology Data Exchange (ETDEWEB)

    Van Horn, Wade D. [Vanderbilt University School of Medicine, Department of Biochemistry and Center for Structural Biology (United States); Ogilvie, Mark E.; Flynn, Peter F. [University of Utah, Department of Chemistry (United States)], E-mail: peter.flynn@utah.edu

    2008-03-15

    Membrane protein structural biology is a rapidly developing field with fundamental importance for elucidating key biological and biophysical processes including signal transduction, intercellular communication, and cellular transport. In addition to the intrinsic interest in this area of research, structural studies of membrane proteins have direct significance on the development of therapeutics that impact human health in diverse and important ways. In this article we demonstrate the potential of investigating the structure of membrane proteins using the reverse micelle forming surfactant dioctyl sulfosuccinate (AOT) in application to the prototypical model ion channel gramicidin A. Reverse micelles are surfactant based nanoparticles which have been employed to investigate fundamental physical properties of biomolecules. The results of this solution NMR based study indicate that the AOT reverse micelle system is capable of refolding and stabilizing relatively high concentrations of the native conformation of gramicidin A. Importantly, pulsed-field-gradient NMR diffusion and NOESY experiments reveal stable gramicidin A homodimer interactions that bridge reverse micelle particles. The spectroscopic benefit of reverse micelle-membrane protein solubilization is also explored, and significant enhancement over commonly used micelle based mimetic systems is demonstrated. These results establish the effectiveness of reverse micelle based studies of membrane proteins, and illustrate that membrane proteins solubilized by reverse micelles are compatible with high resolution solution NMR techniques.

  10. Optimal separation of jojoba protein using membrane processes

    Energy Technology Data Exchange (ETDEWEB)

    Nabetani, Hiroshi; Abbott, T.P.; Kleiman, R. [National Center for Agricultural Utilization Research, Peoria, IL (United States)

    1995-05-01

    The efficiency of a pilot-scale membrane system for purifying and concentrating jojoba protein was estimated. In this system, a jojoba extract was first clarified with a microfiltration membrane. The clarified extract was diafiltrated and the protein was purified with an ultrafiltration membrane. Then the protein solution was concentrated with the ultrafiltration membrane. Permeate flux during microfiltration was essentially independent of solids concentration in the feed, in contrast with the permeate flux during ultrafiltration which was a function of protein concentration. Based on these results, a mathematical model which describes the batchwise concentration process with ultrafiltration membranes was developed. Using this model, the combination of batchwise concentration with diafiltration was optimized, and an industrial-scale process was designed. The effect of ethylenediaminetetraacetic acid (EDTA) on the performance of the membrane system was also investigated. The addition of EDTA increased the concentration of protein in the extract and improved the recovery of protein in the final products. The quality of the final product (color and solubility) was also improved. However, EDTA decreased permeate flux during ultrafiltration.

  11. High membrane protein oxidation in the human cerebral cortex

    Directory of Open Access Journals (Sweden)

    Matthias Granold

    2015-04-01

    Full Text Available Oxidative stress is thought to be one of the main mediators of neuronal damage in human neurodegenerative disease. Still, the dissection of causal relationships has turned out to be remarkably difficult. Here, we have analyzed global protein oxidation in terms of carbonylation of membrane proteins and cytoplasmic proteins in three different mammalian species: aged human cortex and cerebellum from patients with or without Alzheimer's disease, mouse cortex and cerebellum from young and old animals, and adult rat hippocampus and cortex subjected or not subjected to cerebral ischemia. Most tissues showed relatively similar levels of protein oxidation. However, human cortex was affected by severe membrane protein oxidation, while exhibiting lower than average cytoplasmic protein oxidation. In contrast, ex vivo autooxidation of murine cortical tissue primarily induced aqueous protein oxidation, while in vivo biological aging or cerebral ischemia had no major effect on brain protein oxidation. The unusually high levels of membrane protein oxidation in the human cortex were also not predicted by lipid peroxidation, as the levels of isoprostane immunoreactivity in human samples were considerably lower than in rodent tissues. Our results indicate that the aged human cortex is under steady pressure from specific and potentially detrimental membrane protein oxidation. The pronounced difference between humans, mice and rats regarding the primary site of cortical oxidation might have contributed to the unresolved difficulties in translating into therapies the wealth of data describing successful antioxidant neuroprotection in rodents.

  12. Predictive energy landscapes for folding membrane protein assemblies.

    Science.gov (United States)

    Truong, Ha H; Kim, Bobby L; Schafer, Nicholas P; Wolynes, Peter G

    2015-12-28

    We study the energy landscapes for membrane protein oligomerization using the Associative memory, Water mediated, Structure and Energy Model with an implicit membrane potential (AWSEM-membrane), a coarse-grained molecular dynamics model previously optimized under the assumption that the energy landscapes for folding α-helical membrane protein monomers are funneled once their native topology within the membrane is established. In this study we show that the AWSEM-membrane force field is able to sample near native binding interfaces of several oligomeric systems. By predicting candidate structures using simulated annealing, we further show that degeneracies in predicting structures of membrane protein monomers are generally resolved in the folding of the higher order assemblies as is the case in the assemblies of both nicotinic acetylcholine receptor and V-type Na(+)-ATPase dimers. The physics of the phenomenon resembles domain swapping, which is consistent with the landscape following the principle of minimal frustration. We revisit also the classic Khorana study of the reconstitution of bacteriorhodopsin from its fragments, which is the close analogue of the early Anfinsen experiment on globular proteins. Here, we show the retinal cofactor likely plays a major role in selecting the final functional assembly. PMID:26723586

  13. Predictive energy landscapes for folding membrane protein assemblies

    Science.gov (United States)

    Truong, Ha H.; Kim, Bobby L.; Schafer, Nicholas P.; Wolynes, Peter G.

    2015-12-01

    We study the energy landscapes for membrane protein oligomerization using the Associative memory, Water mediated, Structure and Energy Model with an implicit membrane potential (AWSEM-membrane), a coarse-grained molecular dynamics model previously optimized under the assumption that the energy landscapes for folding α-helical membrane protein monomers are funneled once their native topology within the membrane is established. In this study we show that the AWSEM-membrane force field is able to sample near native binding interfaces of several oligomeric systems. By predicting candidate structures using simulated annealing, we further show that degeneracies in predicting structures of membrane protein monomers are generally resolved in the folding of the higher order assemblies as is the case in the assemblies of both nicotinic acetylcholine receptor and V-type Na+-ATPase dimers. The physics of the phenomenon resembles domain swapping, which is consistent with the landscape following the principle of minimal frustration. We revisit also the classic Khorana study of the reconstitution of bacteriorhodopsin from its fragments, which is the close analogue of the early Anfinsen experiment on globular proteins. Here, we show the retinal cofactor likely plays a major role in selecting the final functional assembly.

  14. Isothermal titration calorimetry of membrane proteins - progress and challenges.

    Science.gov (United States)

    Rajarathnam, Krishna; Rösgen, Jörg

    2014-01-01

    Integral membrane proteins, including G protein-coupled receptors (GPCR) and ion channels, mediate diverse biological functions that are crucial to all aspects of life. The knowledge of the molecular mechanisms, and in particular, the thermodynamic basis of the binding interactions of the extracellular ligands and intracellular effector proteins is essential to understand the workings of these remarkable nanomachines. In this review, we describe how isothermal titration calorimetry (ITC) can be effectively used to gain valuable insights into the thermodynamic signatures (enthalpy, entropy, affinity, and stoichiometry), which would be most useful for drug discovery studies, considering that more than 30% of the current drugs target membrane proteins. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding.

  15. Domain formation in membranes caused by lipid wetting of protein.

    Science.gov (United States)

    Akimov, Sergey A; Frolov, Vladimir A J; Kuzmin, Peter I; Zimmerberg, Joshua; Chizmadzhev, Yuri A; Cohen, Fredric S

    2008-05-01

    Formation of rafts and other domains in cell membranes is considered as wetting of proteins by lipids. The membrane is modeled as a continuous elastic medium. Thermodynamic functions of the lipid films that wet proteins are calculated using a mean-field theory of liquid crystals as adapted to biomembranes. This approach yields the conditions necessary for a macroscopic wetting film to form; its thickness could also be determined. It is shown that films of macroscopic thicknesses form around large (tens nanometers in diameter) lipid-protein aggregates; only thin adsorption films form around single proteins or small complexes. The means by which wetting films can facilitate the merger of these aggregates is considered. It is shown that a wetting film prevents a protein from leaving an aggregate. Using experimentally derived values of elastic moduli and spontaneous curvatures as well as height mismatch between aggregates and bulk membrane, we obtained numerical results, which can be compared with the experimental data. PMID:18643096

  16. An overview of membrane transport proteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Andre, B

    1995-12-01

    All eukaryotic cells contain a wide variety of proteins embedded in the plasma and internal membranes, which ensure transmembrane solute transport. It is now established that a large proportion of these transport proteins can be grouped into families apparently conserved throughout organisms. This article presents the data of an in silicio analysis aimed at establishing a preliminary classification of membrane transport proteins in Saccharomyces cerevisiae. This analysis was conducted at a time when about 65% of all yeast genes were available in public databases. In addition to approximately 60 transport proteins whose function was at least partially known, approximately 100 deduced protein sequences of unknown function display significant sequence similarity to membrane transport proteins characterized in yeast and/or other organisms. While some protein families have been well characterized by classical genetic experimental approaches, others have largely if not totally escaped characterization. The proteins revealed by this in silicio analysis also include a putative K+ channel, proteins similar to aquaporins of plant and animal origin, proteins similar to Na+-solute symporters, a protein very similar to electroneural cation-chloride cotransporters, and a putative Na+-H+ antiporter. A new research area is anticipated: the functional analysis of many transport proteins whose existence was revealed by genome sequencing.

  17. Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins

    NARCIS (Netherlands)

    A. Kralt (Annemarie); N.B. Jagalur (Noorjahan ); V. van den Boom (Vincent); R.K. Lokareddy (Ravi K.); A.F.W. van der Steen (Anton); G. Cingolani (Gino); M.W.J. Fornerod (Maarten); L.M. Veenhoff (Liesbeth M.)

    2015-01-01

    textabstractEndoplasmic reticulum-synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these

  18. A unifying mechanism accounts for sensing of membrane curvature by BAR domains, amphipathic helices and membrane-anchored proteins

    DEFF Research Database (Denmark)

    Bhatia, Vikram Kjøller; Hatzakis, Nikos; Stamou, Dimitrios

    2010-01-01

    The discovery of proteins that recognize membrane curvature created a paradigm shift by suggesting that membrane shape may act as a cue for protein localization that is independent of lipid or protein composition. Here we review recent data on membrane curvature sensing by three structurally...

  19. Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

    Science.gov (United States)

    Barros, Marilia; Nanda, Hirsh

    2016-01-01

    ABSTRACT By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane

  20. MreB-Dependent Organization of the E. coli Cytoplasmic Membrane Controls Membrane Protein Diffusion.

    Science.gov (United States)

    Oswald, Felix; Varadarajan, Aravindan; Lill, Holger; Peterman, Erwin J G; Bollen, Yves J M

    2016-03-01

    The functional organization of prokaryotic cell membranes, which is essential for many cellular processes, has been challenging to analyze due to the small size and nonflat geometry of bacterial cells. Here, we use single-molecule fluorescence microscopy and three-dimensional quantitative analyses in live Escherichia coli to demonstrate that its cytoplasmic membrane contains microdomains with distinct physical properties. We show that the stability of these microdomains depends on the integrity of the MreB cytoskeletal network underneath the membrane. We explore how the interplay between cytoskeleton and membrane affects trans-membrane protein (TMP) diffusion and reveal that the mobility of the TMPs tested is subdiffusive, most likely caused by confinement of TMP mobility by the submembranous MreB network. Our findings demonstrate that the dynamic architecture of prokaryotic cell membranes is controlled by the MreB cytoskeleton and regulates the mobility of TMPs. PMID:26958890

  1. Sphingolipid topology and membrane protein nanoclusters

    NARCIS (Netherlands)

    Hötzl, S.

    2009-01-01

    Sphingolipids are an essential class of membrane lipids in eukaryotic cells. Due to their high packing density and their affinity for cholesterol, sphingolipids are able to promote bilayer rigidity and impermeability. Apart from its ability to maintain biomembrane integrity, sphingomyelin (SM) is al

  2. Encapsulated membrane proteins: A simplified system for molecular simulation.

    Science.gov (United States)

    Lee, Sarah C; Khalid, Syma; Pollock, Naomi L; Knowles, Tim J; Edler, Karen; Rothnie, Alice J; R T Thomas, Owen; Dafforn, Timothy R

    2016-10-01

    Over the past 50years there has been considerable progress in our understanding of biomolecular interactions at an atomic level. This in turn has allowed molecular simulation methods employing full atomistic modelling at ever larger scales to develop. However, some challenging areas still remain where there is either a lack of atomic resolution structures or where the simulation system is inherently complex. An area where both challenges are present is that of membranes containing membrane proteins. In this review we analyse a new practical approach to membrane protein study that offers a potential new route to high resolution structures and the possibility to simplify simulations. These new approaches collectively recognise that preservation of the interaction between the membrane protein and the lipid bilayer is often essential to maintain structure and function. The new methods preserve these interactions by producing nano-scale disc shaped particles that include bilayer and the chosen protein. Currently two approaches lead in this area: the MSP system that relies on peptides to stabilise the discs, and SMALPs where an amphipathic styrene maleic acid copolymer is used. Both methods greatly enable protein production and hence have the potential to accelerate atomic resolution structure determination as well as providing a simplified format for simulations of membrane protein dynamics. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:26946242

  3. Role of rab proteins in epithelial membrane traffic

    NARCIS (Netherlands)

    van Ijzendoorn, SCD; Mostov, KE; Hoekstra, D

    2003-01-01

    Small GTPase rab proteins play an important role in various aspects of membrane traffic, including cargo selection, vesicle budding, vesicle motility, tethering, docking, and fusion. Recent data suggest also that rabs, and their divalent effector proteins, organize organelle subdomains and as such m

  4. ARAMEMNON, a novel database for Arabidopsis integral membrane proteins

    DEFF Research Database (Denmark)

    Schwacke, Rainer; Schneider, Anja; van der Graaff, Eric;

    2003-01-01

    A specialized database (DB) for Arabidopsis membrane proteins, ARAMEMNON, was designed that facilitates the interpretation of gene and protein sequence data by integrating features that are presently only available from individual sources. Using several publicly available prediction programs, put...... is accessible at the URL http://aramemnon.botanik.uni-koeln.de....

  5. Nanodisc films for membrane protein studies by neutron reflection

    DEFF Research Database (Denmark)

    Bertram, Nicolas; Laursen, Tomas; Barker, Robert;

    2015-01-01

    Nanodisc films are a promising approach to study the equilibrium conformation of membrane bound proteins in native-like environment. Here we compare nanodisc formation for NADPH-dependent cytochrome P450 oxidoreductase (POR) using two different scaffold proteins, MSP1D1 and MSP1E3D1. Despite the...

  6. Identification of outer membrane proteins of Yersinia pestis through biotinylation

    NARCIS (Netherlands)

    Smither, S.J.; Hill, J.; Baar, B.L.M. van; Hulst, A.G.; Jong, A.L. de; Titball, R.W.

    2007-01-01

    The outer membrane of Gram-negative bacteria contains proteins that might be good targets for vaccines, antimicrobials or detection systems. The identification of surface located proteins using traditional methods is often difficult. Yersinia pestis, the causative agent of plague, was labelled with

  7. Swimbladder membrane protein of an abyssal fish, Coryphaenoides acrolepis.

    Science.gov (United States)

    Mosholder, R S; Josephson, R V; Phleger, C F

    1979-01-01

    Protein components of the membranous foamy tissue collected from the swimbladder of Coryphaenoides acrolepis, a continental slope/deep sea grenadier fish, were partially fractionated and characterized by procedures used successfully for erythrocyte membrane proteins. Methods involving pH and ionic strength adjustment in the presence of EDTA and beta-mercaptoethanol resulted in some protein fractionation but no distinct separation or isolation of membrane proteins. Gel filtration by Sephadex G-100 and Sepharose 2B in the presence of dodecyl sulfate partially fractionated protein species between 18,000 and 150,000 molecular weight (as confirmed by dodecyl sulfate polyacrylamide gel electrophoresis). Low molecular weight proteins were resolvable into a few diffuse and streaky bands by dodecyl sulfate and chloral hydrate polyacrylamide gel electrophoresis, the former giving superior reso-ution. A major fraction of large molecular weight protein (greater than or equal to 40 X 10(6)) was not resolved by any method. A possible explanation for these unusual findings is that decompression due to rapid ascent of the fish from deep ocean caused irreversible alteration of swimbladder membrane proteins. PMID:504363

  8. Proteomic analysis of GPI-anchored membrane proteins

    DEFF Research Database (Denmark)

    Jung, Hye Ryung; Jensen, Ole Nørregaard

    2006-01-01

    Glycosyl-phosphatidyl-inositol-anchored proteins (GPI-APs) represent a subset of post-translationally modified proteins that are tethered to the outer leaflet of the plasma membrane via a C-terminal GPI anchor. GPI-APs are found in a variety of eukaryote species, from pathogenic microorganisms...

  9. Amyloid protein unfolding and insertion kinetics on neuronal membrane mimics

    Science.gov (United States)

    Qiu, Liming; Buie, Creighton; Vaughn, Mark; Cheng, Kwan

    2010-03-01

    Atomistic details of beta-amyloid (Aβ ) protein unfolding and lipid interaction kinetics mediated by the neuronal membrane surface are important for developing new therapeutic strategies to prevent and cure Alzheimer's disease. Using all-atom MD simulations, we explored the early unfolding and insertion kinetics of 40 and 42 residue long Aβ in binary lipid mixtures with and without cholesterol that mimic the cholesterol-depleted and cholesterol-enriched lipid nanodomains of neurons. The protein conformational transition kinetics was evaluated from the secondary structure profile versus simulation time plot. The extent of membrane disruption was examined by the calculated order parameters of lipid acyl chains and cholesterol fused rings as well as the density profiles of water and lipid headgroups at defined regions across the lipid bilayer from our simulations. Our results revealed that both the cholesterol content and the length of the protein affect the protein-insertion and membrane stability in our model lipid bilayer systems.

  10. VAMP-1: a synaptic vesicle-associated integral membrane protein.

    Science.gov (United States)

    Trimble, W S; Cowan, D M; Scheller, R H

    1988-01-01

    Several proteins are associated with, or are integral components of, the lipid bilayer that forms the delineating membrane of neuronal synaptic vesicles. To characterize these molecules, we used a polyclonal antiserum raised against purified cholinergic synaptic vesicles from Torpedo to screen a cDNA expression library constructed from mRNA of the electromotor nucleus. One clone encodes VAMP-1 (vesicle-associated membrane protein 1), a nervous-system-specific protein of 120 amino acids whose primary sequence can be divided into three domains: a proline-rich amino terminus, a highly charged internal region, and a hydrophobic carboxyl-terminal domain that is predicted to comprise a membrane anchor. Tryptic digestion of intact and lysed vesicles suggests that the protein faces the cytoplasm, where it may play a role in packaging, transport, or release of neurotransmitters. Images PMID:3380805

  11. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

    Science.gov (United States)

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-01-01

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer. PMID:26512702

  12. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

    Science.gov (United States)

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-01-01

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  13. Characterization of the major integral protein of vacuolar membrane.

    Science.gov (United States)

    Maeshima, M

    1992-04-01

    The vacuolar membrane of radish (Raphanus sativus) taproot contained a large quantity of a protein of 23 kilodaltons that accounted for more than 25% of the total membrane proteins. The protein, tentatively named VM 23, was purified and characterized. VM 23 tends to aggregate at high temperature even in the presence of 1% sodium dodecyl sulfate. The apparent molecular size of VM 23 was estimated to be about 400 kilodaltons by polyacrylamide gel electrophoresis in the presence of 0.1% Triton X-100. VM 23 was partially extracted from the vacuolar membranes with chloroform:methanol, indicating its high hydrophobicity. The hydrophobic carboxyl modifier N,N'-dicyclohexylcarbodiimide bound covalently to VM 23. The results suggest that VM 23 may act as a secondary transport system coupled with the proton transport. The antibody against radish VM 23 reacted with the major proteins in the vacuolar membranes of mung bean (Vigna radiata) and castor bean (Ricinus communis) hypocotyls and pumpkin (Cucurbita moschata) epicotyl, but not with that of sugar beet (Beta vulgaris) taproot. VM 23 comigrated with vacuolar H(+)-pyrophosphatase on sucrose density gradient centrifugation after sonication of membranes, indicating that it is associated with the vacuolar membrane.

  14. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

    Directory of Open Access Journals (Sweden)

    Marc Lenoir

    2015-10-01

    Full Text Available The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH and Tec homology (TH domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  15. Expression of Extracellular Matrix Proteins in Basal Membranes During Fetal Nephron Development in Mice

    Directory of Open Access Journals (Sweden)

    Beyhan GÜRCÜ

    2016-04-01

    Full Text Available In this study, we investigated the distribution of laminin, collagen type IV, nidogen and fibronectine during metanephric development in fetal mouse kidney by immunohistochemistry. Stain density of basement membranes of tubules, glomerules and mesangial matrix were compared in pre-capillary, immature glomerular and mature glomerular stages of fetal kidney. All the matrix proteins were strongly stained in precapillary stage. In immature glomerular stage, a strong staining was observed for fibronectin. Staining intensity was slightly decreased for the other proteins in this stage. In mature glomerular stage, diminished staining for all proteins was observed similar to the previous stage, except fibronectin. The strongest immunoreactions were found for fibronectin and nidogen in all investigated stages. In general, there was a similar staining intensity for all glycoproteins during maturation except for laminin. It was thought that the distribution of extracellular matrix molecules plays an important role for the kidney development. Interactions amoung these molecules probably crucial on cell behavior like migration, proliferation and differentiation in normal development of the nephron.

  16. Symmetry and size of membrane protein polyhedral nanoparticles

    CERN Document Server

    Li, Di; Haselwandter, Christoph A

    2016-01-01

    In recent experiments [T. Basta et al., Proc. Natl. Acad. Sci. U.S.A. 111, 670 (2014)] lipids and membrane proteins were observed to self-assemble into membrane protein polyhedral nanoparticles (MPPNs) with a well-defined polyhedral protein arrangement and characteristic size. We develop a model of MPPN self-assembly in which the preferred symmetry and size of MPPNs emerge from the interplay of protein-induced lipid bilayer deformations, topological defects in protein packing, and thermal effects. With all model parameters determined directly from experiments, our model correctly predicts the observed symmetry and size of MPPNs. Our model suggests how key lipid and protein properties can be modified to produce a range of MPPN symmetries and sizes in experiments.

  17. A new window into the molecular physiology of membrane proteins.

    Science.gov (United States)

    Landreh, Michael; Robinson, Carol V

    2015-01-15

    Integral membrane proteins comprise ∼25% of the human proteome. Yet, our understanding of their molecular physiology is still in its infancy. This can be attributed to two factors: the experimental challenges that arise from the difficult chemical nature of membrane proteins, and the unclear relationship between their activity and their native environment. New approaches are therefore required to address these challenges. Recent developments in mass spectrometry have shown that it is possible to study membrane proteins in a solvent-free environment and provide detailed insights into complex interactions, ligand binding and folding processes. Interestingly, not only detergent micelles but also lipid bilayer nanodiscs or bicelles can serve as a means for the gentle desolvation of membrane proteins in the gas phase. In this manner, as well as by direct addition of lipids, it is possible to study the effects of different membrane components on the structure and function of the protein components allowing us to add functional data to the least accessible part of the proteome. PMID:25630257

  18. Understanding disordered and membrane protein recognition by molecular dynamics

    OpenAIRE

    Stanley, Nathaniel H., 1983-

    2015-01-01

    This thesis has been about the use of a simulation technique, known as molecular dynamics simulations, to study biophysics in proteins that have historically been difficult to study with other methods. We have studied numerous systems, namely binding to the membrane proteins Fatty acid amide hydrolase (FAAH) and the sphingosine-1-phosphate receptor 1 (S1P1R), and folding in the disordered protein kinase inducible domain (KID). In each case we have been able to analyze processes and uncover be...

  19. High membrane protein oxidation in the human cerebral cortex

    OpenAIRE

    Matthias Granold; Bernd Moosmann; Irina Staib-Lasarzik; Thomas Arendt; Adriana del Rey; Kristin Engelhard; Christian Behl; Parvana Hajieva

    2014-01-01

    Oxidative stress is thought to be one of the main mediators of neuronal damage in human neurodegenerative disease. Still, the dissection of causal relationships has turned out to be remarkably difficult. Here, we have analyzed global protein oxidation in terms of carbonylation of membrane proteins and cytoplasmic proteins in three different mammalian species: aged human cortex and cerebellum from patients with or without Alzheimer's disease, mouse cortex and cerebellum from young and old anim...

  20. Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

    Science.gov (United States)

    Focke, Paul J; Hein, Christopher; Hoffmann, Beate; Matulef, Kimberly; Bernhard, Frank; Dötsch, Volker; Valiyaveetil, Francis I

    2016-08-01

    Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence. PMID:27384110

  1. Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein–protein interfaces

    Energy Technology Data Exchange (ETDEWEB)

    Wylie, Benjamin J. [Columbia University, Department of Chemistry (United States); Dzikovski, Boris G. [Cornell University, National Biomedical Center for Advanced ESR Technology, Department of Chemistry and Chemical Biology (United States); Pawsey, Shane; Caporini, Marc; Rosay, Melanie [Bruker BioSpin Corporation (United States); Freed, Jack H. [Cornell University, National Biomedical Center for Advanced ESR Technology, Department of Chemistry and Chemical Biology (United States); McDermott, Ann E., E-mail: aem5@columbia.edu [Columbia University, Department of Chemistry (United States)

    2015-04-15

    We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces.

  2. Determining nuclear shape: The role of farnesylated nuclear membrane proteins

    OpenAIRE

    Polychronidou, Maria; Großhans, Jörg

    2011-01-01

    Changes in nuclear morphology are observed in diverse developmental processes as well as in pathological conditions. Modification of nuclear membrane and nuclear lamina protein levels results in altered nuclear shapes, as it has been demonstrated in experimental systems ranging from yeast to human cells. The important role of nuclear membrane components in regulating nuclear morphology is additionally highlighted by the abnormally shaped nuclei observed in diseases where nuclear lamina protei...

  3. Erythrocyte membrane protein destabilization versus clinical outcome in 160 Portuguese Hereditary Spherocytosis patients

    OpenAIRE

    Rocha, Susana; Costa, Elísio; Rocha-Pereira, Petronila; Ferreira, Fátima; Cleto, Esmeralda; Barbot, José; Quintanilha, Alexandre; Belo, Luís; Santos-Silva, Alice

    2010-01-01

    Abstract Hereditary Spherocytosis (HS) is a haemolytic anaemia caused by erythrocyte protein membrane defects ? spectrin, ankyrin, band 3 or protein 4.2 ? that lead to membrane destabilization. Ours aims were to evaluate the prevalence of protein deficiencies and the role of membrane proteins or of membrane linked proteins in membrane disturbance and in HS clinical outcome. We studied 215 Portuguese individuals ? 203 from 71 families plus 12 individual unrelated subjects, and found...

  4. Glycan Moieties as Bait to Fish Plasma Membrane Proteins.

    Science.gov (United States)

    Fang, Fei; Zhao, Qun; Sui, Zhigang; Liang, Yu; Jiang, Hao; Yang, Kaiguang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2016-05-17

    Plasma membrane proteome analysis is of significance for screening candidate biomarkers and drug targets. However, due to their low abundance and lack of specific groups that can enable their capture, the plasma membrane proteins (PMPs) are under-represented. On the basis of the fact that PMPs are embedded in or anchored to the phospholipid bilayer of the plasma membrane and the glycan moieties of proteins and lipids located on the plasma membrane are exposed outside of the cell surface, we proposed a strategy to capture PMPs, termed as glycan moieties-directed PMPs enrichment (GMDPE). With the glycan moieties exposed outside of the cells as bait to ensure the selectivity and the phospholipid bilayer as raft to provide the sensitivity, we applied this strategy into the plasma membrane proteome analysis of HeLa cells, and in total, 772 PMPs were identified, increased by 4.5 times compared to those identified by the reported cell surface biotinylation method. Notably, among them, 86 CD antigens and 16 ion channel proteins were confidently identified. All these results demonstrated that our proposed approach has great potential in the large scale plasma membrane proteome profiling.

  5. Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins

    DEFF Research Database (Denmark)

    Elortza, Felix; Nühse, Thomas S; Foster, Leonard J;

    2003-01-01

    Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a functionally and structurally diverse family of post-translationally modified membrane proteins found mostly in the outer leaflet of the plasma membrane in a variety of eukaryotic cells. Although the general role of GPI-APs remains...... development. We here present a general mass spectrometry-based proteomic "shave-and-conquer" strategy that specifically targets GPI-APs. Using a combination of biochemical methods, mass spectrometry, and computational sequence analysis we identified six GPI-APs in a Homo sapiens lipid raft-enriched fraction...... and 44 GPI-APs in an Arabidopsis thaliana membrane preparation, representing the largest experimental dataset of GPI-anchored proteins to date....

  6. Structure and Dynamic Properties of Membrane Proteins using NMR

    DEFF Research Database (Denmark)

    Rösner, Heike; Kragelund, Birthe

    2012-01-01

    conformational changes. Their structural and functional decoding is challenging and has imposed demanding experimental development. Solution nuclear magnetic resonance (NMR) spectroscopy is one of the techniques providing the capacity to make a significant difference in the deciphering of the membrane protein...... structure-function paradigm. The method has evolved dramatically during the last decade resulting in a plethora of new experiments leading to a significant increase in the scientific repertoire for studying membrane proteins. Besides solving the three-dimensional structures using state-of-the-art approaches......-populated states, this review seeks to introduce the vast possibilities solution NMR can offer to the study of membrane protein structure-function analyses with special focus on applicability. © 2012 American Physiological Society. Compr Physiol 2:1491-1539, 2012....

  7. Identification of outer membrane proteins of Mycobacterium tuberculosis.

    Science.gov (United States)

    Song, Houhui; Sandie, Reatha; Wang, Ying; Andrade-Navarro, Miguel A; Niederweis, Michael

    2008-11-01

    The cell wall of mycobacteria includes an unusual outer membrane of extremely low permeability. While Escherichia coli uses more than 60 proteins to functionalize its outer membrane, only two mycobacterial outer membrane proteins (OMPs) are known. The porin MspA of Mycobacterium smegmatis provided the proof of principle that integral mycobacterial OMPs share the beta-barrel structure, the absence of hydrophobic alpha-helices and the presence of a signal peptide with OMPs of gram-negative bacteria. These properties were exploited in a multi-step bioinformatic approach to predict OMPs of M. tuberculosis. A secondary structure analysis was performed for 587 proteins of M. tuberculosis predicted to be exported. Scores were calculated for the beta-strand content and the amphiphilicity of the beta-strands. Reference OMPs of gram-negative bacteria defined threshold values for these parameters that were met by 144 proteins of unknown function of M. tuberculosis. Two of them were verified as OMPs by a novel two-step experimental approach. Rv1698 and Rv1973 were detected only in the total membrane fraction of M. bovis BCG in Western blot experiments, while proteinase K digestion of whole cells showed the surface accessibility of these proteins. These findings established that Rv1698 and Rv1973 are indeed localized in the outer membrane and tripled the number of known OMPs of M. tuberculosis. Significantly, these results provide evidence for the usefulness of the bioinformatic approach to predict mycobacterial OMPs and indicate that M. tuberculosis likely has many OMPs with beta-barrel structure. Our findings pave the way to identify the set of proteins which functionalize the outer membrane of M. tuberculosis. PMID:18439872

  8. The cellular membrane as a mediator for small molecule interaction with membrane proteins.

    Science.gov (United States)

    Mayne, Christopher G; Arcario, Mark J; Mahinthichaichan, Paween; Baylon, Javier L; Vermaas, Josh V; Navidpour, Latifeh; Wen, Po-Chao; Thangapandian, Sundarapandian; Tajkhorshid, Emad

    2016-10-01

    The cellular membrane constitutes the first element that encounters a wide variety of molecular species to which a cell might be exposed. Hosting a large number of structurally and functionally diverse proteins associated with this key metabolic compartment, the membrane not only directly controls the traffic of various molecules in and out of the cell, it also participates in such diverse and important processes as signal transduction and chemical processing of incoming molecular species. In this article, we present a number of cases where details of interaction of small molecular species such as drugs with the membrane, which are often experimentally inaccessible, have been studied using advanced molecular simulation techniques. We have selected systems in which partitioning of the small molecule with the membrane constitutes a key step for its final biological function, often binding to and interacting with a protein associated with the membrane. These examples demonstrate that membrane partitioning is not only important for the overall distribution of drugs and other small molecules into different compartments of the body, it may also play a key role in determining the efficiency and the mode of interaction of the drug with its target protein. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:27163493

  9. Molecular dynamics simulations of biological membranes and membrane proteins using enhanced conformational sampling algorithms.

    Science.gov (United States)

    Mori, Takaharu; Miyashita, Naoyuki; Im, Wonpil; Feig, Michael; Sugita, Yuji

    2016-07-01

    This paper reviews various enhanced conformational sampling methods and explicit/implicit solvent/membrane models, as well as their recent applications to the exploration of the structure and dynamics of membranes and membrane proteins. Molecular dynamics simulations have become an essential tool to investigate biological problems, and their success relies on proper molecular models together with efficient conformational sampling methods. The implicit representation of solvent/membrane environments is reasonable approximation to the explicit all-atom models, considering the balance between computational cost and simulation accuracy. Implicit models can be easily combined with replica-exchange molecular dynamics methods to explore a wider conformational space of a protein. Other molecular models and enhanced conformational sampling methods are also briefly discussed. As application examples, we introduce recent simulation studies of glycophorin A, phospholamban, amyloid precursor protein, and mixed lipid bilayers and discuss the accuracy and efficiency of each simulation model and method. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

  10. Transmembrane protein sorting driven by membrane curvature

    NARCIS (Netherlands)

    H. Strahl; S. Ronneau; B. Solana González; D. Klutsch; C. Schaffner-Barbero; L.W. Hamoen

    2015-01-01

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show th

  11. Major Intrinsic Proteins in Biomimetic Membranes

    DEFF Research Database (Denmark)

    Helix Nielsen, Claus

    2010-01-01

    permeants such as carbon dioxide, nitric oxide, ammonia, hydrogen peroxide and the metalloids antimonite, arsenite, silicic and boric acid depending on the effective restriction mechanism of the protein. The flux properties of MIPs thus lead to the question if MIPs can be used in separation devices...

  12. Codon optimizing for increased membrane protein production

    DEFF Research Database (Denmark)

    Mirzadeh, K.; Toddo, S.; Nørholm, Morten;

    2016-01-01

    Reengineering a gene with synonymous codons is a popular approach for increasing production levels of recombinant proteins. Here we present a minimalist alternative to this method, which samples synonymous codons only at the second and third positions rather than the entire coding sequence...

  13. Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase

    Science.gov (United States)

    Hatahet, Feras; Blazyk, Jessica L.; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E.; Beckwith, Jonathan; Boyd, Dana

    2015-01-01

    Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants. PMID:26598701

  14. Immunochemical Properties of the Major Outer Membrane Protein of Vibrio cholerae

    OpenAIRE

    Kabir, Shahjahan

    1983-01-01

    Antisera to the major outer membrane protein of Vibrio cholerae (molecular weight, 48,000) raised in rabbits (i) agglutinated several strains of V. cholerae and (ii) immunoprecipitated outer membrane proteins prepared from both the biotypes and serotypes of V. cholerae. Antibodies of all isotypes to the major outer membrane protein were detected in immune human sera by enzyme-linked immunosorbent assay. These results suggest that the major outer membrane protein was the common outer membrane ...

  15. Self-assembling peptide and protein nanodiscs for studies of membrane proteins

    DEFF Research Database (Denmark)

    Midtgaard, Søren Roi

    of proteins encoded by the human genome. G-protein coupled receptors mediate the majority of hormone and neurotransmitter signals as well as being responsible for perception of light, smell and taste in the human body, and a number of Nobel prizes has been awarded based on their study. Structural...... membrane proteins. A minimalistic approach was tested where the ApoA1 protein was mimicked my small amphipathic helical peptides. The resulting discs were very similar to ApoA1 based discs in size and in their ability to stabilize incorporated membrane proteins. Furthermore, due to their enhanced dynamical...

  16. Protein receptor-independent plasma membrane remodeling by HAMLET

    DEFF Research Database (Denmark)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.;

    2015-01-01

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This "protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains...... in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range...... of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a "receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET...

  17. Heterologous expression of membrane proteins: choosing the appropriate host.

    Directory of Open Access Journals (Sweden)

    Florent Bernaudat

    Full Text Available BACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals, functions (transporters, receptors, enzymes and topologies (between 0 and 13 transmembrane segments. The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein.

  18. Anti-IL-5 treatment reduces deposition of ECM proteins in the bronchial subepithelial basement membrane of mild atopic asthmatics

    OpenAIRE

    Flood-Page, Patrick; Menzies-Gow, Andrew; Phipps, Simon; Ying, Sun; Wangoo, Arun; Ludwig, Mara S.; Barnes, Neil; Robinson, Douglas; Kay, A. Barry

    2003-01-01

    Eosinophil-derived TGF-β has been implicated in remodeling events in asthma. We hypothesized that reduction of bronchial mucosal eosinophils with anti–IL-5 would reduce markers of airway remodeling. Bronchial biopsies were obtained before and after three infusions of a humanized, anti–IL-5 monoclonal antibody (mepolizumab) in 24 atopic asthmatics in a randomized, double-blind, placebo-controlled study. The thickness and density of tenascin, lumican, and procollagen III in the reticular baseme...

  19. Genome wide analysis indicates genes for basement membrane and cartilage matrix proteins as candidates for hip dysplasia in Labrador Retrievers

    NARCIS (Netherlands)

    Lavrijsen, Ineke; Leegwater, Peter; Martin, AJ; Harris, SJ; Tryfonidou, Marianna; Heuven, Henri; Hazewinkel, Herman

    2014-01-01

    Hip dysplasia, an abnormal laxity of the hip joint, is seen in humans as well as dogs and is one of the most common skeletal disorders in dogs. Canine hip dysplasia is considered multifactorial and polygenic, and a variety of chromosomal regions have been associated with the disorder. We performed a

  20. Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli

    DEFF Research Database (Denmark)

    Toddo, Stephen; Soderstrom, Bill; Palombo, Isolde;

    2012-01-01

    /periplasmic location of the N-terminus of a protein. Here, we show that the bimolecular split-green fluorescent protein complementation system can overcome this limitation and can be used to determine the location of both the N- and C-termini of inner membrane proteins in Escherichia coli....

  1. Decrease in membrane phospholipid unsaturation induces unfolded protein response.

    Science.gov (United States)

    Ariyama, Hiroyuki; Kono, Nozomu; Matsuda, Shinji; Inoue, Takao; Arai, Hiroyuki

    2010-07-16

    Various kinds of fatty acids are distributed in membrane phospholipids in mammalian cells and tissues. The degree of fatty acid unsaturation in membrane phospholipids affects many membrane-associated functions and can be influenced by diet and by altered activities of lipid-metabolizing enzymes such as fatty acid desaturases. However, little is known about how mammalian cells respond to changes in phospholipid fatty acid composition. In this study we showed that stearoyl-CoA desaturase 1 (SCD1) knockdown increased the amount of saturated fatty acids and decreased that of monounsaturated fatty acids in phospholipids without affecting the amount or the composition of free fatty acid and induced unfolded protein response (UPR), evidenced by increased expression of C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) mRNAs and splicing of Xbox-binding protein 1 (XBP1) mRNA. SCD1 knockdown-induced UPR was rescued by various unsaturated fatty acids and was enhanced by saturated fatty acid. Lysophosphatidylcholine acyltransferase 3 (LPCAT3), which incorporates preferentially polyunsaturated fatty acids into phosphatidylcholine, was up-regulated in SCD1 knockdown cells. Knockdown of LPCAT3 synergistically enhanced UPR with SCD1 knockdown. Finally we showed that palmitic acid-induced UPR was significantly enhanced by LPCAT3 knockdown as well as SCD1 knockdown. These results suggest that a decrease in membrane phospholipid unsaturation induces UPR.

  2. Structural investigation of membrane proteins by electron microscopy

    NARCIS (Netherlands)

    Moscicka, Katarzyna Beata

    2009-01-01

    Biological membranes are vital components of all living systems, forming the boundaries of cells and their organelles. They consist of a lipid bilayer and embedded proteins, which are nanomachines that fulfill key functions such as energy conversion, solute transport, secretion, and signal transduct

  3. How curved membranes recruit amphipathic helices and protein anchoring motifs

    DEFF Research Database (Denmark)

    Hatzakis, Nikos; Bhatia, Vikram Kjøller; Larsen, Jannik;

    2009-01-01

    Lipids and several specialized proteins are thought to be able to sense the curvature of membranes (MC). Here we used quantitative fluorescence microscopy to measure curvature-selective binding of amphipathic motifs on single liposomes 50-700 nm in diameter. Our results revealed that sensing is p...

  4. pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins.

    Directory of Open Access Journals (Sweden)

    Priyanka Prakash

    2015-10-01

    Full Text Available Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation.

  5. A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli.

    Science.gov (United States)

    Urfer, Matthias; Bogdanovic, Jasmina; Lo Monte, Fabio; Moehle, Kerstin; Zerbe, Katja; Omasits, Ulrich; Ahrens, Christian H; Pessi, Gabriella; Eberl, Leo; Robinson, John A

    2016-01-22

    Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM.

  6. Increased expression of lysosome membrane protein 2 in glomeruli of patients with idiopathic membranous nephropathy

    NARCIS (Netherlands)

    Rood, I.M.; Merchant, M.L.; Wilkey, D.W.; Zhang, T.; Zabrouskov, V.; Vlag, J. van der; Dijkman, H.B.P.M.; Willemsen, B.K.; Wetzels, J.F.M.; Klein, J.B.; Deegens, J.K.J.

    2015-01-01

    Urinary microvesicles constitute a rich source of membrane-bound and intracellular proteins that may provide important clues of pathophysiological mechanisms in renal disease. In the current study, we analyzed and compared the proteome of urinary microvesicles from patients with idiopathic membranou

  7. A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli.

    Science.gov (United States)

    Urfer, Matthias; Bogdanovic, Jasmina; Lo Monte, Fabio; Moehle, Kerstin; Zerbe, Katja; Omasits, Ulrich; Ahrens, Christian H; Pessi, Gabriella; Eberl, Leo; Robinson, John A

    2016-01-22

    Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM. PMID:26627837

  8. The cholesterol membrane anchor of the Hedgehog protein confers stable membrane association to lipid-modified proteins

    OpenAIRE

    Peters, Carsten; Wolf, Alexander; Wagner, Melanie; Kuhlmann, Jürgen; Waldmann, Herbert

    2004-01-01

    The Hedgehog proteins are potent organizers of animal development. They carry a cholesterol ester at the C terminus of their signaling domain. The membrane anchoring mediated by this lipophilic modification was studied by means of an approach integrating cell biology, biochemistry, biophysics, and organic chemistry techniques. Sterol-modified and fluorescent-labeled Hedgehog-derived peptides and proteins were synthesized and investigated in biophysical and cell-biological assays. These experi...

  9. Chicken Egg Shell Membrane Associated Proteins and Peptides.

    Science.gov (United States)

    Makkar, Sarbjeet; Liyanage, Rohana; Kannan, Lakshmi; Packialakshmi, Balamurugan; Lay, Jack O; Rath, Narayan C

    2015-11-11

    Egg shells are poultry industry byproducts with potential for use in various biological and agricultural applications. We have been interested in the membranes underlying the calcareous shell as a feed supplement, which showed potential to improve immunity and performance of post hatch poultry. Therefore, to determine their protein and peptide profiles, we extracted the egg shell membranes (ESM) from fresh unfertilized eggs with methanol and guanidine hydrochloride (GdHCl) to obtain soluble proteins for analysis by mass spectrometry. The methanol extract was subjected to matrix-assisted laser desorption ionization (MALDI), electrospray ionization (ESI), high-performance reverse phase liquid chromatographic separation (HPLC), and tandem mass spectrometry (MS/MS) to determine its peptide and protein profiles. The GdHCl extract was subjected to ESI-HPLC-MS/MS following trypsin digestion of reduced/alkylated proteins. Nine proteins from the methanol extract and >275 proteins from the GdHCl extract were tentatively identified. The results suggested the presence of several abundant proteins from egg whites, such as ovoalbumin, ovotransferrin, and lysozyme as well as many others associated with antimicrobial, biomechanical, cytoskeletal organizational, cell signaling, and enzyme activities. Collagens, keratin, agrin, and laminin were some of the structural proteins present in the ESM. The methanol-soluble fraction contained several clusterin peptides and defensins, particularly, two isoforms of gallin. The ratios of the two isoforms of gallin differed between the membranes obtained from brown and white eggs. The high abundance of several antimicrobial, immunomodulatory, and other bioactive proteins in the ESM along with its potential to entrap various microbes and antigens may make it a suitable vehicle for oral immunization of post hatch poultry and improve their disease resistance. PMID:26485361

  10. Chicken Egg Shell Membrane Associated Proteins and Peptides.

    Science.gov (United States)

    Makkar, Sarbjeet; Liyanage, Rohana; Kannan, Lakshmi; Packialakshmi, Balamurugan; Lay, Jack O; Rath, Narayan C

    2015-11-11

    Egg shells are poultry industry byproducts with potential for use in various biological and agricultural applications. We have been interested in the membranes underlying the calcareous shell as a feed supplement, which showed potential to improve immunity and performance of post hatch poultry. Therefore, to determine their protein and peptide profiles, we extracted the egg shell membranes (ESM) from fresh unfertilized eggs with methanol and guanidine hydrochloride (GdHCl) to obtain soluble proteins for analysis by mass spectrometry. The methanol extract was subjected to matrix-assisted laser desorption ionization (MALDI), electrospray ionization (ESI), high-performance reverse phase liquid chromatographic separation (HPLC), and tandem mass spectrometry (MS/MS) to determine its peptide and protein profiles. The GdHCl extract was subjected to ESI-HPLC-MS/MS following trypsin digestion of reduced/alkylated proteins. Nine proteins from the methanol extract and >275 proteins from the GdHCl extract were tentatively identified. The results suggested the presence of several abundant proteins from egg whites, such as ovoalbumin, ovotransferrin, and lysozyme as well as many others associated with antimicrobial, biomechanical, cytoskeletal organizational, cell signaling, and enzyme activities. Collagens, keratin, agrin, and laminin were some of the structural proteins present in the ESM. The methanol-soluble fraction contained several clusterin peptides and defensins, particularly, two isoforms of gallin. The ratios of the two isoforms of gallin differed between the membranes obtained from brown and white eggs. The high abundance of several antimicrobial, immunomodulatory, and other bioactive proteins in the ESM along with its potential to entrap various microbes and antigens may make it a suitable vehicle for oral immunization of post hatch poultry and improve their disease resistance.

  11. Interaction of Serum Proteins with Surface of Hemodialysis Fiber Membranes

    Science.gov (United States)

    Afrin, Rehana; Shirako, Yuji; Kishimoto, Kikuo; Ikai, Atsushi

    2012-08-01

    The poly(vinyl pyrrolidone)-covered hydrophilic surface of hollow-fiber membranes (fiber membrane, hereafter) for hemodialysis was mechanically probed using modified tips on an atomic force microscope (AFM) with covalent crosslinkers and several types of serum protein. The retraction part of many of the force extension (F-E) curves obtained with AFM tips coated with serum albumin had a long and smooth extension up to 200-300 nm indicating forced elongation of poly(vinyl pyrrolidone) chains. When fibrinogen-coated tips were used, long extension F-E curves up to 500 nm with multiple peaks were obtained in addition to smooth curves most likely reflecting the unfolding of fibrinogen molecules. The results indicated that individual polymer chains had a significant affinity toward serum proteins. The adhesion frequency of tips coated with serum proteins was lower on the poly(vinyl pyrrolidone) surface than on the uncoated hydrophobic polysulfone surface.

  12. Hydrodynamic collective effects of active proteins in biological membranes

    CERN Document Server

    Koyano, Yuki; Mikhailov, Alexander S

    2016-01-01

    Lipid bilayers forming biological membranes are known to behave as viscous 2D fluids on submicrometer scales; usually they contain a large number of active protein inclusions. Recently, it has been shown [Proc. Nat. Acad. Sci. USA 112, E3639 (2015)] that such active proteins should in- duce non-thermal fluctuating lipid flows leading to diffusion enhancement and chemotaxis-like drift for passive inclusions in biomembranes. Here, a detailed analytical and numerical investigation of such effects is performed. The attention is focused on the situations when proteins are concentrated within lipid rafts. We demonstrate that passive particles tend to become attracted by active rafts and are accumulated inside them.

  13. Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Timmins, P.A. [ILL, Grenoble (France); Pebay-Peyroula, E. [IBS-UJF Grenoble (France)

    1994-12-31

    The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H{sub 2}O/D{sub 2}O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished.

  14. Membrane proteins PmpG and PmpH are major constituents of Chlamydia trachomatis L2 outer membrane complex

    DEFF Research Database (Denmark)

    Mygind, Per H; Christiansen, Gunna; Roepstorff, P;

    2000-01-01

    The outer membrane complex of Chlamydia is involved in the initial adherence and ingestion of Chlamydia by the host cell. In order to identify novel proteins in the outer membrane of Chlamydia trachomatis L2, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. By....... By silver staining of the protein profile, a major protein doublet of 100-110 kDa was detected. In-gel tryptic digestion and matrix-assisted laser desorption/ionization mass spectrometry identified these proteins as the putative outer membrane proteins PmpG and PmpH....

  15. Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with 18O labeling

    Science.gov (United States)

    Zhang, Haizhen; Brown, Roslyn N.; Qian, Wei-Jun; Monroe, Matthew E.; Purvine, Samuel O.; Moore, Ronald J.; Gritsenko, Marina A.; Shi, Liang; Romine, Margaret F; Fredrickson, James K.; Paša-Tolić, Ljiljana; Smith, Richard D.; Lipton, Mary S.

    2010-01-01

    We report a mass spectrometry-based strategy for quantitative analysis of cell surface membrane proteome changes. The strategy includes enrichment of surface membrane proteins using a membrane-impermeable chemical probe followed by stable isotope 18O labeling and LC-MS analysis. We applied this strategy for enriching membrane proteins expressed by Shewanella oneidensis MR-1, a gram-negative bacterium with known metal-reduction capability via extracellular electron transfer between outer membrane proteins and extracellular electron receptors. LC/MS/MS analysis resulted in the identification of about 400 proteins with 79% of them being predicted to be membrane localized. Quantitative aspects of the membrane enrichment were shown by peptide level 16O and 18O labeling of proteins from wild-type and mutant cells (generated from deletion of a type II secretion protein, GspD) prior to LC-MS analysis. Using a chemical probe labeled pure protein as an internal standard for normalization, the quantitative data revealed reduced abundances in ΔgspD mutant cells of many outer membrane proteins including the outer membrane c-cype cytochromes OmcA and MtrC, in agreement with previously investigation demonstrating that these proteins are substrates of the type II secretion system. PMID:20380418

  16. Functionalized membrane supports for covalent protein microsequence analysis

    Energy Technology Data Exchange (ETDEWEB)

    Coull, J.M.; Pappin, D.J.; Mark, J.; Aebersold, R.; Koester, H. (MilliGen/Biosearch, Division of Millipore, Burlington, MA (USA))

    1991-04-01

    Methods were developed for high yield covalent attachment of peptides and proteins to isothiocyanate and arylamine-derivatized poly(vinylidene difluoride) membranes for solid-phase sequence analysis. Solutions of protein or peptide were dried onto 8-mm membrane disks such that the functional groups on the surface and the polypeptide were brought into close proximity. In the case of the isothiocyanate membrane, reaction between polypeptide amino groups and the surface isothiocyanate moieties was promoted by application of aqueous N-methylmorpholine. Attachment of proteins and peptides to the arylamine surface was achieved by application of water-soluble carbodiimide in a pH 5.0 buffer. Edman degradation of covalently bound polypeptides was accomplished with initial and repetitive sequence yields ranging from 33 to 75% and 88.5 to 98.5%, respectively. The yields were independent of the sample load (20 pmol to greater than 1 nmol) for either surface. Significant loss of material was not observed when attachment residues were encountered during sequence runs. Application of bovine beta-lactoglobulin A chain, staphylococcus protein A, or the peptide melittin to the isothiocyanate membrane allowed for extended N-terminal sequence identification (35 residues from 20 pmol of beta-lactoglobulin). A number of synthetic and naturally occurring peptides were sequenced to the C-terminal residue following attachment to the arylamine surface. In one example, 10 micrograms of bovine alpha-casein was digested with staphylococcal protease V8 and the peptides were separated by reverse-phase chromatography. Peptide fractions were then directly applied to arylamine membrane disks for covalent sequence analysis. From as little as 2 pmol of initial signal it was possible to determine substantial sequence information (greater than 10 residues).

  17. Analysis of membrane proteins in metagenomics: networks of correlated environmental features and protein families.

    Science.gov (United States)

    Patel, Prianka V; Gianoulis, Tara A; Bjornson, Robert D; Yip, Kevin Y; Engelman, Donald M; Gerstein, Mark B

    2010-07-01

    Recent metagenomics studies have begun to sample the genomic diversity among disparate habitats and relate this variation to features of the environment. Membrane proteins are an intuitive, but thus far overlooked, choice in this type of analysis as they directly interact with the environment, receiving signals from the outside and transporting nutrients. Using global ocean sampling (GOS) data, we found nearly approximately 900,000 membrane proteins in large-scale metagenomic sequence, approximately a fifth of which are completely novel, suggesting a large space of hitherto unexplored protein diversity. Using GPS coordinates for the GOS sites, we extracted additional environmental features via interpolation from the World Ocean Database, the National Center for Ecological Analysis and Synthesis, and empirical models of dust occurrence. This allowed us to study membrane protein variation in terms of natural features, such as phosphate and nitrate concentrations, and also in terms of human impacts, such as pollution and climate change. We show that there is widespread variation in membrane protein content across marine sites, which is correlated with changes in both oceanographic variables and human factors. Furthermore, using these data, we developed an approach, protein families and environment features network (PEN), to quantify and visualize the correlations. PEN identifies small groups of covarying environmental features and membrane protein families, which we call "bimodules." Using this approach, we find that the affinity of phosphate transporters is related to the concentration of phosphate and that the occurrence of iron transporters is connected to the amount of shipping, pollution, and iron-containing dust.

  18. The Origin and Early Evolution of Membrane Proteins

    Science.gov (United States)

    Pohorille, Andrew; Schweighofter, Karl; Wilson, Michael A.

    2006-01-01

    The origin and early evolution of membrane proteins, and in particular ion channels, are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Prokarya, and Archaea). We discuss the potassium-sodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

  19. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells

    Science.gov (United States)

    Wang, Wei; Yang, Yunze; Wang, Shaopeng; Nagaraj, Vinay J.; Liu, Qiang; Wu, Jie; Tao, Nongjian

    2012-10-01

    Membrane proteins mediate a variety of cellular responses to extracellular signals. Although membrane proteins are studied intensively for their values as disease biomarkers and therapeutic targets, in situ investigation of the binding kinetics of membrane proteins with their ligands has been a challenge. Traditional approaches isolate membrane proteins and then study them ex situ, which does not reflect accurately their native structures and functions. We present a label-free plasmonic microscopy method to map the local binding kinetics of membrane proteins in their native environment. This analytical method can perform simultaneous plasmonic and fluorescence imaging, and thus make it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we determined the distribution of membrane proteins on the surface of single cells and the local binding kinetic constants of different membrane proteins. Furthermore, we studied the polarization of the membrane proteins on the cell surface during chemotaxis.

  20. Pattern Formation by Electrostatic Self-Organization of Membrane Proteins

    Science.gov (United States)

    Boedec, G.; Jaeger, M.; Homble, F.; Leonetti, M.

    2012-07-01

    The electric activity of biological cells and organs such as heart for example is at the origin of various phenomena of pattern formation. The electric membrane potential appears as the order parameter to characterize these spatiotemporal dynamics. A kind of patterns is characterized by a stationary spatial modulation of membrane potential along the cell, breaking a symmetry of the system. They are associated to transcellular currents. A mechanism proposed in literature is based on the coupling of the electric current produced by membrane proteins and their electrophoretic mobilities. Beyond its classical linear stability analysis, the numerical and theoretical analysis of this model offers a variety of spatiotemporal dynamics. Firstly, the background in the modelization of electric phenomena is recalled. Secondly, the analysis is focused on two nonlinear dynamics.

  1. Immunohistochemical study of the membrane skeletal protein, membrane protein palmitoylated 6 (MPP6), in the mouse small intestine.

    Science.gov (United States)

    Kamijo, Akio; Saitoh, Yurika; Ohno, Nobuhiko; Ohno, Shinichi; Terada, Nobuo

    2016-01-01

    The membrane protein palmitoylated (MPP) family belongs to the membrane-associated guanylate kinase (MAGUK) family. MPP1 interacts with the protein 4.1 family member, 4.1R, as a membrane skeletal protein complex in erythrocytes. We previously described the interaction of another MPP family, MPP6, with 4.1G in the mouse peripheral nervous system. In the present study, the immunolocalization of MPP6 in the mouse small intestine was examined and compared with that of E-cadherin, zonula occludens (ZO)-1, and 4.1B, which we previously investigated in intestinal epithelial cells. The immunolocalization of MPP6 was also assessed in the small intestines of 4.1B-deficient (-/-) mice. In the small intestine, Western blotting revealed that the molecular weight of MPP6 was approximately 55-kDa, and MPP6 was immunostained under the cell membranes in the basolateral portions of almost all epithelial cells from the crypts to the villi. The immunostaining pattern of MPP6 in epithelial cells was similar to that of E-cadherin, but differed from that of ZO-1. In intestinal epithelial cells, the immunostained area of MPP6 was slightly different from that of 4.1B, which was restricted to the intestinal villi. The immunolocalization of MPP6 in small intestinal epithelial cells was similar between 4.1B(-/-) mice and 4.1B(+/+) mice. In the immunoprecipitation study, another MAGUK family protein, calcium/calmodulin-dependent serine protein kinase (CASK), was shown to molecularly interact with MPP6. Thus, we herein showed the immunolocalization and interaction proteins of MPP6 in the mouse small intestine, and also that 4.1B in epithelial cells was not essential for the sorting of MPP6.

  2. Slow alpha helix formation during folding of a membrane protein.

    Science.gov (United States)

    Riley, M L; Wallace, B A; Flitsch, S L; Booth, P J

    1997-01-01

    Very little is known about the folding of proteins within biological membranes. A "two-stage" model has been proposed on thermodynamic grounds for the folding of alpha helical, integral membrane proteins, the first stage of which involves formation of transmembrane alpha helices that are proposed to behave as autonomous folding domains. Here, we investigate alpha helix formation in bacteriorhodopsin and present a time-resolved circular dichroism study of the slow in vitro folding of this protein. We show that, although some of the protein's alpha helices form early, a significant part of the protein's secondary structure appears to form late in the folding process. Over 30 amino acids, equivalent to at least one of bacteriorhodopsin's seven transmembrane segments, slowly fold from disordered structures to alpha helices with an apparent rate constant of about 0.012 s-1 at pH 6 or 0.0077 s-1 at pH 8. This is a rate-limiting step in protein folding, which is dependent on the pH and the composition of the lipid bilayer. PMID:8993333

  3. Disrupted yeast mitochondria can import precursor proteins directly through their inner membrane

    OpenAIRE

    1989-01-01

    Import of precursor proteins into the yeast mitochondrial matrix can occur directly across the inner membrane. First, disruption of the outer membrane restores protein import to mitochondria whose normal import sites have been blocked by an antibody against the outer membrane or by a chimeric, incompletely translocated precursor protein. Second, a potential- and ATP-dependent import of authentic or artificial precursor proteins is observed with purified inner membrane vesicles virtually free ...

  4. Influence of nonequilibrium lipid transport, membrane compartmentalization, and membrane proteins on the lateral organization of the plasma membrane

    Science.gov (United States)

    Fan, Jun; Sammalkorpi, Maria; Haataja, Mikko

    2010-01-01

    Compositional lipid domains (lipid rafts) in plasma membranes are believed to be important components of many cellular processes. The mechanisms by which cells regulate the sizes, lifetimes, and spatial localization of these domains are rather poorly understood at the moment. We propose a robust mechanism for the formation of finite-sized lipid raft domains in plasma membranes, the competition between phase separation in an immiscible lipid system and active cellular lipid transport processes naturally leads to the formation of such domains. Simulations of a continuum model reveal that the raft size distribution is broad and the average raft size is strongly dependent on the rates of cellular and interlayer lipid transport processes. We demonstrate that spatiotemporal variations in the recycling may enable the cell to localize larger raft aggregates at specific parts along the membrane. Moreover, we show that membrane compartmentalization may further facilitate spatial localization of the raft domains. Finally, we demonstrate that local interactions with immobile membrane proteins can spatially localize the rafts and lead to further clustering.

  5. Membrane Protein Crystallisation: Current Trends and Future Perspectives.

    Science.gov (United States)

    Parker, Joanne L; Newstead, Simon

    2016-01-01

    Alpha helical membrane proteins are the targets for many pharmaceutical drugs and play important roles in physiology and disease processes. In recent years, substantial progress has been made in determining their atomic structure using X-ray crystallography. However, a major bottleneck still remains; the identification of conditions that give crystals that are suitable for structure determination. Over the past 10 years we have been analysing the crystallisation conditions reported for alpha helical membrane proteins with the aim to facilitate a rational approach to the design and implementation of successful crystallisation screens. The result has been the development of MemGold, MemGold2 and the additive screen MemAdvantage. The associated analysis, summarised and updated in this chapter, has revealed a number of surprisingly successfully strategies for crystallisation and detergent selection. PMID:27553235

  6. Multi-protein assemblies underlie the mesoscale organization of the plasma membrane

    Science.gov (United States)

    Saka, Sinem K.; Honigmann, Alf; Eggeling, Christian; Hell, Stefan W.; Lang, Thorsten; Rizzoli, Silvio O.

    2014-07-01

    Most proteins have uneven distributions in the plasma membrane. Broadly speaking, this may be caused by mechanisms specific to each protein, or may be a consequence of a general pattern that affects the distribution of all membrane proteins. The latter hypothesis has been difficult to test in the past. Here, we introduce several approaches based on click chemistry, through which we study the distribution of membrane proteins in living cells, as well as in membrane sheets. We found that the plasma membrane proteins form multi-protein assemblies that are long lived (minutes), and in which protein diffusion is restricted. The formation of the assemblies is dependent on cholesterol. They are separated and anchored by the actin cytoskeleton. Specific proteins are preferentially located in different regions of the assemblies, from their cores to their edges. We conclude that the assemblies constitute a basic mesoscale feature of the membrane, which affects the patterning of most membrane proteins, and possibly also their activity.

  7. Simulation and analysis of FRET in the study of membrane proteins

    NARCIS (Netherlands)

    Nazarov, P.V.

    2006-01-01

    Membrane proteins play an important role in almost all cell activities. However, the characterization of the structure of membrane proteins in lipid bilayers is still at the frontier of structural biology. While 30-40% of all proteins are situated at or in membranes, yet less than 1% of the known pr

  8. Nonlinear Optical Characterization of Membrane Protein Microcrystals and Nanocrystals.

    Science.gov (United States)

    Newman, Justin A; Simpson, Garth J

    2016-01-01

    Nonlinear optical methods such as second harmonic generation (SHG) and two-photon excited UV fluorescence (TPE-UVF) imaging are promising approaches to address bottlenecks in the membrane protein structure determination pipeline. The general principles of SHG and TPE-UVF are discussed here along with instrument design considerations. Comparisons to conventional methods in high throughput crystallization condition screening and crystal quality assessment prior to X-ray diffraction are also discussed. PMID:27553237

  9. Gangliosides in cell recognition and membrane protein regulation

    OpenAIRE

    Lopez, Pablo H. H.; Schnaar, Ronald L.

    2009-01-01

    Gangliosides, sialic acid-bearing glycosphingolipids, are expressed on all vertebrate cells, and are the major glycans on nerve cells. They are anchored to the plasma membrane through their ceramide lipids with their varied glycans extending into the extracellular space. Through sugar-specific interactions with glycan binding proteins on apposing cells, gangliosides function as receptors in cell-cell recognition, regulating natural killer cell cytotoxicity via Siglec-7 binding, myelin-axon in...

  10. Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

    Science.gov (United States)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

    2015-01-01

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death. PMID:26561036

  11. Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

    Science.gov (United States)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

    2015-11-12

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

  12. Characterization of membrane protein interactions by isothermal titration calorimetry.

    Science.gov (United States)

    Situ, Alan J; Schmidt, Thomas; Mazumder, Parichita; Ulmer, Tobias S

    2014-10-23

    Understanding the structure, folding, and interaction of membrane proteins requires experimental tools to quantify the association of transmembrane (TM) helices. Here, we introduce isothermal titration calorimetry (ITC) to measure integrin αIIbβ3 TM complex affinity, to study the consequences of helix-helix preorientation in lipid bilayers, and to examine protein-induced lipid reorganization. Phospholipid bicelles served as membrane mimics. The association of αIIbβ3 proceeded with a free energy change of -4.61±0.04kcal/mol at bicelle conditions where the sampling of random helix-helix orientations leads to complex formation. At bicelle conditions that approach a true bilayer structure in effect, an entropy saving of >1kcal/mol was obtained from helix-helix preorientation. The magnitudes of enthalpy and entropy changes increased distinctly with bicelle dimensions, indicating long-range changes in bicelle lipid properties upon αIIbβ3 TM association. NMR spectroscopy confirmed ITC affinity measurements and revealed αIIbβ3 association and dissociation rates of 4500±100s(-1) and 2.1±0.1s(-1), respectively. Thus, ITC is able to provide comprehensive insight into the interaction of membrane proteins.

  13. Characterization of Membrane Protein Interactions in Plasma Membrane Derived Vesicles with Quantitative Imaging FRET

    Science.gov (United States)

    Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

    2016-01-01

    CONSPECTUS Here we describe an experimental tool, termed Quantitative Imaging Förster Resonance Energy Transfer (QI-FRET), which enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles which bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), an RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor

  14. Biomimetic Membranes for Multi-Redox Center Proteins

    Directory of Open Access Journals (Sweden)

    Renate L. C. Naumann

    2016-03-01

    Full Text Available His-tag technology was applied for biosensing purposes involving multi-redox center proteins (MRPs. An overview is presented on various surfaces ranging from flat to spherical and modified with linker molecules with nitrile-tri-acetic acid (NTA terminal groups to bind his-tagged proteins in a strict orientation. The bound proteins are submitted to in situ dialysis in the presence of lipid micelles to form a so-called protein-tethered bilayer lipid membrane (ptBLM. MRPs, such as the cytochrome c oxidase (CcO from R. sphaeroides and P. denitrificans, as well as photosynthetic reactions centers (RCs from R. sphaeroides, were thus investigated. Electrochemical and surface-sensitive optical techniques, such as surface plasmon resonance, surface plasmon-enhanced fluorescence, surface-enhanced infrared absorption spectroscopy (SEIRAS and surface-enhanced resonance Raman spectroscopy (SERRS, were employed in the case of the ptBLM structure on flat surfaces. Spherical particles ranging from µm size agarose gel beads to nm size nanoparticles modified in a similar fashion were called proteo-lipobeads (PLBs. The particles were investigated by laser-scanning confocal fluorescence microscopy (LSM and UV/Vis spectroscopy. Electron and proton transfer through the proteins were demonstrated to take place, which was strongly affected by the membrane potential. MRPs can thus be used for biosensing purposes under quasi-physiological conditions.

  15. Protein-lipid interactions in bilayer membranes: a lattice model.

    Science.gov (United States)

    Pink, D A; Chapman, D

    1979-04-01

    A lattice model has been developed to study the effects of intrinsic membrane proteins upon the thermodynamic properties of a lipid bilayer membrane. We assume that only nearest-neighbor van der Waals and steric interactions are important and that the polar group interactions can be represented by effective pressure-area terms. Phase diagrams, the temperature T(0), which locates the gel-fluid melting, the transition enthalpy, and correlations were calculated by mean field and cluster approximations. Average lipid chain areas and chain areas when the lipid is in a given protein environment were obtained. Proteins that have a "smooth" homogeneous surface ("cholesterol-like") and those that have inhomogeneous surfaces or that bind lipids specifically were considered. We find that T(0) can vary depending upon the interactions and that another peak can appear upon the shoulder of the main peak which reflects the melting of a eutectic mixture. The transition enthalpy decreases generally, as was found before, but when a second peak appears departures from this behavior reflect aspects of the eutectic mixture. We find that proteins have significant nonzero probabilities for being adjacent to one another so that no unbroken "annulus" of lipid necessarily exists around a protein. If T(0) does not increase much, or decreases, with increasing c, then lipids adjacent to a protein cannot all be all-trans on the time scale (10(-7) sec) of our system. Around a protein the lipid correlation depth is about one lipid layer, and this increases with c. Possible consequences of ignoring changes in polar group interactions due to clustering of proteins are discussed.

  16. Isolation of a unique membrane protein from Naegleria fowleri.

    Science.gov (United States)

    Réveiller, F L; Suh, S J; Sullivan, K; Cabanes, P A; Marciano-Cabral, F

    2001-01-01

    Naegleria fowleri, an amoeboflagellate, is the causative agent of Primary Amoebic Meningoencephalitis, a fulminating disease of the central nervous system. In order to elucidate the mechanisms of pathogenicity of this amoeba, a cDNA expression library was prepared from N. fowleri RNA. A specific protein was found to be expressed from a cDNA clone designated Mp2CL5. Northern blot analysis showed that the Mp2CL5 mRNA was expressed in pathogenic N. fowleri but was not expressed in non-pathogenic Naegleria species nor in Acanthamoeba. Western blot analysis using anti-N. fowleri antiserum demonstrated that IPTG-induced Escherichia coli Mp2CL5 expressed a 23-kDa recombinant protein. The Mp2CL5 recombinant protein was histidine-tagged and purified to homogeneity from E. coli. A polyclonal rabbit antiserum was prepared against the purified Mp2CL5 recombinant protein. This antibody was used to further characterize the Mp2CL5 native protein expressed by N. fowleri. Western blot analysis in conjunction with immunofluorescence microscopy demonstrated the presence of a native protein of 17 kDa on the plasma membrane of N. fowleri trophozoites. The native N. fowleri protein was expressed in the logarithmic phase of trophozoite growth and the production of this protein increased through the stationary phase of growth. Studies are in progress to examine further its role as a virulence factor.

  17. Accessible Mannitol-Based Amphiphiles (MNAs) for Membrane Protein Solubilisation and Stabilisation.

    Science.gov (United States)

    Hussain, Hazrat; Du, Yang; Scull, Nicola J; Mortensen, Jonas S; Tarrasch, Jeffrey; Bae, Hyoung Eun; Loland, Claus J; Byrne, Bernadette; Kobilka, Brian K; Chae, Pil Seok

    2016-05-17

    Integral membrane proteins are amphipathic molecules crucial for all cellular life. The structural study of these macromolecules starts with protein extraction from the native membranes, followed by purification and crystallisation. Detergents are essential tools for these processes, but detergent-solubilised membrane proteins often denature and aggregate, resulting in loss of both structure and function. In this study, a novel class of agents, designated mannitol-based amphiphiles (MNAs), were prepared and characterised for their ability to solubilise and stabilise membrane proteins. Some of MNAs conferred enhanced stability to four membrane proteins including a G protein-coupled receptor (GPCR), the β2 adrenergic receptor (β2 AR), compared to both n-dodecyl-d-maltoside (DDM) and the other MNAs. These agents were also better than DDM for electron microscopy analysis of the β2 AR. The ease of preparation together with the enhanced membrane protein stabilisation efficacy demonstrates the value of these agents for future membrane protein research. PMID:27072057

  18. The Escherichia coli membrane protein insertase YidC assists in the biogenesis of Penicillin Binding Proteins

    NARCIS (Netherlands)

    de Sousa Borges, Anabela; de Keyzer, Jeanine; Driessen, Arnold J M; Scheffers, Dirk-Jan

    2015-01-01

    Membrane proteins need to be properly inserted and folded in the membrane in order to perform a range of activities that are essential for the survival of bacteria. The Sec translocon and the YidC insertase are responsible for the insertion of the majority of proteins into the cytoplasmic membrane.

  19. Identification of frog photoreceptor plasma and disk membrane proteins by radioiodination

    Energy Technology Data Exchange (ETDEWEB)

    Witt, P.L.; Bownds, M.D.

    1987-03-24

    Several functions have been identified for the plasma membrane of the rod outer segment, including control of light-dependent changes in sodium conductance and a sodium-calcium exchange mechanism. However, little is known about its constituent proteins. Intact rod outer segments substantially free of contaminants were prepared in the dark and purified on a density gradient of Percoll. Surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination, and intact rod outer segments were reisolated. Membrane proteins were identified by polyacrylamide gel electrophoresis and autoradiography. The surface proteins labeled included rhodopsin, the major membrane protein, and 12 other proteins. To compare the protein composition of plasma membrane with that of the internal disk membrane, purified rod outer segments were lysed by hypotonic disruption or freeze-thawing, and plasma plus disk membranes were radioiodinated. In these membrane preparations, rhodopsin was the major iodinated constituent, with 12 other proteins also labeled. Autoradiographic evidence indicated some differences in protein composition between disk and plasma membranes. A quantitative comparison of the two samples showed that labeling of two proteins, 24 kilodaltons (kDa) and 13 kDa, was enriched in the plasma membrane, while labeling of a 220-kDa protein was enriched in the disk membrane. These plasma membrane proteins may be associated with important functions such as the light-sensitive conductance and the sodium-calcium exchanger.

  20. A Shotgun Proteomic Method for the Identification of Membrane-Embedded Proteins and Peptides

    OpenAIRE

    Blackler, Adele R.; Speers, Anna E.; Ladinsky, Mark S.; Wu, Christine C

    2008-01-01

    Integral membrane proteins perform crucial cellular functions and are the targets for the majority of pharmaceutical agents. However, the hydrophobic nature of their membrane-embedded domains makes them difficult to work with. Here, we describe a shotgun proteomic method for the high-throughput analysis of the membrane-embedded transmembrane domains of integral membrane proteins which extends the depth of coverage of the membrane proteome.

  1. Preparation of 2D crystals of membrane proteins for high-resolution electron crystallography data collection.

    Science.gov (United States)

    Abeyrathne, Priyanka D; Chami, Mohamed; Pantelic, Radosav S; Goldie, Kenneth N; Stahlberg, Henning

    2010-01-01

    Electron crystallography is a powerful technique for the structure determination of membrane proteins as well as soluble proteins. Sample preparation for 2D membrane protein crystals is a crucial step, as proteins have to be prepared for electron microscopy at close to native conditions. In this review, we discuss the factors of sample preparation that are key to elucidating the atomic structure of membrane proteins using electron crystallography.

  2. Studies of the molecular effects of a solid support upon lipid membranes and membrane bound proteins

    Science.gov (United States)

    Hartshorn, Christopher M.

    Often, membrane/protein systems are studied and/or utilized on solid supports. The underlying substrate in solid supported lipid bilayer assemblies causes large perturbations to the membrane, but the nature of these effects are not well understood. To gain an understanding, these effects were studied on two fronts: the effect upon the membrane by itself, and then the effects upon a membrane/protein system. First, all-atom molecular dynamics (MD) simulations of DLPC, DMPC, POPC, and DEPC on a hydroxylated nanocrystalline alpha-quartz (011) slab revealed a pronounced thinning effect in the lipid bilayers. It was shown that this thinning effect proceeded by one of two mechanisms: the first through a curling of the terminal methyl groups at the interface of the opposing leaflets, and the second through increased interdigitation of the alkyl chains. Also, with the introduction of the solid support, marked asymmetries in a number of structural properties were reported. These asymmetries included (a) the surface area per lipid, (b) the electron densities of the polar head groups, (c) the radial distributions of the choline groups, and (d) the average orientation of water surrounding the membranes. Next, the free energy perturbation method was used to begin calculating the change in free energy (DeltaGbinding) from a Gramicidin monomer to its dimeric state, which were simulated via MD of supported DLPC, DMPC, and DEPC bilayers. The most notable effect was an asymmetry of the calculated free energies relative to the bilayer side closest to the solid support. In all three systems, there was a large difference in free energy between the Gramicidin monomers that were close to the support and the monomers further from the support.

  3. Intramembrane particles and the organization of lymphocyte membrane proteins.

    Science.gov (United States)

    Kuby, J M; Wofsy, L

    1981-03-01

    An experimental system was developed in which the majority of all lymphocyte cell-surface proteins, regardless of antigenic specificity, could be cross-linked and redistributed in the membrane to determine whether this would induce a corresponding redistribution of intramembrane particles (IMP). Mouse spleen cells were treated with P-diazoniumphenyl- beta-D-lactoside (lac) to modify all exposed cell-surface proteins. Extensive azo- coupling was achieved without significantly reducing cell viability or compromising cellular function in mitogen- or antigen-stimulated cultures. When the lac-modified cell- surface proteins were capped with a sandwich of rabbit antilactoside antibody and fluorescein-goat anti-rabbit Ig, freeze-fracture preparations obtained from these cells revealed no obvious redistribution of IMP on the majority of fracture faces. However, detailed analysis showed a statistically significant 35 percent decrease (P less than 0.01) in average IMP density in the E face of the lac-capped spleen cells compared with control cells, whereas a few E-face micrographs showed intense IMP aggregation. In contrast, there was no significant alteration of P-face IMP densities or distribution. Apparently, the majority of E-face IMP and virtually all P-face IMP densities or distribution. Apparently, the majority of E-face IMP and virtually all P-face IMP do not present accessible antigenic sites on the lymphocyte surface and do not associate in a stable manner with surface protein antigens. This finding suggests that IMP, as observed in freeze-fracture analysis, may not comprise a representative reflection of lymphocyte transmembrane protein molecules and complexes because other evidence establishes: (a) that at least some common lymphocyte surface antigens are indeed exposed portions of transmembrane proteins and (b) that the aggregation of molecules of any surface antigen results in altered organization of contractile proteins at the cytoplasmic face of the membrane.

  4. A membrane protein / signaling protein interaction network for Arabidopsis version AMPv2

    Directory of Open Access Journals (Sweden)

    Sylvie Lalonde

    2010-09-01

    Full Text Available Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway compatible vector. The mating-based split-ubiquitin system was used to screen for potential protein-protein interactions (pPPIs among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases, 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 387 pPPIs between 179 proteins, yielding a scale-free network (r2=0.863. Eighty of 142 transmembrane receptor-like kinases (RLK tested positive, identifying three homomers, 63 heteromers and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

  5. Mapping membrane protein interactions in cell signaling systems.

    Energy Technology Data Exchange (ETDEWEB)

    Light, Yooli Kim; Hadi, Masood Z.; Lane, Pamela; Jacobsen, Richard B.; Hong, Joohee; Ayson, Marites J.; Wood, Nichole L.; Schoeniger, Joseph S.; Young, Malin M.

    2003-12-01

    We proposed to apply a chemical cross-linking, mass spectrometry and modeling method called MS3D to the structure determination of the rhodopsin-transducin membrane protein complex (RTC). Herein we describe experimental progress made to adapt the MS3D approach for characterizing membrane protein systems, and computational progress in experimental design, data analysis and protein structure modeling. Over the past three years, we have developed tailored experimental methods for all steps in the MS3D method for rhodopsin, including protein purification, a functional assay, cross-linking, proteolysis and mass spectrometry. In support of the experimental effort. we have out a data analysis pipeline in place that automatically selects the monoisotopic peaks in a mass spectrometric spectrum, assigns them and stores the results in a database. Theoretical calculations using 24 experimentally-derived distance constraints have resulted in a backbone-level model of the activated form of rhodopsin, which is a critical first step towards building a model of the RTC. Cross-linked rhodopsin-transducin complexes have been isolated via gel electrophoresis and further mass spectrometric characterization of the cross-links is underway.

  6. Comparative transcriptional analysis of Bacillus subtilis cells overproducing either secreted proteins, lipoproteins or membrane proteins

    Directory of Open Access Journals (Sweden)

    Marciniak Bogumiła C

    2012-05-01

    Full Text Available Abstract Background Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1 β-lactamase of Escherichia coli, membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus and lipoproteins (MntA and YcdH of B. subtilis. Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes. Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 β-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue specifically under membrane proteins overproduction. Conclusions The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host.

  7. Immunoprecipitation of membrane proteins of cultured human sarcoma cells.

    Science.gov (United States)

    Grófová, M; Forchhammer, J; Lizonová, A; Popovic, M

    1981-01-01

    Human sarcoma associated antigens (HSAA) have previously been identified by indirect immune fluorescence in human sarcoma cells in culture using sera from patients bearing different types of sarcoma. To further characterize these HSAA, surface proteins of cultured cells were labeled with 125Iodine, [3H]-glucosamine and [35S]-methionine and solubilized. After immunoprecipitation labeled proteins were detected in immune complexes by SDS polyacrylamide gel electrophoresis and autoradiography, which allowed comparison with antigens described by other groups. A surface protein (Mr 96 000) was precipitated with sera from sarcoma bearing patients, and two glycoproteins (Mr 115 000 and 85 000) were preferentially precipitated with antisera from rabbits immunized with membranes from two human sarcoma cell lines. At least two of these proteins were found in each of five human sarcoma cell lines studied (U-4SS, U-3930S, U-20S, B-5GT and B-6FS). None of the proteins were precipitated with three human control sera, and only occasionally a faint band was observed in immunoprecipitates from control cells (B-25F, B-41B, B-42FC, U-2S, and U-393S with the immune sera. These proteins are probably some of the antigens responsible for the immune fluorescence observed in determination of HSAA. However, purification of the proteins and competition experiments are needed before this can be finally established.

  8. Deducing the symmetry of helical assemblies: Applications to membrane proteins.

    Science.gov (United States)

    Coudray, Nicolas; Lasala, Ralph; Zhang, Zhening; Clark, Kathy M; Dumont, Mark E; Stokes, David L

    2016-08-01

    Helical reconstruction represents a convenient and powerful approach for structure determination of macromolecules that assemble into helical arrays. In the case of membrane proteins, formation of tubular crystals with helical symmetry represents an attractive alternative, especially when their small size precludes the use of single-particle analysis. An essential first step for helical reconstruction is to characterize the helical symmetry. This process is often daunting, due to the complexity of helical diffraction and to the low signal-to-noise ratio in images of individual assemblies. Furthermore, the large diameters of the tubular crystals produced by membrane proteins exacerbates the innate ambiguities that, if not resolved, will produce incorrect structures. In this report, we describe a set of tools that can be used to eliminate ambiguities and to validate the choice of symmetry. The first approach increases the signal-to-noise ratio along layer lines by incoherently summing data from multiple helical assemblies, thus producing several candidate indexing schemes. The second approach compares the layer lines from images with those from synthetic models built with the various candidate schemes. The third approach uses unit cell dimensions measured from collapsed tubes to distinguish between these candidate schemes. These approaches are illustrated with tubular crystals from a boron transporter from yeast, Bor1p, and a β-barrel channel from the outer membrane of E. coli, OmpF. PMID:27255388

  9. Atomic-level description of protein-lipid interactions using an accelerated membrane model.

    Science.gov (United States)

    Baylon, Javier L; Vermaas, Josh V; Muller, Melanie P; Arcario, Mark J; Pogorelov, Taras V; Tajkhorshid, Emad

    2016-07-01

    Peripheral membrane proteins are structurally diverse proteins that are involved in fundamental cellular processes. Their activity of these proteins is frequently modulated through their interaction with cellular membranes, and as a result techniques to study the interfacial interaction between peripheral proteins and the membrane are in high demand. Due to the fluid nature of the membrane and the reversibility of protein-membrane interactions, the experimental study of these systems remains a challenging task. Molecular dynamics simulations offer a suitable approach to study protein-lipid interactions; however, the slow dynamics of the lipids often prevents sufficient sampling of specific membrane-protein interactions in atomistic simulations. To increase lipid dynamics while preserving the atomistic detail of protein-lipid interactions, in the highly mobile membrane-mimetic (HMMM) model the membrane core is replaced by an organic solvent, while short-tailed lipids provide a nearly complete representation of natural lipids at the organic solvent/water interface. Here, we present a brief introduction and a summary of recent applications of the HMMM to study different membrane proteins, complementing the experimental characterization of the presented systems, and we offer a perspective of future applications of the HMMM to study other classes of membrane proteins. This article is part of a Special Issue entitled: Membrane proteins edited by J.C. Gumbart and Sergei Noskov. PMID:26940626

  10. Hematopoietic protein-1 regulates the actin membrane skeleton and membrane stability in murine erythrocytes.

    Directory of Open Access Journals (Sweden)

    Maia M Chan

    Full Text Available Hematopoietic protein-1 (Hem-1 is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein complex, which regulates filamentous actin (F-actin polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1⁻/⁻ erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1⁻/⁻ erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A, which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes.

  11. Expression and Purification of SARS Coronavirus Membrane Protein

    Institute of Scientific and Technical Information of China (English)

    戴五星; 雷明军; 吴少庭; 陈智浩; 梁靓; 潘晖榕; 秦莉; 高士同; 袁仕善; 张仁利

    2004-01-01

    To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The re combinants were transformed into Escherichia coli (E. Coli) BL21 (DE3) and induced by Isopropylβ-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0. 992 1 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.

  12. Hydrodynamic collective effects of active proteins in biological membranes

    Science.gov (United States)

    Koyano, Yuki; Kitahata, Hiroyuki; Mikhailov, Alexander S.

    2016-08-01

    Lipid bilayers forming biological membranes are known to behave as viscous two-dimensional fluids on submicrometer scales; usually they contain a large number of active protein inclusions. Recently, it was shown [A. S. Mikhailov and R. Kapral, Proc. Natl. Acad. Sci. USA 112, E3639 (2015), 10.1073/pnas.1506825112] that such active proteins should induce nonthermal fluctuating lipid flows leading to diffusion enhancement and chemotaxislike drift for passive inclusions in biomembranes. Here, a detailed analytical and numerical investigation of such effects is performed. The attention is focused on the situations when proteins are concentrated within lipid rafts. We demonstrate that passive particles tend to become attracted by active rafts and are accumulated inside them.

  13. Production of Membrane Proteins for NMR Studies Using the Condensed Single Protein Production (cSPP) System

    OpenAIRE

    Mao, Lili; Tang, Yuefeng; Vaiphei, S. Thangminlal; Shimazu, Tsutomu; Kim, Sung-Gun; Mani, Rajeswari; Fakhoury, Elias; White, Eileen; Montelione, Gaetano T.; Inouye, Masayori

    2009-01-01

    In the Single Protein Production (SPP) method, all E. coli cellular mRNAs are eliminated by the induction of MazF, an ACA-specific mRNA interferase. When an mRNA for a membrane protein, engineered to have no ACA sequences without altering its amino acid sequence, is induced in the MazF-induced cells, E. coli is converted into a bioreactor producing only the targeted membrane protein. Here we demonstrate that three prokaryotic inner membrane proteins, two prokaryotic outer membrane proteins, a...

  14. Effect of Adsorbed Protein on the Hydraulic Permeability, Membrane and Streaming Potential Values Measured across a Microporous Membrane

    DEFF Research Database (Denmark)

    Benavente, Juana; Jonsson, Gunnar Eigil

    1998-01-01

    The effect of the adsorption of a protein, bovine serum albumin (BSA), on the membrane potential, flux reduction and streaming potential measured across a microporous polysulphone membrane with different NaCl solutions and pH values is studied. From electrokinetic phenomena, information about...... as a "composite" or two-layer membrane, and a comparison of the results obtained with both microporous polysulphone and "composite" (microporous + BSA layer) membranes could permit us to determine some parameters related to the protein sublayer. (C) 1998 Elsevier Science B.V....

  15. Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins*

    Science.gov (United States)

    Galiani, Silvia; Waithe, Dominic; Reglinski, Katharina; Cruz-Zaragoza, Luis Daniel; Garcia, Esther; Clausen, Mathias P.; Schliebs, Wolfgang; Erdmann, Ralf; Eggeling, Christian

    2016-01-01

    Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future. PMID:27311714

  16. Comparison of membrane electroporation and protein denature in response to pulsed electric field with different durations.

    Science.gov (United States)

    Huang, Feiran; Fang, Zhihui; Mast, Jason; Chen, Wei

    2013-05-01

    In this paper, we compared the minimum potential differences in the electroporation of membrane lipid bilayers and the denaturation of membrane proteins in response to an intensive pulsed electric field with various pulse durations. Single skeletal muscle fibers were exposed to a pulsed external electric field. The field-induced changes in the membrane integrity (leakage current) and the Na channel currents were monitored to identify the minimum electric field needed to damage the membrane lipid bilayer and the membrane proteins, respectively. We found that in response to a relatively long pulsed electric shock (longer than the membrane intrinsic time constant), a lower membrane potential was needed to electroporate the cell membrane than for denaturing the membrane proteins, while for a short pulse a higher membrane potential was needed. In other words, phospholipid bilayers are more sensitive to the electric field than the membrane proteins for a long pulsed shock, while for a short pulse the proteins become more vulnerable. We can predict that for a short or ultrashort pulsed electric shock, the minimum membrane potential required to start to denature the protein functions in the cell plasma membrane is lower than that which starts to reduce the membrane integrity.

  17. Polyacrylonitrile-based zwitterionic ultrafiltration membrane with improved anti-protein-fouling capacity

    Science.gov (United States)

    Meng, Hong; Cheng, Qiang; Li, Chunxi

    2014-06-01

    The adhesion of proteins to the surface and pores of ultrafiltration membrane is one of the most important causes of membrane fouling, consequently lead to deterioration of membrane performance. In the present study, a polyacrylonitrile (PAN)-based zwitterionic ultrafiltration membrane was developed to improve its anti-protein-fouling capacity. 3-Dimethylaminopropylamine was first grafted onto the hydrolyzed PAN membrane by activation. Subsequently, carboxylic zwitterions were produced on the membrane surface by quaternization with 3-bromopropionic acid. The zwitterionic membranes were rigorously characterized in terms of chemical composition, morphology, and surface properties indicating that the zwitterion could successfully be bonded onto the PAN membrane without having significant effects on the membrane morphology. The anti-protein-fouling properties of the membrane were tested using static protein adsorption and dynamic-filtration experiments. The experimental results show that, although the hydrophilicity of the zwitterionic membrane decreased, the flux recovery rate of the zwitterion-grafted membrane was much higher than that of the hydrolyzed PAN membrane. Therefore, the formation of zwitterionic hydration layer may effectively prevent the adsorption interaction between protein and membrane surface, which is beneficial for the improvement of the anti-protein-fouling capacity.

  18. Electrophoretic analysis, labeling and isolation of Chlamydomonas reinhardtii flagellum membrane proteins

    Directory of Open Access Journals (Sweden)

    Aleksander F. Sikorski

    2015-05-01

    Full Text Available SDS-polyacrylamide electrophoretic patterns of Chlamydomonas flagellum membrane proteins displayad 6 fractions, 3 PAS-positive among them. The surface radiolabeling of the flagellum membrane suggested an outer surface exposure of fraction '5', and internal localization of fractions '4' and '6'. Application of SDS-polyacrylamide gel electrophoresis and radiolabeled membranes allowed to isolate individual membrane polypeptides.

  19. Membrane protein targeting to the outskirts of the endoplasmic reticulum : A characterization of sorting signals

    NARCIS (Netherlands)

    Kralt, Annemarie

    2015-01-01

    The majority of membrane proteins synthesized in the cell is inserted into the membrane of the endoplasmic reticulum (ER). The ER forms a network that extends from the nuclear envelope (NE), a double membrane surrounding the nucleus, to the cortical ER that underlies the plasma membrane (PM). Locali

  20. Interaction of two different types of membrane proteins with model membranes investigated by FTIR ATR spectroscopy

    Science.gov (United States)

    Siam, M.; Reiter, G.; Schwarzott, M.; Baurecht, D.; Fringeli, U. P.

    1998-06-01

    Polarized FTIR ATR spectroscopy was used to investigate the interaction of mitochondrial creatine kinase (Mi-CK) and intestinal alkaline phosphatase (AP) with model membrane assemblies. Mi-CK was immobilized by adsorption to the negatively charged cardiolipin (CL) leaflet of a supported CL/DPPA bilayer. H-D-exchange of the enzyme and the stability under flowthrough conditions of the protein/membrane assembly were examined. AP, however, was bound to a DPPA Langmuir-Blodgett layer (LBL), followed by the completion of a bilayer-like structure by adsorption of POPC molecules from a vesicular solution. It turned out that the POPC adsorbate exhibited decreased molecular order compared to the POPC molecules on a supported POPC/DPPA bilayer. Enzymatic activity of immobilized AP was determined with p-nitrophenyl phosphate (p-NPP) as substrate and remained unchanged for at least 2 days.

  1. 高脂高糖家兔脑梗死模型基底膜损伤机制的探讨%Mechanism of basement membrane injury in cerebral infarction model in rabbit with hyperglycemia and hyperlipidemia

    Institute of Scientific and Technical Information of China (English)

    徐娉; 庄强

    2008-01-01

    Objective To discuss the mechanism of microvascular endothelial basement membrane injury following cerebral ischemla. Methods Twenty-four New Zealand rabbits were randomly divided into two groups (n=12): normal control group and model group. The latter were fed high-fat, high-sucrose diet, but the former was fed with normal diet. They all were made into the models of cerebral infarction. The change of Laminin (LN) in blood and the expression of LN in brain tissue were observed simultaneously. All groups received pathological examination. Results The levels of blood lipids and blood glucose of rabbit and the content of LN were significantly increased after fed with high fat and high sucrose about 1-2 times in 10 d. The plasma concentration of LN was decreased significantly after the operation of cerebral infarction (P<0.05). Pathological observation revealed that LN was mainly located in cytoplasm of microvascular matrix, and its positive reaction was yellow to brown. Conclusions Injury of the microvascular endothelial basement membrane exists in the early phase of rabbit cerebral ischemia, leading to decrease of LN in extracellular matrix. The early intervention of blood lipids and blood glucose, accompanied by the brain protection with agents and the application of inhibitors of MMPs, will be able to effectively improve the cerebral infarction.%目的 探讨脑缺血后微血管内皮细胞基底膜的损伤机制.方法 24只新西兰家兔采用随机数字表法分为非高脂高糖脑梗死模型对照组(简称空白对照组),高脂高糖脑梗死模型对照组(简称模型对照组),每组12只.空白对照组给予普通饲料,模型对照组给予高脂高糖饲料,均制作成脑梗死模型.监测造模前后血浆中层粘连蛋白(LN)的表达,并观察腩组织中LN表达情况.结果模型对照组高脂高糖模型制作后,家兔血脂血糖增高,与造模前比较差异有统计学意义(P<0.05).脑梗死模型制作后两组血浆中LN表达

  2. Heat Denaturation of Protein Structures and Chlorophyll States in PSII Membranes

    Institute of Scientific and Technical Information of China (English)

    李冬海; 阮翔; 许强; 王可玢; 公衍道; 匡廷云; 赵南明

    2002-01-01

    Heat denaturation is an important technique in the study of the structure and function of photosynthetic proteins. Heat denaturation of photosystem II (PSII) membrane was studied using circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC) and oxygen electrode. Complete loss of oxygen-evolving activity of the PSII membrane was observed at temperatures below 45℃. The decrease of excitonic interaction between chlorophyll molecules occurred more rapidly than the change of the protein secondary structure of the PSII membrane at temperatures above 45℃. The results indicate that the protein secondary structure of the membrane proteins in PSII membranes is more stable than the excitonic interaction between chlorophyll molecules during heat denaturation.

  3. Codon Optimizing for Increased Membrane Protein Production: A Minimalist Approach.

    Science.gov (United States)

    Mirzadeh, Kiavash; Toddo, Stephen; Nørholm, Morten H H; Daley, Daniel O

    2016-01-01

    Reengineering a gene with synonymous codons is a popular approach for increasing production levels of recombinant proteins. Here we present a minimalist alternative to this method, which samples synonymous codons only at the second and third positions rather than the entire coding sequence. As demonstrated with two membrane-embedded transporters in Escherichia coli, the method was more effective than optimizing the entire coding sequence. The method we present is PCR based and requires three simple steps: (1) the design of two PCR primers, one of which is degenerate; (2) the amplification of a mini-library by PCR; and (3) screening for high-expressing clones. PMID:27485329

  4. Modification-specific proteomics of plasma membrane proteins

    DEFF Research Database (Denmark)

    Elortza, Felix; Mohammed, Shabaz; Bunkenborg, Jakob;

    2006-01-01

    that phospholipase D (PLD) treatment of human and plant plasma membrane fractions leads to the release of GPI-anchored proteins that were identified and characterized by capillary liquid chromatography and tandem mass spectrometry. In contrast to phospholipase C, the PLD enzyme is not affected by structural......-recognized as they are candidate cell surface biomarker molecules with potential diagnostic and therapeutic applications in molecular medicine. GPI-APs have also attracted interest in plant biotechnology because of their role in root development and cell remodeling. Using a shave-and-conquer concept, we demonstrate...

  5. X11/Mint Genes Control Polarized Localization of Axonal Membrane Proteins in Vivo

    OpenAIRE

    Garrett G Gross; Lone, G. Mohiddin; Leung, Lok Kwan; Hartenstein, Volker; Guo, Ming

    2013-01-01

    Mislocalization of axonal proteins can result in misassembly and/or miswiring of neural circuits, causing disease. To date, only a handful of genes that control polarized localization of axonal membrane proteins have been identified. Here we report that Drosophila X11/Mint proteins are required for targeting several proteins, including human amyloid precursor protein (APP) and Drosophila APP-like protein (APPL), to axonal membranes and for their exclusion from dendrites of the mushroom body i...

  6. Beyond Membrane Protein Structure: Drug Discovery, Dynamics and Difficulties.

    Science.gov (United States)

    Biggin, Philip C; Aldeghi, Matteo; Bodkin, Michael J; Heifetz, Alexander

    2016-01-01

    Most of the previous content of this book has focused on obtaining the structures of membrane proteins. In this chapter we explore how those structures can be further used in two key ways. The first is their use in structure based drug design (SBDD) and the second is how they can be used to extend our understanding of their functional activity via the use of molecular dynamics. Both aspects now heavily rely on computations. This area is vast, and alas, too large to consider in depth in a single book chapter. Thus where appropriate we have referred the reader to recent reviews for deeper assessment of the field. We discuss progress via the use of examples from two main drug target areas; G-protein coupled receptors (GPCRs) and ion channels. We end with a discussion of some of the main challenges in the area.

  7. Deployment of membrane fusion protein domains during fusion.

    Science.gov (United States)

    Bentz, J; Mittal, A

    2000-01-01

    It is clear that both viral and intracellular membrane fusion proteins contain a minimal set of domains which must be deployed at the appropriate time during the fusion process. An account of these domains and their functions is given here for the four best-described fusion systems: influenza HA, sendai virus F1, HIV gp120/41 and the neuronal SNARE core composed of synaptobrevin (syn), syntaxin (stx) and the N- and C-termini of SNAP25 (sn25), together with the Ca(2+)binding protein synaptotagmin (syt). Membrane fusion begins with the binding of the virion or vesicle to the target membrane via receptors. The committed step in influenza HA- mediated fusion begins with an aggregate of HAs (at least eight) with some of their HA2 N-termini, a.k.a. fusion peptides, embedded into the viral bilayer (Bentz, 2000 a). The hypothesis presented in Bentz (2000 b) is that the conformational change of HA to the extended coiled coil extracts the fusion peptides from the viral bilayer. When this extraction occurs from the center of the site of restricted lipid flow, it exposes acyl chains and parts of the HA transmembrane domains to the aqueous media, i.e. a hydrophobic defect is formed. This is the 'transition state' of the committed step of fusion. It is stabilized by a 'dam' of HAs, which are inhibited from diffusing away by the rest of the HAs in the aggregate and because that would initially expose more acyl chains to water. Recruitment of lipids from the apposed target membrane can heal this hydrophobic defect, initiating lipid mixing and fusion. The HA transmembrane domains are required to be part of the hydrophobic defect, because the HA aggregate must be closely packed enough to restrict lipid flow. This hypothesis provides a simple and direct coupling between the energy released by the formation of the coiled coil to the energy needed to create and stabilize the high energy intermediates of fusion. Several of these essential domains have been described for the viral fusion

  8. INTERACTION OF ALDEHYDES DERIVED FROM LIPID PEROXIDATION AND MEMBRANE PROTEINS.

    Directory of Open Access Journals (Sweden)

    Stefania ePizzimenti

    2013-09-01

    Full Text Available A great variety of compounds are formed during lipid peroxidation of polyunsaturated fatty acids of membrane phospholipids. Among them, bioactive aldehydes, such as 4-hydroxyalkenals, malondialdehyde (MDA and acrolein, have received particular attention since they have been considered as toxic messengers that can propagate and amplify oxidative injury. In the 4-hydroxyalkenal class, 4-hydroxy-2-nonenal (HNE is the most intensively studied aldehyde, in relation not only to its toxic function, but also to its physiological role. Indeed, HNE can be found at low concentrations in human tissues and plasma and participates in the control of biological processes, such as signal transduction, cell proliferation and differentiation. Moreover, at low doses, HNE exerts an anti-cancer effect, by inhibiting cell proliferation, angiogenesis, cell adhesion and by inducing differentiation and/or apoptosis in various tumor cell lines. It is very likely that a substantial fraction of the effects observed in cellular responses, induced by HNE and related aldehydes, be mediated by their interaction with proteins, resulting in the formation of covalent adducts or in the modulation of their expression and/or activity. In this review we focus on membrane proteins affected by lipid peroxidation-derived aldehydes, under physiological and pathological conditions.

  9. Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

    Science.gov (United States)

    Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

    2014-03-01

    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed.

  10. Immunoproteomic Analysis ofBordetella bronchisepticaOuter Membrane Proteins and Identiifcation of New Immunogenic Proteins

    Institute of Scientific and Technical Information of China (English)

    JI Quan-an

    2014-01-01

    Bordetella bronchiseptica is a Gram-negative pathogen that causes acute and chronic respiratory infection in a variety of animals. To identify useful antigen candidates for diagnosis and subunit vaccine ofB. bronchiseptica, immunoproteomic analysis was adopted to analyse outer membrane proteins of it. The outer membrane proteins extracted fromB. bronchiseptica were separated by two-dimensional gel electrophoresis and analyzed by Western blotting for their reactivity with the convalescent serum against two strains. Immunogenic proteins were identiifed by matrix-assisted laser desorption/ionization time of lfight-mass spectrometry (MALDI-TOF-MS), a total of 14 proteins are common immunoreactive proteins, of which 1 was known antigen and 13 were novel immunogenic proteins forB. bronchiseptica. Putative lipoprotein gene was cloned and recombinantly expressed. The recombinant protein induced high titer antibody, but showed low protective indices against challenges with HB (B. bronchiseptica strain isolated from a infected rabbit). The mortality of mice was 80% compared to 100% of positive controls. The identiifcation of these novel antigenic proteins is an important resource for further development of a new diagnostic test and vaccine for B. bronchiseptica.

  11. Cell-free methods to produce structurally intact mammalian membrane proteins.

    Science.gov (United States)

    Shinoda, Takehiro; Shinya, Naoko; Ito, Kaori; Ishizuka-Katsura, Yoshiko; Ohsawa, Noboru; Terada, Takaho; Hirata, Kunio; Kawano, Yoshiaki; Yamamoto, Masaki; Tomita, Taisuke; Ishibashi, Yohei; Hirabayashi, Yoshio; Kimura-Someya, Tomomi; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2016-01-01

    The crystal structures of four membrane proteins, from bacteria or a unicellular alga, have been solved with samples produced by cell-free protein synthesis. In this study, for mammalian membrane protein production, we established the precipitating and soluble membrane fragment methods: membrane proteins are synthesized with the Escherichia coli cell-free system in the presence of large and small membrane fragments, respectively, and are simultaneously integrated into the lipid environments. We applied the precipitating membrane fragment method to produce various mammalian membrane proteins, including human claudins, glucosylceramide synthase, and the γ-secretase subunits. These proteins were produced at levels of about 0.1-1.0 mg per ml cell-free reaction under the initial conditions, and were obtained as precipitates by ultracentrifugation. Larger amounts of membrane proteins were produced by the soluble membrane fragment method, collected in the ultracentrifugation supernatants, and purified directly by column chromatography. For several proteins, the conditions of the membrane fragment methods were further optimized, such as by the addition of specific lipids/detergents. The functional and structural integrities of the purified proteins were confirmed by analyses of their ligand binding activities, size-exclusion chromatography profiles, and/or thermal stabilities. We successfully obtained high-quality crystals of the complex of human claudin-4 with an enterotoxin. PMID:27465719

  12. Optimization of polyvinylidene fluoride (PVDF) membrane fabrication for protein binding using statistical experimental design.

    Science.gov (United States)

    Ahmad, A L; Ideris, N; Ooi, B S; Low, S C; Ismail, A

    2016-01-01

    Statistical experimental design was employed to optimize the preparation conditions of polyvinylidenefluoride (PVDF) membranes. Three variables considered were polymer concentration, dissolving temperature, and casting thickness, whereby the response variable was membrane-protein binding. The optimum preparation for the PVDF membrane was a polymer concentration of 16.55 wt%, a dissolving temperature of 27.5°C, and a casting thickness of 450 µm. The statistical model exhibits a deviation between the predicted and actual responses of less than 5%. Further characterization of the formed PVDF membrane showed that the morphology of the membrane was in line with the membrane-protein binding performance. PMID:27088961

  13. Molecular Signatures of Membrane Protein Complexes Underlying Muscular Dystrophy*

    Science.gov (United States)

    Turk, Rolf; Hsiao, Jordy J.; Smits, Melinda M.; Ng, Brandon H.; Pospisil, Tyler C.; Jones, Kayla S.; Campbell, Kevin P.; Wright, Michael E.

    2016-01-01

    Mutations in genes encoding components of the sarcolemmal dystrophin-glycoprotein complex (DGC) are responsible for a large number of muscular dystrophies. As such, molecular dissection of the DGC is expected to both reveal pathological mechanisms, and provides a biological framework for validating new DGC components. Establishment of the molecular composition of plasma-membrane protein complexes has been hampered by a lack of suitable biochemical approaches. Here we present an analytical workflow based upon the principles of protein correlation profiling that has enabled us to model the molecular composition of the DGC in mouse skeletal muscle. We also report our analysis of protein complexes in mice harboring mutations in DGC components. Bioinformatic analyses suggested that cell-adhesion pathways were under the transcriptional control of NFκB in DGC mutant mice, which is a finding that is supported by previous studies that showed NFκB-regulated pathways underlie the pathophysiology of DGC-related muscular dystrophies. Moreover, the bioinformatic analyses suggested that inflammatory and compensatory mechanisms were activated in skeletal muscle of DGC mutant mice. Additionally, this proteomic study provides a molecular framework to refine our understanding of the DGC, identification of protein biomarkers of neuromuscular disease, and pharmacological interrogation of the DGC in adult skeletal muscle https://www.mda.org/disease/congenital-muscular-dystrophy/research. PMID:27099343

  14. Deposition of bacteriorhodopsin protein in a purple membrane form on nitrocellulose membranes for enhanced photoelectric response.

    Science.gov (United States)

    Kim, Young Jun; Neuzil, Pavel; Nam, Chang-Hoon; Engelhard, Martin

    2012-12-27

    Bacteriorhodopsin protein (bR)-based systems are one of the simplest known biological energy converters. The robust chemical, thermal and electrochemical properties of bR have made it an attractive material for photoelectric devices. This study demonstrates the photoelectric response of a dry bR layer deposited on a nitrocellulose membrane with indium tin oxide (ITO) electrodes. Light-induced electrical current as well as potential and impedance changes of dried bR film were recorded as the function of illumination. We have also tested bR in solution and found that the electrical properties are strongly dependent on light intensity changing locally proton concentration and thus pH of the solution. Experimental data support the assumption that bR protein on a positively charged nitrocellulose membrane (PNM) can be used as highly sensitive photo- and pH detector. Here the bR layer facilitates proton translocation and acts as an ultrafast optoelectric signal transducer. It is therefore useful in applications related to bioelectronics, biosensors, bio-optics devices and current carrying junction devices.

  15. Deposition of Bacteriorhodopsin Protein in a Purple Membrane Form on Nitrocellulose Membranes for Enhanced Photoelectric Response

    Directory of Open Access Journals (Sweden)

    Chang-Hoon Nam

    2012-12-01

    Full Text Available Bacteriorhodopsin protein (bR-based systems are one of the simplest known biological energy converters. The robust chemical, thermal and electrochemical properties of bR have made it an attractive material for photoelectric devices. This study demonstrates the photoelectric response of a dry bR layer deposited on a nitrocellulose membrane with indium tin oxide (ITO electrodes. Light-induced electrical current as well as potential and impedance changes of dried bR film were recorded as the function of illumination. We have also tested bR in solution and found that the electrical properties are strongly dependent on light intensity changing locally proton concentration and thus pH of the solution. Experimental data support the assumption that bR protein on a positively charged nitrocellulose membrane (PNM can be used as highly sensitive photo- and pH detector. Here the bR layer facilitates proton translocation and acts as an ultrafast optoelectric signal transducer. It is therefore useful in applications related to bioelectronics, biosensors, bio-optics devices and current carrying junction devices.

  16. Human nectin-like 1, a novel membrane protein interacting with protein 4.1 R

    Institute of Scientific and Technical Information of China (English)

    YANG Hongbo; YUAN Jiangang; PENG Xiaozhong; ZHOU Yan; HU Xiaoyan; XU Yaqin; YIN Bin; ZHU Shengtao; FAN Wenhong; FAN Ming; QIANG Boqin

    2005-01-01

    Human nectin-like 1 (NECL1) full-length cDNA was cloned by bioinformatics method when searching for candidate membrane proteins interacting with members of protein 4.1 family. The cytoplasmic and extracellular regions of NECL1 were expressed in and purified from E. coli, and the polyclonal antibody was produced. Interaction between the cytoplasmic region of NECL1 and the 30 kD membrane binding domain of protein 4.1 on red blood cell (4. 1R) was demonstrated by IAsys-biosensor system and GST pull-down experiment. Results of biotin-labeled peptide ELISA further demonstrated the key amino acids for the binding. The interaction research of NECL1's cytoplasmic domain provides basis for further study of the functions of NECL1 in nervous system.

  17. Identification of salt-tolerant Sinorhizobium sp. strain BL3 membrane proteins based on proteomics

    DEFF Research Database (Denmark)

    Tanthanuch, Waraporn; Tittabutr, Panlada; Mohammed, Shabaz;

    2010-01-01

    Sinorhizobium sp. BL3 is a salt-tolerant strain that can fix atmospheric nitrogen in symbiosis with leguminous host plants under salt-stress conditions. Since cell membranes are the first barrier to environmental change, it is interesting to explore the membrane proteins within this protective...... functional categories, the two biggest of which were energy production and conversion, and proteins not in clusters of orthologous groups (COGs). In addition, a comparative analysis of membrane proteins between salt-stressed and non-stressed BL3 cells was conducted using a membrane enrichment method and off...... barrier under salt stress. The protein contents of membrane-enriched fractions obtained from BL3 were analyzed by nanoflow liquid chromatography interfaced with electrospray ionization tandem mass spectrometry. A total of 105 membrane proteins were identified. These proteins could be classified into 17...

  18. The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins

    Science.gov (United States)

    Adachi, Jun; Kumar, Chanchal; Zhang, Yanling; Olsen, Jesper V; Mann, Matthias

    2006-01-01

    Background Urine is a desirable material for the diagnosis and classification of diseases because of the convenience of its collection in large amounts; however, all of the urinary proteome catalogs currently being generated have limitations in their depth and confidence of identification. Our laboratory has developed methods for the in-depth characterization of body fluids; these involve a linear ion trap-Fourier transform (LTQ-FT) and a linear ion trap-orbitrap (LTQ-Orbitrap) mass spectrometer. Here we applied these methods to the analysis of the human urinary proteome. Results We employed one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography for protein separation and fractionation. Fractionated proteins were digested in-gel or in-solution, and digests were analyzed with the LTQ-FT and LTQ-Orbitrap at parts per million accuracy and with two consecutive stages of mass spectrometric fragmentation. We identified 1543 proteins in urine obtained from ten healthy donors, while essentially eliminating false-positive identifications. Surprisingly, nearly half of the annotated proteins were membrane proteins according to Gene Ontology (GO) analysis. Furthermore, extracellular, lysosomal, and plasma membrane proteins were enriched in the urine compared with all GO entries. Plasma membrane proteins are probably present in urine by secretion in exosomes. Conclusion Our analysis provides a high-confidence set of proteins present in human urinary proteome and provides a useful reference for comparing datasets obtained using different methodologies. The urinary proteome is unexpectedly complex and may prove useful in biomarker discovery in the future. PMID:16948836

  19. Homeostatic restitution of cell membranes. Nuclear membrane lipid biogenesis and transport of protein from cytosol to intranuclear spaces.

    Directory of Open Access Journals (Sweden)

    Amalia Slomiany, Maria Grabska, Bronislaw L. Slomiany

    2006-01-01

    Full Text Available Our studies on homeostatic restitution of cellular and subcellular membranes showed that vesicular intracellular transport is engaged in systematic and coordinated replacement of lipids and proteins in the membranes of the secretory, non-dividing epithelial cells (Slomiany et al., J. Physiol. Pharmacol. 2004; 55: 837-860. In this report, we present evidence on the homeostatic restitution of lipids in the biomembranes that constitute nuclear envelopes. We investigated nuclear membranes lipid synthesis by employing purified intact nuclei (IN, the outer nuclear membrane (ONM, the inner nuclear membrane (INM and the cell cytosol (CC. In contrast to Endoplasmic Reticulum (ER which in the presence of CC generates new biomembrane that forms ER vesicles transporting ER products to Golgi, the IN, ONM and INM are not producing transport vesicles. Instead, the newly synthesized lipids remain in the nuclear membranes. The membranes (INM, ONM of IN incubated with CC become enriched with newly synthesized phosphatidylcholine (PC, phosphatidylinositol (PI, phosphatidylinositol phosphates (PIPs and phosphatidic acid (PA. The incubation of separated ONM and INM with CC also enriched the membranes with IN specific lipids identified above. Moreover, the incubation of IN or its membranes with CC afforded retention of numerous CC proteins on the nuclear membrane. Here, we concentrated on 30kDa CC protein that displayed affinity to nuclear membrane PIP2. The 30kDa CC protein bound to PIP2 of IN, INM, and ONM. With IN, initially the PIP2-30kDa CC protein complex was detected on ONM, after 30-120 min of incubation, was found on INM and in nuclear contents. At the same time when the 30 kDa protein was released from INM and found in nuclear contents, the PIP2 of INM and ONM became undetectable, while the lipid extract from the membrane displaced from IN contained labeled PI only. Since ONM is an uninterrupted continuum of ER and INM, we speculate that the synthesis of

  20. Toward high-resolution computational design of the structure and function of helical membrane proteins.

    Science.gov (United States)

    Barth, Patrick; Senes, Alessandro

    2016-06-01

    The computational design of α-helical membrane proteins is still in its infancy but has already made great progress. De novo design allows stable, specific and active minimal oligomeric systems to be obtained. Computational reengineering can improve the stability and function of naturally occurring membrane proteins. Currently, the major hurdle for the field is the experimental characterization of the designs. The emergence of new structural methods for membrane proteins will accelerate progress. PMID:27273630

  1. The Membrane- and Soluble-Protein Helix-Helix Interactome: Similar Geometry via Different Interactions

    OpenAIRE

    Zhang, Shao-Qing; Kulp, Daniel W.; Schramm, Chaim A.; Mravic, Marco; Samish, Ilan; DeGrado, William F

    2015-01-01

    Alpha-helices are a basic unit of protein secondary structure and therefore the interaction between helices is crucial to understanding tertiary and higher-order folds. Comparing subtle variations in the structural and sequence motifs between membrane and soluble proteins sheds light on the different constraints faced by each environment and elucidates the complex puzzle of membrane protein folding. Here, we demonstrate that membrane and water-soluble helix pairs share a small number of simil...

  2. Benchmark data for identifying multi-functional types of membrane proteins.

    Science.gov (United States)

    Wan, Shibiao; Mak, Man-Wai; Kung, Sun-Yuan

    2016-09-01

    Identifying membrane proteins and their multi-functional types is an indispensable yet challenging topic in proteomics and bioinformatics. In this article, we provide data that are used for training and testing Mem-ADSVM (Wan et al., 2016. "Mem-ADSVM: a two-layer multi-label predictor for identifying multi-functional types of membrane proteins" [1]), a two-layer multi-label predictor for predicting multi-functional types of membrane proteins. PMID:27294176

  3. Towards understanding of Nipah virus attachment protein assembly and the role of protein affinity and crowding for membrane curvature events.

    Energy Technology Data Exchange (ETDEWEB)

    Stachowiak, Jeanne C.; Hayden, Carl C.; Negrete, Oscar.; Davis, Ryan Wesley; Sasaki, Darryl Y

    2013-10-01

    Pathogenic viruses are a primary threat to our national security and to the health and economy of our world. Effective defense strategies to combat viral infection and spread require the development of understanding of the mechanisms that these pathogens use to invade the host cell. We present in this report results of our research into viral particle recognition and fusion to cell membranes and the role that protein affinity and confinement in lipid domains plays in membrane curvature in cellular fusion and fission events. Herein, we describe 1) the assembly of the G attachment protein of Nipah virus using point mutation studies to define its role in viral particle fusion to the cell membrane, 2) how lateral pressure of membrane bound proteins induce curvature in model membrane systems, and 3) the role of membrane curvature in the selective partitioning of molecular receptors and specific affinity of associated proteins.

  4. Flux recovery of ceramic tubular membranes fouled with whey proteins: Some aspects of membrane cleaning

    OpenAIRE

    Popović Svetlana S.; Milanović Spasenija D.; Iličić Mirela D.; Lukić Nataša Lj.; Šijački Ivana M.

    2008-01-01

    Efficiency of membrane processes is greatly affected by the flux reduction due to the deposits formation at the surface and/or in the pores of the membrane. Efficiency of membrane processes is affected by cleaning procedure applied to regenerate flux. In this work, flux recovery of ceramic tubular membranes with 50 and 200 nm pore size was investigated. The membranes were fouled with reconstituted whey solution for 1 hour. After that, the membranes were rinsed with clean water and then cleane...

  5. Two Rab proteins, vesicle-associated membrane protein 2 (VAMP-2) and secretory carrier membrane proteins (SCAMPs), are present on immunoisolated parietal cell tubulovesicles.

    Science.gov (United States)

    Calhoun, B C; Goldenring, J R

    1997-01-01

    The tubulovesicles of gastric parietal cells sequester H+/K+-ATPase molecules within resting parietal cells. Stimulation of parietal cell secretion elicits delivery of intracellular H+/K+-ATPase to the apically oriented secretory canaliculus. Previous investigations have suggested that this process requires the regulated fusion of intracellular tubulovesicles with the canalicular target membrane. We have sought to investigate the presence of critical putative regulators of vesicle fusion on immunoisolated gastric parietal cell tubulovesicles. Highly purified tubulovesicles were prepared by gradient fractionation and immunoisolation on magnetic beads coated with monoclonal antibodies against the alpha subunit of H+/K+-ATPase. Western blot analysis revealed the presence of Rab11, Rab25, vesicle-associated membrane protein 2 (VAMP-2) and secretory carrier membrane proteins (SCAMPs) on immunoisolated vesicles. The same cohort of proteins was recovered on vesicles immunoisolated with monoclonal antibodies against SCAMPs and VAMP-2. In contrast, whereas immunoreactivities for syntaxin 1A/1B and synaptosome-associated protein (SNAP-25) were present in gradient-isolated vesicles, none of the immunoreactivity was associated with immunoisolated vesicles. The observation of VAMP-2 and two Rab proteins on immunoisolated H+/K+-ATPase-containing tubulovesicles supports the role for tubulovesicles in a regulated vesicle fusion process. In addition, the presence of SCAMPs along with Rab11 and Rab25 implicates the tubulovesicles as a critical apical recycling vesicle population. PMID:9230141

  6. Higher-order assemblies of BAR domain proteins for shaping membranes.

    Science.gov (United States)

    Suetsugu, Shiro

    2016-06-01

    Most cellular organelles contain lipid bilayer membranes. The earliest characterization of cellular organelles was performed by electron microscopy observation of such membranes. However, the precise mechanisms for shaping the membrane in particular subcellular organelles is poorly understood. Classically, the overall cellular shape, i.e. the shape of the plasma membrane, was thought to be governed by the reorganization of cytoskeletal components such as actin and microtubules. The plasma membrane contains various submicron structures such as clathrin-coated pits, caveolae, filopodia and lamellipodia. These subcellular structures are either invaginations or protrusions and are associated with the cytoskeleton. Therefore, it could be hypothesized that there are membrane-binding proteins that cooperates with cytoskeleton in shaping of plasma membrane organelles. Proteins with the Bin-Amphiphysin-Rvs (BAR) domain connect a variety of membrane shapes to actin filaments. The BAR domains themselves bend the membranes by their rigidity and then mold the membranes into tubules through their assembly as spiral polymers, which are thought to be involved in the various submicron structures. Membrane tubulation by polymeric assembly of the BAR domains is supposed to be regulated by binding proteins, binding lipids and the mechanical properties of the membrane. This review gives an overview of BAR protein assembly, describes the significance of the assembly and discusses how to study the assembly in the context of membrane and cellular morphology. The technical problems encountered in microscopic observation of BAR domain assembly are also discussed. PMID:26884618

  7. Higher-order assemblies of BAR domain proteins for shaping membranes.

    Science.gov (United States)

    Suetsugu, Shiro

    2016-06-01

    Most cellular organelles contain lipid bilayer membranes. The earliest characterization of cellular organelles was performed by electron microscopy observation of such membranes. However, the precise mechanisms for shaping the membrane in particular subcellular organelles is poorly understood. Classically, the overall cellular shape, i.e. the shape of the plasma membrane, was thought to be governed by the reorganization of cytoskeletal components such as actin and microtubules. The plasma membrane contains various submicron structures such as clathrin-coated pits, caveolae, filopodia and lamellipodia. These subcellular structures are either invaginations or protrusions and are associated with the cytoskeleton. Therefore, it could be hypothesized that there are membrane-binding proteins that cooperates with cytoskeleton in shaping of plasma membrane organelles. Proteins with the Bin-Amphiphysin-Rvs (BAR) domain connect a variety of membrane shapes to actin filaments. The BAR domains themselves bend the membranes by their rigidity and then mold the membranes into tubules through their assembly as spiral polymers, which are thought to be involved in the various submicron structures. Membrane tubulation by polymeric assembly of the BAR domains is supposed to be regulated by binding proteins, binding lipids and the mechanical properties of the membrane. This review gives an overview of BAR protein assembly, describes the significance of the assembly and discusses how to study the assembly in the context of membrane and cellular morphology. The technical problems encountered in microscopic observation of BAR domain assembly are also discussed.

  8. Mapping of unfolding states of integral helical membrane proteins by GPS-NMR and scattering techniques

    DEFF Research Database (Denmark)

    Calcutta, Antonello; Jessen, Christian Moestrup; Behrens, Manja Annette;

    2012-01-01

    Membrane proteins are vital for biological function, and their action is governed by structural properties critically depending on their interactions with the membranes. This has motivated considerable interest in studies of membrane protein folding and unfolding. Here the structural changes...... induced by unfolding of an integral membrane protein, namely TFE-induced unfolding of KcsA solubilized by the n-dodecyl ß-d-maltoside (DDM) surfactant is investigated by the recently introduced GPS-NMR (Global Protein folding State mapping by multivariate NMR) (Malmendal et al., PlosONE 5, e10262 (2010...

  9. Neutron scattering studies on protein dynamics using the human myelin peripheral membrane protein P2

    Directory of Open Access Journals (Sweden)

    Laulumaa Saara

    2015-01-01

    Full Text Available Myelin is a multilayered proteolipid membrane structure surrounding selected axons in the vertebrate nervous system, which allows the rapid saltatory conduction of nerve impulses. Deficits in myelin formation and maintenance may lead to chronic neurological disease. P2 is an abundant myelin protein from peripheral nerves, binding between two apposing lipid bilayers. We studied the dynamics of the human myelin protein P2 and its mutated P38G variant in hydrated powders using elastic incoherent neutron scattering. The local harmonic vibrations at low temperatures were very similar for both samples, but the mutant protein had increased flexibility and softness close to physiological temperatures. The results indicate that a drastic mutation of proline to glycine at a functional site can affect protein dynamics, and in the case of P2, they may explain functional differences between the two proteins.

  10. The HOPS/Class C Vps Complex Tethers High-Curvature Membranes via a Direct Protein-Membrane Interaction.

    Science.gov (United States)

    Ho, Ruoya; Stroupe, Christopher

    2016-10-01

    Membrane tethering is a physical association of two membranes before their fusion. Many membrane tethering factors have been identified, but the interactions that mediate inter-membrane associations remain largely a matter of conjecture. Previously, we reported that the homotypic fusion and protein sorting/Class C vacuolar protein sorting (HOPS/Class C Vps) complex, which has two binding sites for the yeast vacuolar Rab GTPase Ypt7p, can tether two low-curvature liposomes when both membranes bear Ypt7p. Here, we show that HOPS tethers highly curved liposomes to Ypt7p-bearing low-curvature liposomes even when the high-curvature liposomes are protein-free. Phosphorylation of the curvature-sensing amphipathic lipid-packing sensor (ALPS) motif from the Vps41p HOPS subunit abrogates tethering of high-curvature liposomes. A HOPS complex without its Vps39p subunit, which contains one of the Ypt7p binding sites in HOPS, lacks tethering activity, though it binds high-curvature liposomes and Ypt7p-bearing low-curvature liposomes. Thus, HOPS tethers highly curved membranes via a direct protein-membrane interaction. Such high-curvature membranes are found at the sites of vacuole tethering and fusion. There, vacuole membranes bend sharply, generating large areas of vacuole-vacuole contact. We propose that HOPS localizes via the Vps41p ALPS motif to these high-curvature regions. There, HOPS binds via Vps39p to Ypt7p in an apposed vacuole membrane. PMID:27307091

  11. Measure Guideline: Basement Insulation Basics

    Energy Technology Data Exchange (ETDEWEB)

    Aldrich, R.; Mantha, P.; Puttagunta, S.

    2012-10-01

    This guideline is intended to describe good practices for insulating basements in new and existing homes, and is intended to be a practical resources for building contractors, designers, and also to homeowners.

  12. Protein-Backbone Thermodynamics across the Membrane Interface.

    Science.gov (United States)

    Bereau, Tristan; Kremer, Kurt

    2016-07-01

    The thermodynamics of insertion of a protein in a membrane depends on the fine interplay between backbone and side-chain contributions interacting with the lipid environment. Using computer simulations, we probe how different descriptions of the backbone glycyl unit affect the thermodynamics of insertion of individual residues, dipeptides, and entire transmembrane helices. Due to the lack of reference data, we first introduce an efficient methodology to estimate atomistic potential of mean force (PMF) curves from a series of representative and uncorrelated coarse-grained (CG) snapshots. We find strong discrepancies between two CG models, Martini and PLUM, against reference atomistic PMFs and experiments. Atomistic simulations suggest a weak free energy of insertion between water and a POPC membrane for the glycyl unit, in overall agreement with experimental results despite severe assumptions in our calculations. We show that refining the backbone contribution in PLUM significantly improves the PMF of insertion of the WALP16 transmembrane peptide. An improper balance between the glycyl backbone and the attached side chain will lead to energetic artifacts, rationalizing Martini's overstabilization of WALP's adsorbed interfacial state. It illustrates difficulties associated with free-energy-based parametrizations of single-residue models, as the relevant free energy of partitioning used for force-field parametrization does not arise from the entire residue but rather the solvent-accessible chemical groups. PMID:27138459

  13. Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins.

    Science.gov (United States)

    Vasilev, Kalin V; Gallo, Eugenio; Shank, Nathaniel; Jarvik, Jonathan W

    2016-01-01

    We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay. PMID:27055753

  14. Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing.

    Science.gov (United States)

    Mehler, Michaela; Eckert, Carl Elias; Busche, Alena; Kulhei, Jennifer; Michaelis, Jonas; Becker-Baldus, Johanna; Wachtveitl, Josef; Dötsch, Volker; Glaubitz, Clemens

    2015-11-13

    Protein trans-splicing using split inteins is well established as a useful tool for protein engineering. Here we show, for the first time, that this method can be applied to a membrane protein under native conditions. We provide compelling evidence that the heptahelical proteorhodopsin can be assembled from two separate fragments consisting of helical bundles A and B and C, D, E, F, and G via a splicing site located in the BC loop. The procedure presented here is on the basis of dual expression and ligation in vivo. Global fold, stability, and photodynamics were analyzed in detergent by CD, stationary, as well as time-resolved optical spectroscopy. The fold within lipid bilayers has been probed by high field and dynamic nuclear polarization-enhanced solid-state NMR utilizing a (13)C-labeled retinal cofactor and extensively (13)C-(15)N-labeled protein. Our data show unambiguously that the ligation product is identical to its non-ligated counterpart. Furthermore, our data highlight the effects of BC loop modifications onto the photocycle kinetics of proteorhodopsin. Our data demonstrate that a correctly folded and functionally intact protein can be produced in this artificial way. Our findings are of high relevance for a general understanding of the assembly of membrane proteins for elucidating intramolecular interactions, and they offer the possibility of developing novel labeling schemes for spectroscopic applications.

  15. BCL::MP-Fold: membrane protein structure prediction guided by EPR restraints

    CERN Document Server

    Fischer, Axel Walter; Woetzel, Nils; Karakas, Mert; Weiner, Brian; Meiler, Jens

    2015-01-01

    For many membrane proteins the determination of their topology remains a challenge for methods like X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Electron paramagnetic resonance (EPR) spectroscopy has evolved as an alternative technique to study structure and dynamics of membrane proteins. The present study demonstrates the feasibility of membrane protein topology determination using limited EPR distance and accessibility measurements. The BCL::MP-Fold (BioChemical Library membrane protein fold) algorithm assembles secondary structure elements (SSEs) in the membrane using a Monte Carlo Metropolis (MCM) approach. Sampled models are evaluated using knowledge-based potential functions and agreement with the EPR data and a knowledge-based energy function. Twenty-nine membrane proteins of up to 696 residues are used to test the algorithm. The RMSD100 value of the most accurate model is better than 8{\\AA} for twenty-seven, better than 6{\\AA} for twenty-two and better than 4{\\AA} for fifte...

  16. Understanding leaf membrane protein extraction to develop a food-grade process

    NARCIS (Netherlands)

    Tamayo Tenorio, Angelica; Boom, Remko M.; Goot, van der Atze Jan

    2017-01-01

    Leaf membrane proteins are an underutilised protein fraction for food applications. Proteins from leaves can contribute to a more complete use of resources and help to meet the increasing protein demand. Leaf protein extraction and purification is applied by other disciplines, such as proteomics.

  17. The role of hydrophobic interactions in positioning of peripheral proteins in membranes

    Directory of Open Access Journals (Sweden)

    Lomize Mikhail A

    2007-06-01

    Full Text Available Abstract Background Three-dimensional (3D structures of numerous peripheral membrane proteins have been determined. Biological activity, stability, and conformations of these proteins depend on their spatial positions with respect to the lipid bilayer. However, these positions are usually undetermined. Results We report the first large-scale computational study of monotopic/peripheral proteins with known 3D structures. The optimal translational and rotational positions of 476 proteins are determined by minimizing energy of protein transfer from water to the lipid bilayer, which is approximated by a hydrocarbon slab with a decadiene-like polarity and interfacial regions characterized by water-permeation profiles. Predicted membrane-binding sites, protein tilt angles and membrane penetration depths are consistent with spin-labeling, chemical modification, fluorescence, NMR, mutagenesis, and other experimental studies of 53 peripheral proteins and peptides. Experimental membrane binding affinities of peripheral proteins were reproduced in cases that did not involve a helix-coil transition, specific binding of lipids, or a predominantly electrostatic association. Coordinates of all examined peripheral proteins and peptides with the calculated hydrophobic membrane boundaries, subcellular localization, topology, structural classification, and experimental references are available through the Orientations of Proteins in Membranes (OPM database. Conclusion Positions of diverse peripheral proteins and peptides in the lipid bilayer can be accurately predicted using their 3D structures that represent a proper membrane-bound conformation and oligomeric state, and have membrane binding elements present. The success of the implicit solvation model suggests that hydrophobic interactions are usually sufficient to determine the spatial position of a protein in the membrane, even when electrostatic interactions or specific binding of lipids are substantial. Our

  18. Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs.

    Science.gov (United States)

    Roy, Jahnabi; Pondenis, Holly; Fan, Timothy M; Das, Aditi

    2015-10-20

    Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na(+)/K(+)-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs.

  19. Expression Screening of Integral Membrane Proteins by Fusion to Fluorescent Reporters.

    Science.gov (United States)

    Bird, Louise E; Nettleship, Joanne E; Järvinen, Valtteri; Rada, Heather; Verma, Anil; Owens, Raymond J

    2016-01-01

    The production of recombinant integral membrane proteins for structural and functional studies remains technically challenging due to their relatively low levels of expression. To address this problem, screening strategies have been developed to identify the optimal membrane sequence and expression host for protein production. A common approach is to genetically fuse the membrane protein to a fluorescent reporter, typically Green Fluorescent Protein (GFP) enabling expression levels, localization and detergent solubilisation to be assessed. Initially developed for screening the heterologous expression of bacterial membrane proteins in Escherichia coli, the method has been extended to eukaryotic hosts, including insect and mammalian cells. Overall, GFP-based expression screening has made a major impact on the number of membrane protein structures that have been determined in the last few years. PMID:27553231

  20. Randomly organized lipids and marginally stable proteins: a coupling of weak interactions to optimize membrane signaling.

    Science.gov (United States)

    Rice, Anne M; Mahling, Ryan; Fealey, Michael E; Rannikko, Anika; Dunleavy, Katie; Hendrickson, Troy; Lohese, K Jean; Kruggel, Spencer; Heiling, Hillary; Harren, Daniel; Sutton, R Bryan; Pastor, John; Hinderliter, Anne

    2014-09-01

    Eukaryotic lipids in a bilayer are dominated by weak cooperative interactions. These interactions impart highly dynamic and pliable properties to the membrane. C2 domain-containing proteins in the membrane also interact weakly and cooperatively giving rise to a high degree of conformational plasticity. We propose that this feature of weak energetics and plasticity shared by lipids and C2 domain-containing proteins enhance a cell's ability to transduce information across the membrane. We explored this hypothesis using information theory to assess the information storage capacity of model and mast cell membranes, as well as differential scanning calorimetry, carboxyfluorescein release assays, and tryptophan fluorescence to assess protein and membrane stability. The distribution of lipids in mast cell membranes encoded 5.6-5.8bits of information. More information resided in the acyl chains than the head groups and in the inner leaflet of the plasma membrane than the outer leaflet. When the lipid composition and information content of model membranes were varied, the associated C2 domains underwent large changes in stability and denaturation profile. The C2 domain-containing proteins are therefore acutely sensitive to the composition and information content of their associated lipids. Together, these findings suggest that the maximum flow of signaling information through the membrane and into the cell is optimized by the cooperation of near-random distributions of membrane lipids and proteins. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

  1. Life at the border: Adaptation of proteins to anisotropic membrane environment

    Science.gov (United States)

    Pogozheva, Irina D; Mosberg, Henry I; Lomize, Andrei L

    2014-01-01

    This review discusses main features of transmembrane (TM) proteins which distinguish them from water-soluble proteins and allow their adaptation to the anisotropic membrane environment. We overview the structural limitations on membrane protein architecture, spatial arrangement of proteins in membranes and their intrinsic hydrophobic thickness, co-translational and post-translational folding and insertion into lipid bilayers, topogenesis, high propensity to form oligomers, and large-scale conformational transitions during membrane insertion and transport function. Special attention is paid to the polarity of TM protein surfaces described by profiles of dipolarity/polarizability and hydrogen-bonding capacity parameters that match polarity of the lipid environment. Analysis of distributions of Trp resides on surfaces of TM proteins from different biological membranes indicates that interfacial membrane regions with preferential accumulation of Trp indole rings correspond to the outer part of the lipid acyl chain region—between double bonds and carbonyl groups of lipids. These “midpolar” regions are not always symmetric in proteins from natural membranes. We also examined the hydrophobic effect that drives insertion of proteins into lipid bilayer and different free energy contributions to TM protein stability, including attractive van der Waals forces and hydrogen bonds, side-chain conformational entropy, the hydrophobic mismatch, membrane deformations, and specific protein–lipid binding. PMID:24947665

  2. Effect of membrane protein concentration on binding of 3H-imipramine in human platelets

    International Nuclear Information System (INIS)

    Binding of 3H-imipramine to platelet membranes has been implicated as a marker for depression. Comparing 3H-IMI binding between depressed patients and normal subjects we observed an increase in the dissociation constant Kd with increasing membrane protein. This phenomenon was studied more rigorously in five normal subjects. Platelet membranes were prepared and adjusted to four concentrations of protein ranging from 100 to 800 micrograms/ml. The 3H-IMI binding parameters of maximum binding sites number (Bmax) and Kd were obtained by Scatchard analysis at each membrane concentration. A positive linear relationship was found between K/sub d/ values and the concentration of membrane protein in the assay, but no change was observed in Bmax. The variability in Kd values reported in the literature may be accounted for in part by the different concentrations of membrane protein used in various studies

  3. Pearling instability of membrane tubes driven by curved proteins and actin polymerization

    Science.gov (United States)

    Jelerčič, U.; Gov, N. S.

    2015-12-01

    Membrane deformation inside living cells is crucial for the proper shaping of various intracellular organelles and is necessary during the fission/fusion processes that allow membrane recycling and transport (e.g. endocytosis). Proteins that induce membrane curvature play a key role in such processes, mostly by adsorbing to the membrane and forming a scaffold that deforms the membrane according to the curvature of the proteins. In this paper we explore the possibility of membrane tube destabilization through a pearling mechanism enabled by the combined effects of the adsorbed curved proteins and the actin polymerization that they recruit. The pearling instability can serve as the initiation for fission of the tube into vesicles. We find that adsorbed curved proteins are more likely to stabilize the tubes, while the actin polymerization can provide the additional constrictive force needed for the robust instability. We discuss the relevance of the theoretical results to in vivo and in vitro experiments.

  4. Pearling instability of membrane tubes driven by curved proteins and actin polymerization

    CERN Document Server

    Jelerčič, Urška

    2014-01-01

    Membrane deformation inside living cells is crucial for the proper shaping of various intracellular organelles and is necessary during the fission/fusion processes that allow membrane recycling and transport (e.g. endocytosis). Proteins that induce membrane curvature play a key role in such processes, mostly by adsorbing to the membrane and forming a scaffold that deforms the membrane according to the curvature of the proteins. In this paper we explore the possibility of membrane tube destabilisation through a pearling mechanism enabled by the combined effects of the adsorbed curved proteins and the actin polymerization they may recruit. The pearling instability can furthermore serve as the initiation for fission of the tube into vesicles. We find that adsorbed proteins are more likely to stabilise the tubes, while the actin polymerization can provide the additional constrictive force needed for the robust instability. We discuss the relevance of the theoretical results to in-vivo and in-vitro experiments.

  5. Transmembrane protein topology mapping by the substituted cysteine accessibility method (SCAM™): Application to lipid-specific membrane protein topogenesis

    OpenAIRE

    Bogdanov, Mikhail; Zhang, Wei; Xie, Jun; Dowhan, William

    2005-01-01

    We provide an overview of lipid-dependent polytopic membrane protein topogenesis, with particular emphasis on Escherichia coli strains genetically altered in their lipid composition and strategies for experimentally determining the transmembrane organization of proteins. A variety of reagents and experimental strategies are described including the use of lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by substituted cy...

  6. Postirradiation decrease in the protein shielding of phosphatidyl ethanolamine in the erythrocyte membranes

    International Nuclear Information System (INIS)

    Using trinitrobenzene sulfoacids the authors showed that 60 minutes after irradiation of rats (800 rad), the protein shielding of phosphatidyl ethanolamine in the erythrocyte membranes decreased. Such an action of ionizing radiation is suggested to be connected with the structural rearrangement of the membranes and changes in the lipid/protein interactions

  7. Computer simulation of the phase behavior of a model membrane protein: Annexin V

    NARCIS (Netherlands)

    Bates, M.A.; Noro, M.G.; Frenkel, D.

    2002-01-01

    The bulk thermodynamic properties of membrane proteins originate from a complex combination of molecular interactions. We propose a simple model based on the pair interactions between a model membrane protein, annexin V. The experimental observations of a honeycomb (p6) and a triangular (p3) phase a

  8. Interaction between La(III) and proteins on the plasma membrane of horseradish

    Science.gov (United States)

    Yang, Guang-Mei; Chu, Yun-Xia; Lv, Xiao-Fen; Zhou, Qing; Huang, Xiao-Hua

    2012-06-01

    Lanthanum (La) is an important rare earth element in the ecological environment of plant. The proteins on the plasma membrane control the transport of molecules into and out of cell. It is very important to investigate the effect of La(III) on the proteins on the plasma membrane in the plant cell. In the present work, the interaction between La(III) and proteins on the plasma membrane of horseradish was investigated using optimization of the fluorescence microscopy and fluorescence spectroscopy. It is found that the fluorescence of the complex system of protoplasts and 1-aniline Kenai-8-sulfonic acid in horseradish treated with the low concentration of La(III) is increased compared with that of the control horseradish. The opposite effect is observed in horseradish treated with the high concentration of La(III). These results indicated that the low concentration of La(III) can interact with the proteins on the plasma membrane of horseradish, causing the improvement in the structure of proteins on the plasma membrane. The high concentration of La(III) can also interact with the proteins on the plasma membrane of horseradish, leading to the destruction of the structure of proteins on the plasma membrane. We demonstrate that the proteins on the plasma membrane are the targets of La(III) action on plant cell.

  9. Isolation of monodisperse nanodisc-reconstituted membrane proteins using free flow electrophoresis

    DEFF Research Database (Denmark)

    Justesen, Bo Højen; Laursen, Tomas; Weber, Gerhard;

    2013-01-01

    Free flow electrophoresis is used for rapid and high-recovery isolation of homogeneous preparations of functionally active membrane proteins inserted into nanodiscs. The approach enables isolation of integral and membrane anchored proteins and is also applicable following introduction of, e...

  10. A New Class of Amphiphiles Bearing Rigid Hydrophobic Groups for Solubilization and Stabilization of Membrane Proteins

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Rasmussen, Søren G F; Rana, Rohini R;

    2012-01-01

    Non-traditional amphiphiles: Conferring aqueous solubility on membrane proteins generally requires the use of a detergent or other amphiphilic agent. A new class of amphiphiles was synthesized, based on steroidal lipophilic groups, and evaluated with several membrane proteins. The results show th...

  11. SUN proteins facilitate the removal of membranes from chromatin during nuclear envelope breakdown

    OpenAIRE

    Turgay Y; Champion L; Balazs C; Held M; Toso A; Gerlich DW; Meraldi P; Kutay U.

    2014-01-01

    SUN proteins reside in the inner nuclear membrane and form complexes with KASH proteins of the outer nuclear membrane that connect the nuclear envelope (NE) to the cytoskeleton. These complexes have well-established functions in nuclear anchorage and migration in interphase, but little is known about their involvement in mitotic processes. Our analysis demonstrates that simultaneous depletion of human SUN1 and SUN2 delayed removal of membranes from chromatin during NE breakdown (NEBD) and imp...

  12. A delocalized proton-binding site within a membrane protein.

    Science.gov (United States)

    Wolf, Steffen; Freier, Erik; Gerwert, Klaus

    2014-07-01

    The role of protein-bound water molecules in protein function and catalysis is an emerging topic. Here, we studied the solvation of an excess proton by protein-bound water molecules and the contribution of the surrounding amino acid residues at the proton release site of the membrane protein bacteriorhodopsin. It hosts an excess proton within a protein-bound water cluster, which is hydrogen bonded to several surrounding amino acids. Indicative of delocalization is a broad continuum absorbance experimentally observed by time-resolved Fourier transform infrared spectroscopy. In combination with site-directed mutagenesis, the involvement of several amino acids (especially Glu-194 and Glu-204) in the delocalization was elaborated. Details regarding the contributions of the glutamates and water molecules to the delocalization mode in biomolecular simulations are controversial. We carried out quantum mechanics/molecular mechanics (QM/MM) self-consistent charge density functional tight-binding simulations for all amino acids that have been experimentally shown to be involved in solvation of the excess proton, and systematically investigated the influence of the quantum box size. We compared calculated theoretical infrared spectra with experimental ones as a measure for the correct description of excess proton delocalization. A continuum absorbance can only be observed for small quantum boxes containing few amino acids and/or water molecules. Larger quantum boxes, including all experimentally shown involved amino acids, resulted in narrow absorbance bands, indicating protonation of a single binding site in contradiction to experimental results. We conclude that small quantum boxes seem to reproduce representative extreme cases of proton delocalization modes: proton delocalization only on water molecules or only between Glu-194 and Glu-204. Extending the experimental spectral region to lower wave numbers, a water-delocalized proton reproduces the observed continuum

  13. Training-induced changes in membrane transport proteins of human skeletal muscle

    DEFF Research Database (Denmark)

    Juel, C.

    2006-01-01

    Training improves human physical performance by inducing structural and cardiovascular changes, metabolic changes, and changes in the density of membrane transport proteins. This review focuses on the training-induced changes in proteins involved in sarcolemmal membrane transport. It is concluded...... that the same type of training affects many transport proteins, suggesting that all transport proteins increase with training, and that both sprint and endurance training in humans increase the density of most membrane transport proteins. There seems to be an upper limit for these changes: intense...... training for 6-8 weeks substantially increases the density of membrane proteins, whereas years of training (as performed by athletes) have no further effect. Studies suggest that training-induced changes at the protein level are important functionally. The underlying factors responsible for these changes...

  14. Large-scale identification of membrane proteins with properties favorable for crystallization.

    Science.gov (United States)

    Kim, Jared; Kagawa, Allison; Kurasaki, Kellie; Ataie, Niloufar; Cho, Il Kyu; Li, Qing X; Ng, Ho Leung

    2015-11-01

    Membrane protein crystallography is notoriously difficult due to challenges in protein expression and issues of degradation and structural stability. We have developed a novel method for large-scale screening of native sources for integral membrane proteins that have intrinsic biochemical properties favorable for crystallization. Highly expressed membrane proteins that are thermally stable and nonaggregating in detergent solutions were identified by mass spectrometry from Escherichia coli, Saccharomyces cerevisiae, and Sus scrofa cerebrum. Many of the membrane proteins identified had been crystallized previously, supporting the promise of the approach. Most identified proteins have known functions and include high-value targets such as transporters and ATPases. To validate the method, we recombinantly expressed and purified the yeast protein, Yop1, which is responsible for endoplasmic reticulum curvature. We demonstrate that Yop1 can be purified with the detergent dodecylmaltoside without aggregating.

  15. Identification and extraction of Pasteurella haemolytica membrane proteins.

    OpenAIRE

    Squire, P G; Smiley, D W; Croskell, R B

    1984-01-01

    The inner and outer membranes of Pasteurella haemolytica were separated by sucrose density gradient centrifugation after plasmolysis of the cells in 20% sucrose and fragmentation in a French pressure cell. Assays of the two membrane fractions for 2-keto-3-deoxyoctonate, succinate dehydrogenase, and NADH dehydrogenase and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that each of the two membrane fractions was purified fivefold relative to the other. The outer membrane...

  16. Defining the Free-Energy Landscape of Curvature-Inducing Proteins on Membrane Bilayers

    CERN Document Server

    Tourdot, Richard W; Radhakrishnan, Ravi

    2015-01-01

    Curvature-sensing and curvature-remodeling proteins are known to reshape cell membranes, and this remodeling event is essential for key biophysical processes such as tubulation, exocytosis, and endocytosis. Curvature-inducing proteins can act as curvature sensors as well as induce curvature in cell membranes to stabilize emergent high curvature, non-spherical, structures such as tubules, discs, and caveolae. A definitive understanding of the interplay between protein recruitment and migration, the evolution of membrane curvature, and membrane morphological transitions is emerging but remains incomplete. Here, within a continuum framework and using the machinery of Monte Carlo simulations, we introduce and compare three free-energy methods to delineate the free-energy landscape of curvature-inducing proteins on bilayer membranes. We demonstrate the utility of the Widom test-particle/field insertion methodology in computing the excess chemical potentials associated with curvature-inducing proteins on the membra...

  17. Structural elucidation of the interaction between neurodegenerative disease-related tau protein with model lipid membranes

    Science.gov (United States)

    Jones, Emmalee M.

    A protein's sequence of amino acids determines how it folds. That folded structure is linked to protein function, and misfolding to dysfunction. Protein misfolding and aggregation into beta-sheet rich fibrillar aggregates is connected with over 20 neurodegenerative diseases, including Alzheimer's disease (AD). AD is characterized in part by misfolding, aggregation and deposition of the microtubule associated tau protein into neurofibrillary tangles (NFTs). However, two questions remain: What is tau's fibrillization mechanism, and what is tau's cytotoxicity mechanism? Tau is prone to heterogeneous interactions, including with lipid membranes. Lipids have been found in NFTs, anionic lipid vesicles induced aggregation of the microtubule binding domain of tau, and other protein aggregates induced ion permeability in cells. This evidence prompted our investigation of tau's interaction with model lipid membranes to elucidate the structural perturbations those interactions induced in tau protein and in the membrane. We show that although tau is highly charged and soluble, it is highly surface active and preferentially interacts with anionic membranes. To resolve molecular-scale structural details of tau and model membranes, we utilized X-ray and neutron scattering techniques. X-ray reflectivity indicated tau aggregated at air/water and anionic lipid membrane interfaces and penetrated into membranes. More significantly, membrane interfaces induced tau protein to partially adopt a more compact conformation with density similar to folded protein and ordered structure characteristic of beta-sheet formation. This suggests possible membrane-based mechanisms of tau aggregation. Membrane morphological changes were seen using fluorescence microscopy, and X-ray scattering techniques showed tau completely disrupts anionic membranes, suggesting an aggregate-based cytotoxicity mechanism. Further investigation of protein constructs and a "hyperphosphorylation" disease mimic helped

  18. Biomimetic triblock copolymer membrane arrays: a stable template for functional membrane proteins

    DEFF Research Database (Denmark)

    Gonzalez-Perez, A.; Jensen, Karin Bagger Stibius; Vissing, Thomas;

    2009-01-01

    It is demonstrated that biomimetic stable triblock copolymer membrane arrays can be prepared using a scaffold containing 64 apertures of 300 μm diameter each. The membranes were made from a stock solution of block copolymers with decane as a solvent using a new deposition method. By using decane,...... to what is observed in black lipid membranes. The ion-channel gramicidin A was successfully incorporated into the membrane in a functional form....

  19. Independent mobility of proteins and lipids in the plasma membrane of Escherichia coli.

    Science.gov (United States)

    Nenninger, Anja; Mastroianni, Giulia; Robson, Alexander; Lenn, Tchern; Xue, Quan; Leake, Mark C; Mullineaux, Conrad W

    2014-06-01

    Fluidity is essential for many biological membrane functions. The basis for understanding membrane structure remains the classic Singer-Nicolson model, in which proteins are embedded within a fluid lipid bilayer and able to diffuse laterally within a sea of lipid. Here we report lipid and protein diffusion in the plasma membrane of live cells of the bacterium Escherichia coli, using Fluorescence Recovery after Photobleaching (FRAP) and Total Internal Reflection Fluorescence (TIRF) microscopy to measure lateral diffusion coefficients. Lipid and protein mobility within the membrane were probed by visualizing an artificial fluorescent lipid and a simple model membrane protein consisting of a single membrane-spanning alpha-helix with a Green Fluorescent Protein (GFP) tag on the cytoplasmic side. The effective viscosity of the lipid bilayer is strongly temperature-dependent, as indicated by changes in the lipid diffusion coefficient. Surprisingly, the mobility of the model protein was unaffected by changes in the effective viscosity of the bulk lipid, and TIRF microscopy indicates that it clusters in segregated, mobile domains. We suggest that this segregation profoundly influences the physical behaviour of the protein in the membrane, with strong implications for bacterial membrane function and bacterial physiology.

  20. Fluorometric functional assay for ion channel proteins in lipid nanovesicle membranes

    Energy Technology Data Exchange (ETDEWEB)

    Patti, J T [Department of Bioengineering, University of California, Los Angeles (United States); Montemagno, C D [College of Engineering, University of Cincinnati, Cincinnati (United States)

    2007-08-15

    Voltage-gated membrane proteins function as biomolecular transistors, making them attractive components for biologically based nanodevices. A functional assay for purified channel proteins is described and demonstrated with sodium selective, voltage-gated NaChBac ion channels. Purified NaChBac proteins were incorporated into a nanovesicle system utilizing oxonol VI, a fluorescent indicator of trans-membrane voltage. The ionophore valinomycin was used to trigger a change in membrane potential, allowing the observation of sodium permeability using a fluorometer. This method is suitable for concurrently testing a large population of purified proteins prior to incorporation in nanodevices.

  1. Fluorometric functional assay for ion channel proteins in lipid nanovesicle membranes

    Science.gov (United States)

    Patti, J. T.; Montemagno, C. D.

    2007-08-01

    Voltage-gated membrane proteins function as biomolecular transistors, making them attractive components for biologically based nanodevices. A functional assay for purified channel proteins is described and demonstrated with sodium selective, voltage-gated NaChBac ion channels. Purified NaChBac proteins were incorporated into a nanovesicle system utilizing oxonol VI, a fluorescent indicator of trans-membrane voltage. The ionophore valinomycin was used to trigger a change in membrane potential, allowing the observation of sodium permeability using a fluorometer. This method is suitable for concurrently testing a large population of purified proteins prior to incorporation in nanodevices.

  2. Shape deformation of lipid membranes by banana-shaped protein rods: Comparison with isotropic inclusions and membrane rupture

    Science.gov (United States)

    Noguchi, Hiroshi

    2016-05-01

    The assembly of curved protein rods on fluid membranes is studied using implicit-solvent meshless membrane simulations. As the rod curvature increases, the rods on a membrane tube assemble along the azimuthal direction first and subsequently along the longitudinal direction. Here, we show that both transition curvatures decrease with increasing rod stiffness. For comparison, curvature-inducing isotropic inclusions are also simulated. When the isotropic inclusions have the same bending rigidity as the other membrane regions, the inclusions are uniformly distributed on the membrane tubes and vesicles even for large spontaneous curvature of the inclusions. However, the isotropic inclusions with much larger bending rigidity induce shape deformation and are concentrated on the region of a preferred curvature. For high rod density, high rod stiffness, and/or low line tension of the membrane edge, the rod assembly induces vesicle rupture, resulting in the formation of a high-genus vesicle. A gradual change in the curvature suppresses this rupture. Hence, large stress, compared to the edge tension, induced by the rod assembly is the key factor determining rupture. For rod curvature with the opposite sign to the vesicle curvature, membrane rupture induces inversion of the membrane, leading to division into multiple vesicles as well as formation of a high-genus vesicle.

  3. Probing Peptide and Protein Insertion in a Biomimetic S-Layer Supported Lipid Membrane Platform

    Directory of Open Access Journals (Sweden)

    Samar Damiati

    2015-01-01

    Full Text Available The most important aspect of synthetic lipid membrane architectures is their ability to study functional membrane-active peptides and membrane proteins in an environment close to nature. Here, we report on the generation and performance of a biomimetic platform, the S-layer supported lipid membrane (SsLM, to investigate the structural and electrical characteristics of the membrane-active peptide gramicidin and the transmembrane protein α-hemolysin in real-time using a quartz crystal microbalance with dissipation monitoring in combination with electrochemical impedance spectroscopy. A shift in membrane resistance is caused by the interaction of α-hemolysin and gramicidin with SsLMs, even if only an attachment onto, or functional channels through the lipid membrane, respectively, are formed. Moreover, the obtained results did not indicate the formation of functional α-hemolysin pores, but evidence for functional incorporation of gramicidin into this biomimetic architecture is provided.

  4. Pinkbar is an epithelial-specific BAR domain protein that generates planar membrane structures

    Energy Technology Data Exchange (ETDEWEB)

    Pykäläinen, Anette; Boczkowska, Malgorzata; Zhao, Hongxia; Saarikangas, Juha; Rebowski, Grzegorz; Jansen, Maurice; Hakanen, Janne; Koskela, Essi V.; Peränen, Johan; Vihinen, Helena; Jokitalo, Eija; Salminen, Marjo; Ikonen, Elina; Dominguez, Roberto; Lappalainen, Pekka (Helsinki); (Penn)

    2013-05-29

    Bin/amphipysin/Rvs (BAR)-domain proteins sculpt cellular membranes and have key roles in processes such as endocytosis, cell motility and morphogenesis. BAR domains are divided into three subfamilies: BAR- and F-BAR-domain proteins generate positive membrane curvature and stabilize cellular invaginations, whereas I-BAR-domain proteins induce negative curvature and stabilize protrusions. We show that a previously uncharacterized member of the I-BAR subfamily, Pinkbar, is specifically expressed in intestinal epithelial cells, where it localizes to Rab13-positive vesicles and to the plasma membrane at intercellular junctions. Notably, the BAR domain of Pinkbar does not induce membrane tubulation but promotes the formation of planar membrane sheets. Structural and mutagenesis analyses reveal that the BAR domain of Pinkbar has a relatively flat lipid-binding interface and that it assembles into sheet-like oligomers in crystals and in solution, which may explain its unique membrane-deforming activity.

  5. Screening and identification of neutralizated single-chain antibody of anti-glomerular basement membrane antibody%中和抗肾小球基底膜抗体的单链抗体的筛选与鉴定

    Institute of Scientific and Technical Information of China (English)

    肖静; 刘章锁; 王沛; 黄留玉; 宋宏彬; 赵明辉

    2010-01-01

    Objective To screen a human single-chain variable fragments(scFv)against antiGBM antibody.Methods Using phage display technique,the phage antibody library was panned by antiglomerular basement membrane(GBM)antibody which was coated in a micro-titer plate,one clone was found to have high affinity to anti-GBM antibody.The DNA sequence of the positive clone was determined.Results Along with the increase of rounds anti-GBM antibody specific phage antibody was highly enriched and screening efficiency was increased 137 folds than the firest round.ELISA and competition inhibition assay showed that the scFv had a specific combination character with anti-GBM antibody.DNA sequencing confirmed that the whole gene of scFv was 750 bp,and in accordance with humanized single-chain variable region antibody sequence structure.Conclusion The results suggested that the scFv fragment to anti-GBM antibody could be successfully selected by recombinant phage antibody technique,which will laid an experimental foundation for further research of the therapy of Goodpasture syndrome.%目的 制备人抗肾小球基底膜(GBM)抗体的特异性人源化单链可变区抗体.方法 采用噬菌体表面展示技术,获得一个与人抗GBM抗体结合活性较强的单链可变区抗体片段的阳性克隆,并对该克隆进行DNA序列测定分析.结果 对噬菌体单链可变区抗体库经过3轮筛选后,与第1轮相比富集了137倍.噬菌体抗体与人抗GBM抗体的结合活性其中有35株克隆ELISA的吸光度较高.对这些噬菌体抗体进行交叉反应后,确定其中有10株交叉反应较弱.确定1株(C31)阳性克隆提取质粒,进行DNA序列测定,大小为750 bp,并符合人源化单链可变区抗体的序列结构.结论 应用噬菌体展示技术成功获得人-抗GBM抗体的单链可变区抗体基因,为临床上治疗Goodpasture综合征奠定实验基础.

  6. Lipid recognition propensities of amino acids in membrane proteins from atomic resolution data

    Directory of Open Access Journals (Sweden)

    Morita Mizuki

    2011-12-01

    Full Text Available Abstract Background Protein-lipid interactions play essential roles in the conformational stability and biological functions of membrane proteins. However, few of the previous computational studies have taken into account the atomic details of protein-lipid interactions explicitly. Results To gain an insight into the molecular mechanisms of the recognition of lipid molecules by membrane proteins, we investigated amino acid propensities in membrane proteins for interacting with the head and tail groups of lipid molecules. We observed a common pattern of lipid tail-amino acid interactions in two different data sources, crystal structures and molecular dynamics simulations. These interactions are largely explained by general lipophilicity, whereas the preferences for lipid head groups vary among individual proteins. We also found that membrane and water-soluble proteins utilize essentially an identical set of amino acids for interacting with lipid head and tail groups. Conclusions We showed that the lipophilicity of amino acid residues determines the amino acid preferences for lipid tail groups in both membrane and water-soluble proteins, suggesting that tightly-bound lipid molecules and lipids in the annular shell interact with membrane proteins in a similar manner. In contrast, interactions between lipid head groups and amino acids showed a more variable pattern, apparently constrained by each protein's specific molecular function.

  7. Lipid recognition propensities of amino acids in membrane proteins from atomic resolution data

    International Nuclear Information System (INIS)

    Protein-lipid interactions play essential roles in the conformational stability and biological functions of membrane proteins. However, few of the previous computational studies have taken into account the atomic details of protein-lipid interactions explicitly. To gain an insight into the molecular mechanisms of the recognition of lipid molecules by membrane proteins, we investigated amino acid propensities in membrane proteins for interacting with the head and tail groups of lipid molecules. We observed a common pattern of lipid tail-amino acid interactions in two different data sources, crystal structures and molecular dynamics simulations. These interactions are largely explained by general lipophilicity, whereas the preferences for lipid head groups vary among individual proteins. We also found that membrane and water-soluble proteins utilize essentially an identical set of amino acids for interacting with lipid head and tail groups. We showed that the lipophilicity of amino acid residues determines the amino acid preferences for lipid tail groups in both membrane and water-soluble proteins, suggesting that tightly-bound lipid molecules and lipids in the annular shell interact with membrane proteins in a similar manner. In contrast, interactions between lipid head groups and amino acids showed a more variable pattern, apparently constrained by each protein's specific molecular function

  8. Solid state NMR: The essential technology for helical membrane protein structural characterization

    Science.gov (United States)

    Cross, Timothy A.; Ekanayake, Vindana; Paulino, Joana; Wright, Anna

    2014-02-01

    NMR spectroscopy of helical membrane proteins has been very challenging on multiple fronts. The expression and purification of these proteins while maintaining functionality has consumed countless graduate student hours. Sample preparations have depended on whether solution or solid-state NMR spectroscopy was to be performed - neither have been easy. In recent years it has become increasingly apparent that membrane mimic environments influence the structural result. Indeed, in these recent years we have rediscovered that Nobel laureate, Christian Anfinsen, did not say that protein structure was exclusively dictated by the amino acid sequence, but rather by the sequence in a given environment (Anfinsen, 1973) [106]. The environment matters, molecular interactions with the membrane environment are significant and many examples of distorted, non-native membrane protein structures have recently been documented in the literature. However, solid-state NMR structures of helical membrane proteins in proteoliposomes and bilayers are proving to be native structures that permit a high resolution characterization of their functional states. Indeed, solid-state NMR is uniquely able to characterize helical membrane protein structures in lipid environments without detergents. Recent progress in expression, purification, reconstitution, sample preparation and in the solid-state NMR spectroscopy of both oriented samples and magic angle spinning samples has demonstrated that helical membrane protein structures can be achieved in a timely fashion. Indeed, this is a spectacular opportunity for the NMR community to have a major impact on biomedical research through the solid-state NMR spectroscopy of these proteins.

  9. Liposome-based assays to study membrane-associated protein networks.

    Science.gov (United States)

    Niehage, Christian; Stange, Christoph; Anitei, Mihaela; Hoflack, Bernard

    2014-01-01

    Transport carriers regulate the bidirectional flow of membrane between the compartments of the secretory and endocytic pathways. Their biogenesis relies on the recruitment of a number of cytosolic proteins and protein complexes on specific membrane microdomains with defined protein and lipid compositions. The timely assembly of these cellular machines onto membranes involves multiple protein-protein and protein-lipid interactions and is necessary to select membrane proteins and lipids into nascent carriers, to bend the flat membrane of the donor compartment, to change the shape of this nascent carrier into a tubular-vesicular structure, and to operate its scission from the donor compartment. A challenge in this field of membrane cell biology has been to identify these machineries and to understand their precise function, in particular by studying their spatial and temporal dynamics during carrier biogenesis. During the past years, liposome-based synthetic biology fully recapitulating the fidelity of carrier biogenesis as seen in vivo has proved to be instrumental to identify these key cytosolic components using mass spectrometry and their dynamics using fluorescence microscopy. We describe here the methods to isolate on synthetic membranes the protein networks needed for carrier biogenesis, to identify them using label-free quantitative proteomics, and to visualize their dynamics on giant unilamellar vesicles. PMID:24359957

  10. Protective effect of black tea on integral membrane proteins in rat liver.

    Science.gov (United States)

    Szachowicz-Petelska, Barbara; Skrzydlewska, Elżbieta; Figaszewski, Zbigniew

    2013-01-01

    Ethanol intoxication is accompanied by oxidative stress formation. Consequently, it leads to disturbances in cellular metabolism that can alter the structure and function of cell membrane components. Black tea displays antioxidant properties, protects membrane phospholipids and may protect integral membrane proteins. In the present study, we examined whether black tea induces changes in the liver integral membrane proteins of 12-months old rats chronically intoxicated with ethanol. To estimate qualitatively and quantitatively the levels of the liver integral membrane proteins, the proteins were selectively hydrolyzed by trypsin, the obtained peptides were resolved by HPLC and the levels of specific amino acids within the individual peptides were determined. All of the obtained peptides contained phenylalanine (Phe), cysteine (Cys) and lysine (Lys). Compared to the control group, rats in the ethanol intoxication group showed decreased liver levels of integral membrane proteins as well as fewer trypsin-hydrolyzed peptides and amino acids in the hydrolyzed peptides. Administration of black tea to ethanol-intoxicated rats partially protected proteins against the structural changes caused by ethanol. Black tea prevented decreases in the levels of cysteine (in about 90% of cases), lysine (in about 60% of cases), phenylalanine (in about 70% of cases) and examined peptides (in about 60% of cases). The liver protein level was higher (by about 18%) in rats who received black tea and ethanol than in those who received ethanol alone. In conclusion, black tea partially protects the composition and level of rat liver cell integral membrane proteins against changes caused by ethanol intoxication.

  11. Protein complexes in bacterial and yeast mitochondrial membranes differ in their sensitivity towards dissociation by SDS.

    Science.gov (United States)

    Gubbens, Jacob; Slijper, Monique; de Kruijff, Ben; de Kroon, Anton I P M

    2008-12-01

    Previously, a 2D gel electrophoresis approach was developed for the Escherichia coli inner membrane, which detects membrane protein complexes that are stable in sodium dodecyl sulfate (SDS) at room temperature, and dissociate under the influence of trifluoroethanol [R. E. Spelbrink et al., J. Biol. Chem. 280 (2005), 28742-8]. Here, the method was applied to the evolutionarily related mitochondrial inner membrane that was isolated from the yeast Saccharomyces cerevisiae. Surprisingly, only very few proteins were found to be dissociated by trifluoroethanol of which Lpd1p, a component of multiple protein complexes localized in the mitochondrial matrix, is the most prominent. Usage of either milder or more stringent conditions did not yield any additional proteins that were released by fluorinated alcohols. This strongly suggests that membrane protein complexes in yeast are less stable in SDS solution than their E. coli counterparts, which might be due to the overall reduced hydrophobicity of mitochondrial transmembrane proteins. PMID:18817900

  12. Membrane-Protein Interactions in a Generic Coarse-Grained Model for Lipid Bilayers

    CERN Document Server

    West, Beate; Schmid, Friederike

    2008-01-01

    We study membrane-protein interactions and membrane-mediated protein-protein interactions by Monte Carlo simulations of a generic coarse-grained model for lipid bilayers with cylindrical hydrophobic inclusions. The strength of the hydrophobic force and the hydrophobic thickness of the proteins are systematically varied. The results are compared with analytical predictions of two popular analytical theories: The Landau-de Gennes theory and the elastic theory. The elastic theory provides an excellent description of the fluctuation spectra of pure membranes and successfully reproduces the deformation profiles of membranes around single proteins. However, its prediction for the potential of mean force between proteins is not compatible with the simulation data for large distances. The simulations show that the lipid-mediated interactions are governed by five competing factors: Direct interactions, lipid-induced depletion interactions, lipid bridging, lipid packing, and a smooth long-range contribution. The mechan...

  13. Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins

    CERN Document Server

    Ramakrishnan, N; Radhakrishnan, Ravi

    2015-01-01

    Biological membranes constitute boundaries of cells and cell organelles. Physico-chemical mechanisms at the atomic scale are dictated by protein-lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across the various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. The suite of methods discussed here can be tailored to applicatio...

  14. Identification of Thylakoid Membrane Protein Complexes by Using a BN-Chip/MS Approach

    Institute of Scientific and Technical Information of China (English)

    Longquan Fan; Yinghong Pan

    2012-01-01

    Thylakoid membrane protein complexes of wheat (Triticum aestivum Linn.)play crucial roles in growth and crop production.Knowledge of the composition and structure of protein complexes,as well as protein interactions,will result in a much deeper understanding of metabolic pathways and cellular processes than protein identities alone,especially if the complexes can be separated in the native forms.Whereas the analysis of membrane protein complexes is a significant challenge due to their hydrophobic properties and relatively low abundance.A rapid and efficient method of identifying membrane protein complexes will greatly facilitate the investigation of agriculture.The present work developed an BN-Chip/MS approach for exhaustive separation and identification of protein complexes,by combining using blue-native polyacrylamide gel electrophoresis (BN-PAGE) and chip-based high-performance liquid chromatography quadruple time-of-flight tandem mass spectrometry (HPLC-Chip/ESI-QT-OF-MS,Chip/MS).By using this approach,seventy-five nonredundant proteins of wheat thylakoid membrane complexes were identified from digested 13 bands of BN-gel.When the protocol of BN separation was not used,only 37 nonredundant proteins had been identified and among of them 9 proteins were uniquely identi? ed.This BN-Chip/MS approach is rapid and efficient for identifying protein complexes in wheat thylakoid membranes,and also providing reliable foundations for further functional research of wheat chloroplast and for identifying protein complexes of other species.

  15. Relationship between the changes in ischemia/reperfusion cerebro-microvessel basement membrane injury and gelatinase system in senile rat%老龄大鼠脑缺血/再灌注致脑微血管基底膜损伤的变化及与明胶酶系的关系

    Institute of Scientific and Technical Information of China (English)

    李建生; 刘轲; 刘敬霞; 王明航; 赵跃武; 刘正国

    2008-01-01

    Objective To study the relationship of cerebro-microvessel basement membrane injury and gelatinase system after cerebral ischemia/reperfusion(I/R)in aged rats.Methods Cerebral I/R injury model was reproduced by intraluminal silk ligature thrombosis of the middle cerebral artery occlusion (MCAO).Rats were divided randomly into sham control and I/R groups in young rats Cischemia 3 hours (13 h)and reperfusion 6 hours(I/R 6 h),12 hours(1/R 12 h),24 hours(I/R 24 h),3 days(1/R 3 d),6 days(I/R 6 d)],and sham control group and I/R group in aged rats(1 3 h and I/R 6 h,I/R 12 h,I/R 24 h,1/R 3 d,1/R 6 d).The change in cerebro-cortex microvessel basement membrane structure,basement membrane type Ⅳ collagen(Col Ⅳ)and laminin(LN)contents,matrix metalloproteinases(MMPs)and tissue inhibitor of metalloproteinases(TIMPs)expression in every group were determined with immunohis tochemical method and zymogram analysis.Results With the inerease in age,Col Ⅳ and LN contents of the microvessel basement membrane were increased,and MMP-2 and MMP-9 expressions were stronger.With prolongation of I/R.the degradation of microvessel basement membrane components(Col Ⅳ and LN)was positively correlated with the duration of cerebral I/R.MMP-2 expression was increased gradually,and MMP-9 and TIMP-1 expression increased at the beginning and decreased subsequently.Col Ⅳ(I 3 h,I/R 6 h,I/R 12 h),LN(1 3 h,I/R 6-24 h),MMP-2(I 3 h,1/R 6 h-6 d)and MMP-9(1 3 h,I/R 6-24 h)expression level in aged rats with I/R injury were higher,and TIMP-1(I/R 24 h)expression was lower than those in young rats(P<0.05 or P<0.01).In addition,changes in MMP-2 and MMP-9 contents as determined by zymogram analysis method coincided with their immunoexpression.Conclusion With the increase of age,alteration in membrane components of eerebro-microvessel basement membrane in rats is related with MMPs and TlMP.Cerebro-mierovesseI basement membrane injury is more serious in aged rats than that of young rats.Changes in

  16. Present and future of membrane protein structure determination by electron crystallography.

    Science.gov (United States)

    Ubarretxena-Belandia, Iban; Stokes, David L

    2010-01-01

    Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This chapter describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins.

  17. Mass spectrometric analysis of integral membrane proteins: application to complete mapping of bacteriorhodopsins and rhodopsin.

    OpenAIRE

    Ball, L. E.; Oatis, J. E.; Dharmasiri, K.; Busman, M.; Wang, J.; Cowden, L. B.; Galijatovic, A.; N. Chen; Crouch, R K; Knapp, D R

    1998-01-01

    Integral membrane proteins have not been readily amenable to the general methods developed for mass spectrometric (or internal Edman degradation) analysis of soluble proteins. We present here a sample preparation method and high performance liquid chromatography (HPLC) separation system which permits online HPLC-electrospray ionization mass spectrometry (ESI-MS) and -tandem mass spectrometry (MS/MS) analysis of cyanogen bromide cleavage fragments of integral membrane proteins. This method has...

  18. Using tryptophan fluorescence to measure the stability of membrane proteins folded in liposomes

    OpenAIRE

    Moon, C. Preston; Fleming, Karen G.

    2011-01-01

    Accurate measurements of the thermodynamic stability of folded membrane proteins require methods for monitoring their conformation that are free of experimental artifacts. For tryptophan fluorescence emission experiments with membrane proteins folded into liposomes, there are two significant sources of artifacts: the first is light scattering by the liposomes; the second is the nonlinear relationship of some tryptophan spectral parameters with changes in protein conformation. Both of these so...

  19. Proteomic Analysis of Outer Membrane Proteins from Salmonella Enteritidis Strains with Different Sensitivity to Human Serum

    Science.gov (United States)

    Dudek, Bartłomiej; Krzyżewska, Eva; Kapczyńska, Katarzyna; Rybka, Jacek; Pawlak, Aleksandra; Korzekwa, Kamila; Klausa, Elżbieta; Bugla-Płoskońska, Gabriela

    2016-01-01

    Differential analysis of outer membrane composition of S. Enteritidis strains, resistant to 50% normal human serum (NHS) was performed in order to find factors influencing the resistance to higher concentrations of NHS. Ten S. Enteritidis clinical strains, resistant to 50% NHS, all producing very long lipopolysaccharide, were subjected to the challenge of 75% NHS. Five extreme strains: two resistant and three sensitive to 75% NHS, were chosen for the further analysis of outer membrane proteins composition. Substantial differences were found in the levels of particular outer membrane proteins between resistant and sensitive strains, i.e. outer membrane protease E (PgtE) was present mainly in resistant strains, while sensitive strains possessed a high level of flagellar hook-associated protein 2 (FliD) and significantly higher levels of outer membrane protein A (OmpA). PMID:27695090

  20. Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

    Science.gov (United States)

    Smith, Elizabeth Myhra

    The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

  1. Interplay between hydrophobicity and the positive-inside rule in determining membrane-protein topology.

    Science.gov (United States)

    Elazar, Assaf; Weinstein, Jonathan Jacob; Prilusky, Jaime; Fleishman, Sarel Jacob

    2016-09-13

    The energetics of membrane-protein interactions determine protein topology and structure: hydrophobicity drives the insertion of helical segments into the membrane, and positive charges orient the protein with respect to the membrane plane according to the positive-inside rule. Until recently, however, quantifying these contributions met with difficulty, precluding systematic analysis of the energetic basis for membrane-protein topology. We recently developed the dsTβL method, which uses deep sequencing and in vitro selection of segments inserted into the bacterial plasma membrane to infer insertion-energy profiles for each amino acid residue across the membrane, and quantified the insertion contribution from hydrophobicity and the positive-inside rule. Here, we present a topology-prediction algorithm called TopGraph, which is based on a sequence search for minimum dsTβL insertion energy. Whereas the average insertion energy assigned by previous experimental scales was positive (unfavorable), the average assigned by TopGraph in a nonredundant set is -6.9 kcal/mol. By quantifying contributions from both hydrophobicity and the positive-inside rule we further find that in about half of large membrane proteins polar segments are inserted into the membrane to position more positive charges in the cytoplasm, suggesting an interplay between these two energy contributions. Because membrane-embedded polar residues are crucial for substrate binding and conformational change, the results implicate the positive-inside rule in determining the architectures of membrane-protein functional sites. This insight may aid structure prediction, engineering, and design of membrane proteins. TopGraph is available online (topgraph.weizmann.ac.il).

  2. TRIM Proteins in Therapeutic Membrane Repair of Muscular Dystrophy

    OpenAIRE

    Alloush, Jenna; Weisleder, Noah

    2013-01-01

    Muscular dystrophy represents a major unmet medical need as only palliative treatments exist for these debilitating diseases. Since multiple forms of muscular dystrophy arise from compromised sarcolemmal membrane integrity a therapeutic approach that can target this loss of membrane barrier function could be applicable to a number of these distinct genetic diseases. One pathway that presents an excellent opportunity to affect compromised membrane integrity is the process that the cell uses to...

  3. REDOR solid-state NMR as a probe of the membrane locations of membrane-associated peptides and proteins

    Science.gov (United States)

    Jia, Lihui; Liang, Shuang; Sackett, Kelly; Xie, Li; Ghosh, Ujjayini; Weliky, David P.

    2015-04-01

    Rotational-echo double-resonance (REDOR) solid-state NMR is applied to probe the membrane locations of specific residues of membrane proteins. Couplings are measured between protein 13CO nuclei and membrane lipid or cholesterol 2H and 31P nuclei. Specific 13CO labeling is used to enable unambiguous assignment and 2H labeling covers a small region of the lipid or cholesterol molecule. The 13CO-31P and 13CO-2H REDOR respectively probe proximity to the membrane headgroup region and proximity to specific insertion depths within the membrane hydrocarbon core. One strength of the REDOR approach is use of chemically-native proteins and membrane components. The conventional REDOR pulse sequence with 100 kHz 2H π pulses is robust with respect to the 2H quadrupolar anisotropy. The 2H T1's are comparable to the longer dephasing times (τ's) and this leads to exponential rather than sigmoidal REDOR buildups. The 13CO-2H buildups are well-fitted to A × (1 - e-γτ) where A and γ are fitting parameters that are correlated as the fraction of molecules (A) with effective 13CO-2H coupling d = 3γ/2. The REDOR approach is applied to probe the membrane locations of the "fusion peptide" regions of the HIV gp41 and influenza virus hemagglutinin proteins which both catalyze joining of the viral and host cell membranes during initial infection of the cell. The HIV fusion peptide forms an intermolecular antiparallel β sheet and the REDOR data support major deeply-inserted and minor shallowly-inserted molecular populations. A significant fraction of the influenza fusion peptide molecules form a tight hairpin with antiparallel N- and C-α helices and the REDOR data support a single peptide population with a deeply-inserted N-helix. The shared feature of deep insertion of the β and α fusion peptide structures may be relevant for fusion catalysis via the resultant local perturbation of the membrane bilayer. Future applications of the REDOR approach may include samples that contain cell

  4. Plasma membrane lipid–protein interactions affect signaling processes in sterol-biosynthesis mutants in Arabidopsis thaliana

    OpenAIRE

    Zauber, Henrik; Burgos, Asdrubal; Garapati, Prashanth; Schulze, Waltraud X.

    2014-01-01

    The plasma membrane is an important organelle providing structure, signaling and transport as major biological functions. Being composed of lipids and proteins with different physicochemical properties, the biological functions of membranes depend on specific protein-protein and protein-lipid interactions. Interactions of proteins with their specific sterol and lipid environment were shown to be important factors for protein recruitment into sub-compartmental structures of the plasma membrane...

  5. Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

    Directory of Open Access Journals (Sweden)

    McClafferty Heather

    2005-01-01

    Full Text Available Abstract Background Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes. Results C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands. Conclusions C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP

  6. Coordination of Pancreatic HCO3- Secretion by Protein-Protein Interaction between Membrane Transporters

    Directory of Open Access Journals (Sweden)

    Lee MG

    2001-07-01

    Full Text Available Increasing evidence suggests that protein-protein interaction is essential in many biological processes including epithelial transport. In this report, we discuss the significance of protein interactions to HCO(3(- secretion in pancreatic duct cells. In pancreatic ducts HCO(3(- secretion is mediated by cystic fibrosis transmembrane conductance regulator (CFTR activated luminal Cl(-/HCO(3(- exchange activity and HCO(3(- absorption is achieved by Na(+-dependent mechanisms including Na(+/H(+ exchanger 3 (NHE3. We found biochemical and functional association between CFTR and NHE3. In addition, protein binding through PDZ modules is needed for this regulatory interaction. CFTR affected NHE3 activities in two ways. Acutely, CFTR augmented the cAMP-dependent inhibition of NHE3. In a chronic mechanism, CFTR increases the luminal expression of Na(+/H(+ exchange in pancreatic duct cells. These findings reveal that protein complexes in the plasma membrane of pancreatic duct cells are highly organized for efficient HCO(3(- secretion.

  7. Improving Anti-Protein-Fouling Property of Polyacrylonitrile Ultrafiltration Membrane by Grafting Sulfobetaine Zwitterions

    Directory of Open Access Journals (Sweden)

    Hong Meng

    2014-01-01

    Full Text Available Zwitterions show great superiority in the field of polymer membrane surface functionalization, as the synthesis process is simple, the adaptability of functional groups is strong, and zwitterions with strong hydration capacity in aqueous solutions can inhibit protein adsorption. In this study, a polyacrylonitrile ultrafiltration membrane was modified to improve anti-protein-fouling capacity by grafting short-chain sulfonic type zwitterions. 3-Dimethylaminopropylamine was first grafted onto hydrolyzed polyacrylonitrile (PAN membrane by the activation of 1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride (EDC. Subsequently, sulfobetaine zwitterions emerged on the membrane surface by quaternization of 1,3-propane sultone. The sulfobetaine zwitterionic membranes were analyzed for surface chemical composition, hydrophilic properties, and surface and cross-sectional structure of the membrane, by a combination of Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, contact angle measurement, and scanning electron microscopy. Static protein adsorption and dynamic filtration experiments were undertaken to show that the modified membrane had excellent resistance to protein adsorption. It was found that the molecular weight cutoff of the substrate membrane had great influence on the flux recovery rate of the modified membrane.

  8. A novel ER J-protein DNAJB12 accelerates ER-associated degradation of membrane proteins including CFTR.

    Science.gov (United States)

    Yamamoto, Yo-hei; Kimura, Taiji; Momohara, Shuku; Takeuchi, Masato; Tani, Tokio; Kimata, Yukio; Kadokura, Hiroshi; Kohno, Kenji

    2010-01-01

    Cytosolic Hsc70/Hsp70 are known to contribute to the endoplasmic reticulum (ER)-associated degradation of membrane proteins. However, at least in mammalian cells, its partner ER-localized J-protein for this cellular event has not been identified. Here we propose that this missing protein is DNAJB12. Protease protection assay and immunofluorescence study revealed that DNAJB12 is an ER-localized single membrane-spanning protein carrying a J-domain facing the cytosol. Using co-immunoprecipitation assay, we found that DNAJB12 is able to bind Hsc70 and thus can recruit Hsc70 to the ER membrane. Remarkably, cellular overexpression of DNAJB12 accelerated the degradation of misfolded membrane proteins including cystic fibrosis transmembrane conductance regulator (CFTR), but not a misfolded luminal protein. The DNAJB12-dependent degradation of CFTR was compromised by a proteasome inhibitor, lactacystin, suggesting that this process requires the ubiquitin-proteasome system. Conversely, knockdown of DNAJB12 expression attenuated the degradation of CFTR. Thus, DNAJB12 is a novel mammalian ER-localized J-protein that plays a vital role in the quality control of membrane proteins.

  9. Myelin membrane assembly is driven by a phase transition of myelin basic proteins into a cohesive protein meshwork.

    Science.gov (United States)

    Aggarwal, Shweta; Snaidero, Nicolas; Pähler, Gesa; Frey, Steffen; Sánchez, Paula; Zweckstetter, Markus; Janshoff, Andreas; Schneider, Anja; Weil, Marie-Theres; Schaap, Iwan A T; Görlich, Dirk; Simons, Mikael

    2013-01-01

    Rapid conduction of nerve impulses requires coating of axons by myelin. To function as an electrical insulator, myelin is generated as a tightly packed, lipid-rich multilayered membrane sheath. Knowledge about the mechanisms that govern myelin membrane biogenesis is required to understand myelin disassembly as it occurs in diseases such as multiple sclerosis. Here, we show that myelin basic protein drives myelin biogenesis using weak forces arising from its inherent capacity to phase separate. The association of myelin basic protein molecules to the inner leaflet of the membrane bilayer induces a phase transition into a cohesive mesh-like protein network. The formation of this protein network shares features with amyloid fibril formation. The process is driven by phenylalanine-mediated hydrophobic and amyloid-like interactions that provide the molecular basis for protein extrusion and myelin membrane zippering. These findings uncover a physicochemical mechanism of how a cytosolic protein regulates the morphology of a complex membrane architecture. These results provide a key mechanism in myelin membrane biogenesis with implications for disabling demyelinating diseases of the central nervous system.

  10. Myelin membrane assembly is driven by a phase transition of myelin basic proteins into a cohesive protein meshwork.

    Directory of Open Access Journals (Sweden)

    Shweta Aggarwal

    Full Text Available Rapid conduction of nerve impulses requires coating of axons by myelin. To function as an electrical insulator, myelin is generated as a tightly packed, lipid-rich multilayered membrane sheath. Knowledge about the mechanisms that govern myelin membrane biogenesis is required to understand myelin disassembly as it occurs in diseases such as multiple sclerosis. Here, we show that myelin basic protein drives myelin biogenesis using weak forces arising from its inherent capacity to phase separate. The association of myelin basic protein molecules to the inner leaflet of the membrane bilayer induces a phase transition into a cohesive mesh-like protein network. The formation of this protein network shares features with amyloid fibril formation. The process is driven by phenylalanine-mediated hydrophobic and amyloid-like interactions that provide the molecular basis for protein extrusion and myelin membrane zippering. These findings uncover a physicochemical mechanism of how a cytosolic protein regulates the morphology of a complex membrane architecture. These results provide a key mechanism in myelin membrane biogenesis with implications for disabling demyelinating diseases of the central nervous system.

  11. GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane

    Science.gov (United States)

    Sevcsik, Eva; Brameshuber, Mario; Fölser, Martin; Weghuber, Julian; Honigmann, Alf; Schütz, Gerhard J.

    2015-04-01

    The organization of proteins and lipids in the plasma membrane has been the subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored-mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase nor result in any enrichment of nanoscopic-ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane.

  12. Outer Membrane Proteins of Brucella abortus Vaccinal and Field Strains and their Immune Response in Buffaloes

    OpenAIRE

    Rukhshanda Munir*, M. Afzal1, M. Hussain2, S. M. S. Naqvi3 and A. Khanum3

    2010-01-01

    Outer membrane proteins (OMPs) of three strains of B. abortus i.e. S19, RB51 and a local field isolate of biotype 1 were isolated through disrupting cells to generate membranes by centrifugation and sodium lauryl sarcosinate solubilisation of inner membrane proteins. Distinct OMP profiles of each strain were seen on SDS-PAGE. SDS-PAGE analysis of S19 and field isolate revealed eight protein bands in each strain. The OMPs of S19 had molecular masses 89.0, 73.0, 53.7, 49.0, 38.0, 27.0, 22.3, a...

  13. Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

    Institute of Scientific and Technical Information of China (English)

    YUAN Ye; WANG Xiuli; GUO Sheping; QIU xuemei

    2011-01-01

    Gram-negative vibrio parahaemolyticus is a common pathogen in humans and marine animals.The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host.Thus,the outer membrane proteins are an ideal target for vaccines.We amplified a complete outer membrane protein gene (ompW) from V.parahaemolyticus ATCC 17802.We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells.The gene coded for a protein that was 42.78 kDa.We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting,respectively.Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V.parahaemolyticus.In addition,the purified OmpW protein can be used for further functional and structural studies.

  14. Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

    Science.gov (United States)

    Yuan, Ye; Wang, Xiuli; Guo, Sheping; Qiu, Xuemei

    2011-06-01

    Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V. parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.

  15. Advances in structural and functional analysis of membrane proteins by electron crystallography.

    Science.gov (United States)

    Wisedchaisri, Goragot; Reichow, Steve L; Gonen, Tamir

    2011-10-12

    Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane proteins by electron crystallography.

  16. Membrane protein thermodynamic stability may serve as the energy sink for sorting in the periplasm.

    Science.gov (United States)

    Moon, C Preston; Zaccai, Nathan R; Fleming, Patrick J; Gessmann, Dennis; Fleming, Karen G

    2013-03-12

    Thermodynamic stabilities are pivotal for understanding structure-function relationships of proteins, and yet such determinations are rare for membrane proteins. Moreover, the few measurements that are available have been conducted under very different experimental conditions, which compromises a straightforward extraction of physical principles underlying stability differences. Here, we have overcome this obstacle and provided structure-stability comparisons for multiple membrane proteins. This was enabled by measurements of the free energies of folding and the m values for the transmembrane proteins PhoP/PhoQ-activated gene product (PagP) and outer membrane protein W (OmpW) from Escherichia coli. Our data were collected in the same lipid bilayer and buffer system we previously used to determine those parameters for E. coli outer membrane phospholipase A (OmpLA). Biophysically, our results suggest that the stabilities of these proteins are strongly correlated to the water-to-bilayer transfer free energy of the lipid-facing residues in their transmembrane regions. We further discovered that the sensitivities of these membrane proteins to chemical denaturation, as judged by their m values, was consistent with that previously observed for water-soluble proteins having comparable differences in solvent exposure between their folded and unfolded states. From a biological perspective, our findings suggest that the folding free energies for these membrane proteins may be the thermodynamic sink that establishes an energy gradient across the periplasm, thus driving their sorting by chaperones to the outer membranes in living bacteria. Binding free energies of these outer membrane proteins with periplasmic chaperones support this energy sink hypothesis.

  17. Nonbilayer lipids affect peripheral and integral membrane proteins via changes in the lateral pressure profile.

    Science.gov (United States)

    van den Brink-van der Laan, Els; Killian, J Antoinette; de Kruijff, Ben

    2004-11-01

    Nonbilayer lipids can be defined as cone-shaped lipids with a preference for nonbilayer structures with a negative curvature, such as the hexagonal phase. All membranes contain these lipids in large amounts. Yet, the lipids in biological membranes are organized in a bilayer. This leads to the question: what is the physiological role of nonbilayer lipids? Different models are discussed in this review, with a focus on the lateral pressure profile within the membrane. Based on this lateral pressure model, predictions can be made for the effect of nonbilayer lipids on peripheral and integral membrane proteins. Recent data on the catalytic domain of Leader Peptidase and the potassium channel KcsA are discussed in relation to these predictions and in relation to the different models on the function of nonbilayer lipids. The data suggest a general mechanism for the interaction between nonbilayer lipids and membrane proteins via the membrane lateral pressure. PMID:15519321

  18. One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

    DEFF Research Database (Denmark)

    Lee, Yu-Chen; Block, Gregory; Chen, Huiwen;

    2008-01-01

    We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane...... proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment...... was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5'-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5'-nucleotidase activity with 33% recovery...

  19. High pressure modulated transport and signaling functions of membrane proteins in models and in vivo

    International Nuclear Information System (INIS)

    Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property

  20. High pressure modulated transport and signaling functions of membrane proteins in models and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Vogel, R F; Linke, K; Teichert, H; Ehrmann, M A [Technische Universitaet Muenchen, Technische Mikrobiologie, Weihenstephaner Steig 16, 85350 Freising (Germany)], E-mail: rudi.vogel@wzw.tum.de

    2008-07-15

    Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property.

  1. pH Dependence of Chlorophyll States, Protein Structures and Function of the PSII Membranes

    Institute of Scientific and Technical Information of China (English)

    李冬海; 阮翔; 许强; 王可玢; 公衍道; 匡廷云; 张秀芳; 赵南明

    2003-01-01

    The effect of varying pH on the photosystem II (PSII) membrane was studied using absorption and steady-state fluorescence spectroscopy, and using a variable fluorescence technique.pH variations induced significant changes in the chlorophyll states of the PSII membrane, but no effect was seen on the chlorophyll fluorescence parameter F′v/F′m.For acidic pH conditions, protein structures of the PSII membrane were slightly altered, whilst at alkaline pH levels, large changes in the protein structure of the PSII membrane were detected.The results indicate that the microenvironment around Cys in the PSII membrane is very susceptible to alkaline pH conditions, and that in the acid (4≤pH7) regions, pH variation has no effect on the protein structures of the PSII reaction center (RC).

  2. Flux recovery of ceramic tubular membranes fouled with whey proteins: Some aspects of membrane cleaning

    Directory of Open Access Journals (Sweden)

    Popović Svetlana S.

    2008-01-01

    Full Text Available Efficiency of membrane processes is greatly affected by the flux reduction due to the deposits formation at the surface and/or in the pores of the membrane. Efficiency of membrane processes is affected by cleaning procedure applied to regenerate flux. In this work, flux recovery of ceramic tubular membranes with 50 and 200 nm pore size was investigated. The membranes were fouled with reconstituted whey solution for 1 hour. After that, the membranes were rinsed with clean water and then cleaned with sodium hydroxide solutions or formulated detergents (combination of P3 Ultrasil 67 and P3 Ultrasil 69. Flux recovery after the rinsing step was not satisfactory although fouling resistance reduction was significant so that chemical cleaning was necessary. In the case of 50 nm membrane total flux recovery was achieved after cleaning with 1.0% (w/w sodium hydroxide solution. In the case of 200 nm membrane total flux recovery was not achieved irrespective of the cleaning agent choice and concentration. Cleaning with commercial detergent was less efficient than cleaning with the sodium hydroxide solution.

  3. Engineering of the E. coli Outer Membrane Protein FhuA to overcome the Hydrophobic Mismatch in Thick Polymeric Membranes

    Directory of Open Access Journals (Sweden)

    Fioroni Marco

    2011-03-01

    Full Text Available Abstract Background Channel proteins like the engineered FhuA Δ1-159 often cannot insert into thick polymeric membranes due to a mismatch between the hydrophobic surface of the protein and the hydrophobic surface of the polymer membrane. To address this problem usually specific block copolymers are synthesized to facilitate protein insertion. Within this study in a reverse approach we match the protein to the polymer instead of matching the polymer to the protein. Results To increase the FhuA Δ1-159 hydrophobic surface by 1 nm, the last 5 amino acids of each of the 22 β-sheets, prior to the more regular periplasmatic β-turns, were doubled leading to an extended FhuA Δ1-159 (FhuA Δ1-159 Ext. The secondary structure prediction and CD spectroscopy indicate the β-barrel folding of FhuA Δ1-159 Ext. The FhuA Δ1-159 Ext insertion and functionality within a nanocontainer polymeric membrane based on the triblock copolymer PIB1000-PEG6000-PIB1000 (PIB = polyisobutylene, PEG = polyethyleneglycol has been proven by kinetic analysis using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine. Identical experiments with the unmodified FhuA Δ1-159 report no kinetics and presumably no insertion into the PIB1000-PEG6000-PIB1000 membrane. Furthermore labeling of the Lys-NH2 groups present in the FhuA Δ1-159 Ext channel, leads to controllability of in/out flux of substrates and products from the nanocontainer. Conclusion Using a simple "semi rational" approach the protein's hydrophobic transmembrane region was increased by 1 nm, leading to a predicted lower hydrophobic mismatch between the protein and polymer membrane, minimizing the insertion energy penalty. The strategy of adding amino acids to the FhuA Δ1-159 Ext hydrophobic part can be further expanded to increase the protein's hydrophobicity, promoting the efficient embedding into thicker/more hydrophobic block copolymer membranes.

  4. Lysinuric protein intolerance mutation is expressed in the plasma membrane of cultured skin fibroblasts.

    OpenAIRE

    Smith, D. W.; Scriver, C R; Tenenhouse, H S; Simell, O.

    1987-01-01

    Lysinuric protein intolerance (LPI) is an autosomal recessive phenotype consistent with impaired transport of cationic amino acids at the basolateral membrane of intestinal and renal epithelia. On the assumption that the basolateral membrane of epithelial cells and plasma membrane of parenchymal cells are functional analogues, we studied transport of cationic amino acids by cultured skin fibroblasts from LPI and control subjects matched for age, sex, and site of biopsy. We measured Na+-indepe...

  5. A general theory of non-equilibrium dynamics of lipid-protein fluid membranes

    DEFF Research Database (Denmark)

    Lomholt, Michael Andersen; Hansen, Per Lyngs; Miao, L.

    2005-01-01

    We present a general and systematic theory of non-equilibrium dynamics of multi-component fluid membranes, in general, and membranes containing transmembrane proteins, in particular. Developed based on a minimal number of principles of statistical physics and designed to be a meso/macroscopic-sca......-equilibrium phenomena in a range of membrane systems, as discussions in the paper of a few limit cases demonstrate. © EDP Sciences / Società Italiana di Fisica / Springer-Verlag 2005....

  6. Architecture of beta-barrel membrane proteins: analysis of trimeric porins.

    OpenAIRE

    Seshadri, K.; Garemyr, R.; Wallin, E.; von Heijne, G; Elofsson, A.

    1998-01-01

    We have analyzed the known three-dimensional structures of trimeric porins from bacterial outer membranes. The distribution of surface-exposed residues in a direction perpendicular to the membrane is similar to that in helical membrane proteins, with aliphatic residues concentrated in the central 20 A of the bilayer. Outside these residues is a layer of aromatic residues, followed by polar and charged residues. Residues in the trimer interface are more conserved than residues not in the inter...

  7. Comparative proteomic analysis of plasma membrane proteins between human osteosarcoma and normal osteoblastic cell lines

    International Nuclear Information System (INIS)

    Osteosarcoma (OS) is the most common primary malignant tumor of bone in children and adolescents. However, the knowledge in diagnostic modalities has progressed less. To identify new biomarkers for the early diagnosis of OS as well as for potential novel therapeutic candidates, we performed a sub-cellular comparative proteomic research. An osteosarcoma cell line (MG-63) and human osteoblastic cells (hFOB1.19) were used as our comparative model. Plasma membrane (PM) was obtained by aqueous two-phase partition. Proteins were analyzed through iTRAQ-based quantitative differential LC/MS/MS. The location and function of differential proteins were analyzed through GO database. Protein-protein interaction was examined through String software. One of differentially expressed proteins was verified by immunohistochemistry. 342 non-redundant proteins were identified, 68 of which were differentially expressed with 1.5-fold difference, with 25 up-regulated and 43 down-regulated. Among those differential proteins, 69% ware plasma membrane, which are related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc., and interaction with each other. One protein--CD151 located in net nodes was verified to be over-expressed in osteosarcoma tissue by immunohistochemistry. It is the first time to use plasma membrane proteomics for studying the OS membrane proteins according to our knowledge. We generated preliminary but comprehensive data about membrane protein of osteosarcoma. Among these, CD151 was further validated in patient samples, and this small molecule membrane might be a new target for OS research. The plasma membrane proteins identified in this study may provide new insight into osteosarcoma biology and potential diagnostic and therapeutic biomarkers

  8. Role of amphipathic helix of a herpesviral protein in membrane deformation and T cell receptor downregulation.

    Directory of Open Access Journals (Sweden)

    Chan-Ki Min

    2008-11-01

    Full Text Available Lipid rafts are membrane microdomains that function as platforms for signal transduction and membrane trafficking. Tyrosine kinase interacting protein (Tip of T lymphotropic Herpesvirus saimiri (HVS is targeted to lipid rafts in T cells and downregulates TCR and CD4 surface expression. Here, we report that the membrane-proximal amphipathic helix preceding Tip's transmembrane (TM domain mediates lipid raft localization and membrane deformation. In turn, this motif directs Tip's lysosomal trafficking and selective TCR downregulation. The amphipathic helix binds to the negatively charged lipids and induces liposome tubulation, the TM domain mediates oligomerization, and cooperation of the membrane-proximal helix with the TM domain is sufficient for localization to lipid rafts and lysosomal compartments, especially the mutivesicular bodies. These findings suggest that the membrane-proximal amphipathic helix and TM domain provide HVS Tip with the unique ability to deform the cellular membranes in lipid rafts and to downregulate TCRs potentially through MVB formation.

  9. delta-Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains.

    Science.gov (United States)

    Bourova, Lenka; Kostrnova, Alexandra; Hejnova, Lucie; Moravcova, Zuzana; Moon, Hyo-Eun; Novotny, Jiri; Milligan, Graeme; Svoboda, Petr

    2003-04-01

    Low-density membrane fragments (domains) were separated from the bulk of plasma membranes of human embryonic kidney (HEK)293 cells expressing a delta-opioid (DOP) receptor-Gi1alpha fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein-linked Gi1alpha and endogenous pertussis-toxin sensitive G proteins was measured as d-Ala2, d-Leu5-enkephalin stimulated high-affinity GTPase or guanosine-5'-[gamma-35S]triphosphate ([35S]GTPgammaS) binding. The maximum d-Ala2-d-Leu5 enkephalin (DADLE)-stimulated GTPase was two times higher in low-density membrane fragments than in bulk of plasma membranes; 58 and 27 pmol/mg/min, respectively. The same difference was obtained for [35S]GTPgammaS binding. Contrarily, the low-density domains contained no more than half the DOP receptor binding sites (Bmax = 6.6 pmol/mg versus 13.6 pmol/mg). Thus, when corrected for expression levels of the receptor, low-density domains exhibited four times higher agonist-stimulated GTPase and [35S]GTPgammaS binding than the bulk plasma membranes. The regulator of G protein signaling RGS1, enhanced further the G protein functional activity but did not remove the difference between domain-bound and plasma membrane pools of G protein. The potency of the agonist in functional studies and the affinity of specific [3H]DADLE binding to the receptor were, however, the same in both types of membranes - EC50 = 4.5 +/- 0.1 x 10(-8) and 3.2 +/- 1.4 x 10(-8) m for GTPase; Kd = 1.2 +/- 0.1 and 1.3 +/- 0.1 nm for [3H]DADLE radioligand binding assay. Similar results were obtained when sodium bicarbonate was used for alkaline isolation of membrane domains. By contrast, detergent-insensitive membrane domains isolated following treatment of cells with Triton X100 exhibited no DADLE-stimulated GTPase or GTPgammaS binding. Functional coupling between the DOP receptor

  10. High sensitivity identification of membrane proteins by MALDI TOF-MASS spectrometry using polystyrene beads.

    Science.gov (United States)

    Bensalem, Noura; Masscheleyn, Sandrine; Mozo, Julien; Vallée, Benoit; Brouillard, Franck; Trudel, Stéphanie; Ricquier, Daniel; Edelman, Aleksander; Guerrera, Ida Chiara; Miroux, Bruno

    2007-04-01

    Membrane proteins play a large variety of functions in life and represent 30% of all genomes sequenced. Due to their hydrophobic nature, they are tightly bound to their biological membrane, and detergents are always required to extract and isolate them before identification by mass spectrometry (MS). The latter, however remains difficult. Peptide mass fingerprinting methods using techniques such as MALDI-TOF MS, for example, have become an important analytical tool in the identification of proteins. However, PMF of membrane proteins is a real challenge for at least three reasons. First, membrane proteins are naturally present at low levels; second, most of the detergents strongly inhibit proteases and have deleterious effects on MALDI spectra; and third, despite the presence of detergent, membrane proteins are unstable and often aggregate. We took the mitochondrial uncoupling protein 1 (UCP1) as a model and showed that differential acetonitrile extraction of tryptic peptides combined with the use of polystirene Bio-Beads triggered high resolution of the MALDI-TOF identification of mitochondrial membrane proteins solubilized either with Triton-X100 or CHAPS detergents. PMID:17355127

  11. Generation and Identification of Monoclonal Antibody Against Porcine Adipocyte Plasma Membrane Proteins

    Institute of Scientific and Technical Information of China (English)

    CAO Jin-ling; CHEN Jian-jie; WANG Zhi-rui; WANG Jun-dong

    2007-01-01

    Production of monoclonal antibody against porcine adipocyte plasma membrane proteins to explore a new way of controlling body fat deposition and improving carcass quality is discussed in this article. Membrane proteins of pig adipocyte plasma membrane proteins were extracted with the help of sucrose density gradient centrifugation, and two kinds of proteins were obtained. The monoclonal antibody (designated 3B2 and 3F3) of IgGl and IgG2b subclass against adipocyte membrane proteins were produced by immunization, with adipocyte membrane proteins as an antigen, and its titer was 1:105 detected by enzyme-linked immunoadsorbent assay (ELISA). The cell strains were identified by analyzing the number of chromosomes, the heat stability, the acid and alkali, the types and subtypes of immnoglobulin, and its peculiarities and affinities. Through identification, the chromosome number of hybridoma cell strains was from 80 to 100 and the strains formed good hybridomas colonies. The strains' affinity constants were 4.63×109 and 3.75×109 (mol L-1)-1, respectively. At the same time, the McAb secreted was stable to environmental factors, such as, temperature, acid, alkali and so on. The monoclonal antibodies had been obtained and their specificity to porcine adipocyte plasma membrane proteins had been identified.

  12. Cloning of immunodominant membrane protein genes of phytoplasmas and their in planta expression.

    Science.gov (United States)

    Kakizawa, Shigeyuki; Oshima, Kenro; Ishii, Yoshiko; Hoshi, Ayaka; Maejima, Kensaku; Jung, Hee-Young; Yamaji, Yasuyuki; Namba, Shigetou

    2009-04-01

    Phytoplasmas are plant pathogenic bacteria that cause devastating yield losses in diverse crops worldwide. Although the understanding of the pathogen biology is important in agriculture, the inability to culture phytoplasmas has hindered their full characterization. Previous studies demonstrated that immunodominant membrane proteins could be classified into three types, immunodominant membrane protein (Imp), immunodominant membrane protein A (IdpA), and antigenic membrane protein (Amp), and they are nonhomologous to each other. Here, cloning and sequencing of imp-containing genomic fragments were performed for several groups of phytoplasma including the aster yellows and rice yellow dwarf groups, for which an imp sequence has not previously been reported. Sequence comparison analysis revealed that Imps are highly variable among phytoplasmas, and clear positive selection was observed in several Imps, suggesting that Imp has important roles in host-phytoplasma interactions. As onion yellows (OY) phytoplasma was known to have Amp as the immunodominant membrane protein, the protein accumulation level of Imp in planta was measured compared with that of Amp. The resulting accumulation of Imp was calculated as approximately one-tenth that of Amp, being consistent with the immunodominant property of Amp in OY. It is suggested that an ancestral type of immunodominant membrane protein could be Imp, and subsequently the expression level of Amp or IdpA is increased in several phytoplasma groups. PMID:19222574

  13. Protein trafficking, ergosterol biosynthesis and membrane physics impact recombinant protein secretion in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Mattanovich Diethard

    2011-11-01

    Full Text Available Abstract Background The increasing availability of 'omics' databases provide important platforms for yeast engineering strategies since they offer a lot of information on the physiology of the cells under diverse growth conditions, including environmental stresses. Notably, only a few of these approaches have considered a performance under recombinant protein production conditions. Recently, we have identified a beneficial effect of low oxygen availability on the expression of a human Fab fragment in Pichia pastoris. Transcriptional analysis and data mining allowed for the selection of potential targets for strain improvement. A first selection of these candidates has been evaluated as recombinant protein secretion enhancers. Results Based on previous transcriptomics analyses, we selected 8 genes for co-expression in the P. pastoris strain already secreting a recombinant Fab fragment. Notably, WSC4 (which is involved in trafficking through the ER has been identified as a novel potential target gene for strain improvement, with up to a 1.2-fold increase of product yield in shake flask cultures. A further transcriptomics-based strategy to modify the yeast secretion system was focused on the ergosterol pathway, an aerobic process strongly affected by oxygen depletion. By specifically partially inhibiting ergosterol synthesis with the antifungal agent fluconazole (inhibiting Erg11p, we tried to mimic the hypoxic conditions, in which the cellular ergosterol content was significantly decreased. This strategy led to an improved Fab yield (2-fold without impairing cellular growth. Since ergosterol shortage provokes alterations in the plasma membrane composition, an important role of this cellular structure in protein secretion is suggested. This hypothesis was additionally supported by the fact that the addition of non-ionic surfactants also enhanced Fab secretion. Conclusions The current study presents a systems biotechnology-based strategy for the

  14. A scalable, GFP-based pipeline for membrane protein overexpression screening and purification

    NARCIS (Netherlands)

    Drew, David; Slotboom, Dirk-Jan; Friso, Giulia; Reda, Torsten; Genevaux, Pierre; Rapp, Mikaela; Meindl-Beinker, Nadja M.; Lambert, Wietske; Lerch, Mirjam; Daley, Daniel O.; Wijk, Klaas-Jan van; Hirst, Judy; Kunji, Edmund; Gier, Jan-Willem de

    2005-01-01

    We describe a generic, GFP-based pipeline for membrane protein overexpression and purification in Escherichia coli. We exemplify the use of the pipeline by the identification and characterization of E. coli YedZ, a new, membrane-integral flavocytochrome. The approach is scalable and suitable for hig

  15. Phosphatidylglycerol is involved in protein translocation across Escherichia coli inner membranes.

    NARCIS (Netherlands)

    T. de Vrije; R.L. de Swart (Rik); W. Dowhan; J. Tommassen (Jan); B. de Kruijff

    1988-01-01

    textabstractNewly synthesized proteins to be exported out of the cytoplasm of bacterial cells have to pass across the inner membrane. In Gram-negative bacteria ATP, a membrane potential, the products of the sec genes and leader peptidases (enzymes which cleave the N-terminal signal peptides of the p

  16. On the interaction between intrinsic proteins and phosphatidylglycerol in the membrane of Acholeplasma laidlawii

    NARCIS (Netherlands)

    Bevers, E.M.; Wang, H.H.; Kamp, J.A.F. op den; Deenen, L.L.M. van

    1979-01-01

    About 30% of the phosphatidylglycerol in oleic acid-enriched Acholeplasma laidlawii membranes are not hydrolyzed at temperatures below 10 °C by phospholipase A2 from porcine pancreas. Removal of 53% of the membrane proteins by proteolysis did not reduce the size of this inaccessible phosphatidylglyc

  17. Viroporins, Examples of the Two-Stage Membrane Protein Folding Model

    Directory of Open Access Journals (Sweden)

    Luis Martinez-Gil

    2015-06-01

    Full Text Available Viroporins are small, α-helical, hydrophobic virus encoded proteins, engineered to form homo-oligomeric hydrophilic pores in the host membrane. Viroporins participate in multiple steps of the viral life cycle, from entry to budding. As any other membrane protein, viroporins have to find the way to bury their hydrophobic regions into the lipid bilayer. Once within the membrane, the hydrophobic helices of viroporins interact with each other to form higher ordered structures required to correctly perform their porating activities. This two-step process resembles the two-stage model proposed for membrane protein folding by Engelman and Poppot. In this review we use the membrane protein folding model as a leading thread to analyze the mechanism and forces behind the membrane insertion and folding of viroporins. We start by describing the transmembrane segment architecture of viroporins, including the number and sequence characteristics of their membrane-spanning domains. Next, we connect the differences found among viroporin families to their viral genome organization, and finalize focusing on the pathways used by viroporins in their way to the membrane and on the transmembrane helix-helix interactions required to achieve proper folding and assembly.

  18. Membrane fusion induced by the major lipid-binding domain of the cytoskeletal protein talin

    NARCIS (Netherlands)

    Isenberg, G; Doerhoefer, S; Hoekstra, D; Goldmann, WH

    2002-01-01

    Secondary structure predictions have led to the identification of a major membrane-anchoring domain of the cytoskeletal protein talin spanning from amino acid 385 to 406. Using a synthetically derived peptide of this region, researchers have shown that it inserts into POPC/POPG phospholipid membrane

  19. Peptides and membrane fusion : Towards an understanding of the molecular mechanism of protein-induced fusion

    NARCIS (Netherlands)

    Pecheur, EI; Sainte-Marie, J; Bienvenue, A; Hoekstra, D

    1999-01-01

    Processes such as endo- or exocytosis, membrane recycling, fertilization and enveloped viruses infection require one or more critical membrane fusion reactions. A key feature in viral and cellular fusion phenomena is the involvement of specific fusion proteins. Among the few well-characterized fusio

  20. Changes in the anisotropy of oriented membrane dynamics induced by myelin basic protein

    Science.gov (United States)

    Natali, F.; Gliozzi, A.; Rolandi, R.; Relini, A.; Cavatorta, P.; Deriu, A.; Fasano, A.; Riccio, P.

    We report recent results showing the evidence of the effect induced by physiological amounts of myelin basic protein (MBP) on the dynamics of dimyristoyl L-a-phosphatidic acid (DMPA) membranes. Incoherent elastic neutron scattering scans, performed over a wide temperature range, have shown that the anisotropy of motions in oriented membranes is significantly enhanced by the presence of MBP.

  1. Changes in the anisotropy of oriented membrane dynamics induced by myelin basic protein

    Energy Technology Data Exchange (ETDEWEB)

    Natali, F. [OGG-INFM, Grenoble (France); Gliozzi, A.; Rolandi, R.; Relini, A. [Dipartimento di Fisica and Istituto Nazionale per la Fisica della Materia, Universita di Genova (Italy); Cavatorta, P.; Deriu, A. [Dipartimento di Fisica and Istituto Nazionale per la Fisica della Materia, Universita di Parma (Italy); Fasano, A. [Dipartimento di Biochimica e Biologia Molecolare, Universita di Bari (Italy); Riccio, P. [Dipartimento di Biologia D.B.A.F., Universita della Basilicata, Potenza (Italy)

    2002-07-01

    We report recent results showing the evidence of the effect induced by physiological amounts of myelin basic protein (MBP) on the dynamics of dimyristoyl L-a-phosphatidic acid (DMPA) membranes. Incoherent elastic neutron scattering scans, performed over a wide temperature range, have shown that the anisotropy of motions in oriented membranes is significantly enhanced by the presence of MBP. (orig.)

  2. MAL Is a Regulator of the Recruitment of Myelin Protein PLP to Membrane Microdomains

    NARCIS (Netherlands)

    Bijlard, Marjolein; de Jonge, Jenny C.; Klunder, Bert; Nomden, Anita; Hoekstra, Dick; Baron, Wia

    2016-01-01

    In oligodendrocytes (OLGs), an indirect, transcytotic pathway is mediating transport of de novo synthesized PLP, a major myelin specific protein, from the apical-like plasma membrane to the specialized basolateral-like myelin membrane to prevent its premature compaction. MAL is a well-known regulato

  3. Long Unfolded Linkers Facilitate Membrane Protein Import Through the Nuclear Pore Complex

    NARCIS (Netherlands)

    Meinema, Anne C.; Laba, Justyna K.; Hapsari, Rizqiya A.; Otten, Renee; Mulder, Frans A. A.; Kralt, Annemarie; van den Bogaart, Geert; Lusk, C. Patrick; Poolman, Bert; Veenhoff, Liesbeth M.

    2011-01-01

    Active nuclear import of soluble cargo involves transport factors that shuttle cargo through the nuclear pore complex (NPC) by binding to phenylalanine-glycine (FG) domains. How nuclear membrane proteins cross through the NPC to reach the inner membrane is presently unclear. We found that at least a

  4. Monitoring the native phosphorylation state of plasma membrane proteins from a single mouse cerebellum

    DEFF Research Database (Denmark)

    Schindler, J.; Ye, J. Y.; Jensen, Ole Nørregaard;

    2013-01-01

    Neuronal processing in the cerebellum involves the phosphorylation and dephosphorylation of various plasma membrane proteins such as AMPA or NMDA receptors. Despite the importance of changes in phosphorylation pattern, no global phospho-proteome analysis has yet been performed. As plasma membrane...

  5. Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling.

    Science.gov (United States)

    Larance, Mark; Kirkwood, Kathryn J; Tinti, Michele; Brenes Murillo, Alejandro; Ferguson, Michael A J; Lamond, Angus I

    2016-07-01

    We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754).

  6. Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling*

    Science.gov (United States)

    Larance, Mark; Kirkwood, Kathryn J.; Tinti, Michele; Brenes Murillo, Alejandro; Ferguson, Michael A. J.; Lamond, Angus I.

    2016-01-01

    We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754). PMID:27114452

  7. RNAi-mediated downregulation of poplar plasma membrane intrinsic proteins (PIPs) changes plasma membrane proteome composition and affects leaf physiology.

    Science.gov (United States)

    Bi, Zhen; Merl-Pham, Juliane; Uehlein, Norbert; Zimmer, Ina; Mühlhans, Stefanie; Aichler, Michaela; Walch, Axel Karl; Kaldenhoff, Ralf; Palme, Klaus; Schnitzler, Jörg-Peter; Block, Katja

    2015-10-14

    Plasma membrane intrinsic proteins (PIPs) are one subfamily of aquaporins that mediate the transmembrane transport of water. To reveal their function in poplar, we generated transgenic poplar plants in which the translation of PIP genes was downregulated by RNA interference investigated these plants with a comprehensive leaf plasma membrane proteome and physiome analysis. First, inhibition of PIP synthesis strongly altered the leaf plasma membrane protein composition. Strikingly, several signaling components and transporters involved in the regulation of stomatal movement were differentially regulated in transgenic poplars. Furthermore, hormonal crosstalk related to abscisic acid, auxin and brassinosteroids was altered, in addition to cell wall biosynthesis/cutinization, the organization of cellular structures and membrane trafficking. A physiological analysis confirmed the proteomic results. The leaves had wider opened stomata and higher net CO2 assimilation and transpiration rates as well as greater mesophyll conductance for CO2 (gm) and leaf hydraulic conductance (Kleaf). Based on these results, we conclude that PIP proteins not only play essential roles in whole leaf water and CO2 flux but have important roles in the regulation of stomatal movement. PMID:26248320

  8. Identification of chromatophore membrane protein complexes formed under different nitrogen availability conditions in Rhodospirillum rubrum

    DEFF Research Database (Denmark)

    Selao, Tiago Toscano; Branca, Rui; Chae, Pil Seok;

    2011-01-01

    The chromatophore membrane of the photosynthetic diazotroph Rhodospirillum rubrum is of vital importance for a number of central processes, including nitrogen fixation. Using a novel amphiphile, we have identified protein complexes present under different nitrogen availability conditions by the u...

  9. Stability and structure of the membrane protein transporter Ffh is modulated by substrates and lipids

    DEFF Research Database (Denmark)

    Reinau, Marika Ejby; Otzen, Daniel

    2009-01-01

    The cytosolic protein Ffh transports membrane proteins from the ribosome to the inner membrane in complex with 4.5S RNA. Here we show that native Ffh binds to the hydrophobic probe ANS in a 1 Ffh:3 ANS stoichiometry, revealing a hydrophobic binding site. Thermal precipitation of Ffh is shifted...... the apoprotein. Escherichia coli lipid and DOPG (and to a smaller extent DOPC) increase Ffh's α-helical content, possibly related to Ffh's role in guiding membrane proteins to the membrane. Binding is largely mediated by electrostatic interactions but does not protect Ffh against trypsinolysis. We conclude...... that Ffh is a structurally flexible and dynamic protein whose stability is significantly modulated by the environment. © 2009 Elsevier Inc. All rights reserved....

  10. Outer membrane proteins induced by iron deficiency in Anabaena sp.PCC 7120

    Institute of Scientific and Technical Information of China (English)

    Yanling Dong; Xudong Xu

    2009-01-01

    Iron deficiency can induce cyanobacteria to synthesize siderophore receptor proteins on the outer membrane to enhance the uptake of iron. In this study, an outer membrane of high purity was prepared from Anabaena sp. PCC 7120 based on aqueous polymer two-phase partitioning and discontinuous sucrose density ultra-centrifugation, and the induction of outer membrane proteins by iron deficiency was investigated using 2-D gel electrophoresis. At least five outer membrane proteins were newly synthesized or significantly up-regulated in cells transferred to iron-deficient conditions, which were all identified to be siderophore receptor proteins according to MALDI-TOF-MS analyses. Bacterial luciferase reporter genes luxAB were employed to monitor the transcription of the encoding genes. The genes were induced by iron deficiency at the transcriptional level in different responsive modes. Luciferase activity expressed from an iron-regulated promoter may be used as a bioreporter for utilizable iron in natural water samples.

  11. Novel Methods To Detect Membrane Proteins | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Institute on Aging's Laboratory of Cardiovascular Sciences is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize modules to aid the study of mammalian membrane proteins.

  12. A novel regulatory mechanism for whey acidic protein gene expression.

    OpenAIRE

    Chen, L.H.; Bissell, M J

    1989-01-01

    When primary mouse mammary epithelial cells (PMME) are cultured on a basement membrane type matrix, they undergo extensive morphogenesis leading to the formation of 3-dimensional alveoli-like spherical structures surrounding a closed lumen. We show for the first time that cells cultured on basement membrane-type matrix express high levels of whey acidic protein (WAP) mRNA and secrete the protein into the lumen. The expression of WAP appears to be dependent upon the formation of the alveoli-li...

  13. Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions.

    Science.gov (United States)

    Tsachaki, Maria; Birk, Julia; Egert, Aurélie; Odermatt, Alex

    2015-07-01

    Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide significant insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic co-factor pools and substrates. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a simple method for defining intracellular localization and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green-fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11β-hydroxysteroid dehydrogenase (11β-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The results revealed a luminal orientation of the catalytic site for three enzymes, i.e. 17β-HSD6, 7 and 12. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and on the role of the luminal co-factor pool.

  14. Association of lipids with integral membrane surface proteins of Mycoplasma hyorhinis

    International Nuclear Information System (INIS)

    Triton X-114 (TX-114)-phase fractionation was used to identify and characterize integral membrane surface proteins of the wall-less procaryote Mycoplasma hyorhinis GDL. Phase fractionation of mycoplasmas followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed selective partitioning of approximately 30 [35S]methionine-labeled intrinsic membrane proteins into the TX-114 phase. Similar analysis of [3H]palmitate-labeled cells showed that approximately 20 proteins of this organism were associated with lipid, all of which also efficiently partitioned as integral membrane components into the detergent phase. Immunoblotting and immunoprecipitation of TX-114-phase proteins from 125I-surface-labeled cells with four monoclonal antibodies to distinct surface epitopes of M. hyorhinis identified surface proteins p120, p70, p42, and p23 as intrinsic membrane components. Immunoprecipitation of [3H]palmitate-labeled TX-114-phase proteins further established that surface proteins p120, p70, and p23 (a molecule that mediates complement-dependent mycoplasmacidal monoclonal antibody activity) were among the lipid-associated proteins of this organism. Two of these proteins, p120 and p123, were acidic (pI less than or equal to 4.5), as shown by two-dimensional isoelectric focusing. This study established that M. hyorhinis contains an abundance of integral membrane proteins tightly associated with lipids and that many of these proteins are exposed at the external surface of the single limiting plasma membrane. Monoclonal antibodies are reported that will allow detailed analysis of the structure and processing of lipid-associated mycoplasma proteins

  15. Vibrio cholerae expresses iron-regulated outer membrane proteins in vivo.

    OpenAIRE

    Sciortino, C V; Finkelstein, R A

    1983-01-01

    A comparison was made, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of the outer membrane proteins of four strains of Vibrio cholerae grown in vivo in infant rabbits and in vitro in low-iron and iron-supplemented defined media. In vivo-grown V. cholerae expressed novel outer membrane-associated proteins which, in part, were similar to those observed on V. cholerae grown in vitro under conditions of iron deprivation.

  16. Analysis of lysosomal membrane proteins exposed to melanin in HeLa cells

    OpenAIRE

    Bang, Seung Hyuck; Park, Dong Jun; Kim, Yang-Hoon; Min, Jiho

    2016-01-01

    Objectives There have been developed to use targeting ability for antimicrobial, anticancerous, gene therapy and cosmetics through analysis of various membrane proteins isolated from cell organelles. Methods It was examined about the lysosomal membrane protein extracted from lysosome isolated from HeLa cell treated by 100 ppm melanin for 24 hours in order to find associated with targeting ability to melanin using by 2-dimensional electrophoresis. Results The result showed 14 up-regulated (1.5...

  17. Probing Membrane Protein Structure Using Water Polarization Transfer Solid-State NMR

    OpenAIRE

    Williams, Jonathan K.; Hong, Mei

    2014-01-01

    Water plays an essential role in the structure and function of proteins, lipid membranes and other biological macromolecules. Solid-state NMR heteronuclear-detected 1H polarization transfer from water to biomolecules is a versatile approach for studying water-protein, water-membrane, and water-carbohydrate interactions in biology. We review radiofrequency pulse sequences for measuring water polarization transfer to biomolecules, the mechanisms of polarization transfer, and the application of ...

  18. Transmembrane Protein Diffusion in Gel-Supported Dual-Leaflet Membranes

    OpenAIRE

    Wang, Chih-Ying; Hill, Reghan J.

    2014-01-01

    Tools to measure transmembrane-protein diffusion in lipid bilayer membranes have advanced in recent decades, providing a need for predictive theoretical models that account for interleaflet leaflet friction on tracer mobility. Here we address the fully three-dimensional flows driven by a (nonprotruding) transmembrane protein embedded in a dual-leaflet membrane that is supported above and below by soft porous supports (e.g., hydrogel or extracellular matrix), each of which has a prescribed per...

  19. Mapping lipid and detergent molecules at the surface of membrane proteins.

    Science.gov (United States)

    Cogdell, Richard J; Gardiner, Alastair T; Roszak, Aleksander W; Stončius, Sigitas; Kočovský, Pavel; Isaacs, Neil W

    2011-06-01

    Electron-density maps for the crystal structures of membrane proteins often show features suggesting binding of lipids and/or detergent molecules on the hydrophobic surface, but usually it is difficult to identify the bound molecules. In our studies, heavy-atom-labelled phospholipids and detergents have been used to unequivocally identify these binding sites at the surfaces of test membrane proteins, the reaction centres from Rhodobacter sphaeroides and Blastochloris viridis. The generality of this method is discussed in the present article.

  20. A simple detection method for low-affinity membrane protein interactions by baculoviral display.

    Directory of Open Access Journals (Sweden)

    Toshiko Sakihama

    Full Text Available BACKGROUND: Membrane protein interactions play an important role in cell-to-cell recognition in various biological activities such as in the immune or neural system. Nevertheless, there has remained the major obstacle of expression of the membrane proteins in their active form. Recently, we and other investigators found that functional membrane proteins express on baculovirus particles (budded virus, BV. In this study, we applied this BV display system to detect interaction between membrane proteins important for cell-to-cell interaction in immune system. METHODOLOGY/PRINCIPAL FINDINGS: We infected Sf9 cells with recombinant baculovirus encoding the T cell membrane protein CD2 or its ligand CD58 and recovered the BV. We detected specific interaction between CD2-displaying BV and CD58-displaying BV by an enzyme-linked immunosorbent assay (ELISA. Using this system, we also detected specific interaction between two other membrane receptor-ligand pairs, CD40-CD40 ligand (CD40L, and glucocorticoid-induced TNFR family-related protein (GITR-GITR ligand (GITRL. Furthermore, we observed specific binding of BV displaying CD58, CD40L, or GITRL to cells naturally expressing their respective receptors by flowcytometric analysis using anti-baculoviral gp64 antibody. Finally we isolated CD2 cDNA from a cDNA expression library by magnetic separation using CD58-displaying BV and anti-gp64 antibody. CONCLUSIONS: We found the BV display system worked effectively in the detection of the interaction of membrane proteins. Since various membrane proteins and their oligomeric complexes can be displayed on BV in the native form, this BV display system should prove highly useful in the search for natural ligands or to develop screening systems for therapeutic antibodies and/or compounds.