Membrane Protein Of Molecular Weight Research Articles

Validating Antibodies for Quantitative Western Blot Measurements with Microwestern Array

Fluorescence-based western blots are quantitative in principal, but require determining linear range for each antibody. Here, we use microwestern array to rapidly evaluate suitable conditions for quantitative western blotting, with up to 192 antibody/dilution/replicate combinations on a single standard size gel with a seven-point, two-fold lysate dilution series (~100-fold range). Pilot experiments demonstrate a high proportion of investigated antibodies (17/24) are suitable for quantitative use; however this sample of antibodies is not yet comprehensive across companies, molecular weights, and other important antibody properties, so the ubiquity of this property cannot yet be determined. In some cases microwestern struggled with higher molecular weight membrane proteins, so the technique may not be uniformly applicable to all validation tasks. Linear range for all validated antibodies is at least 8-fold, and up to two orders of magnitude. Phospho-specific and total antibodies do not have discernable trend differences in linear range or limit of detection. Total antibodies generally required higher working concentrations, but more comprehensive antibody panels are required to better establish whether this trend is general or not. Importantly, we demonstrate that results from microwestern analyses scale to normal “macro” western for a subset of antibodies.

The role of the AMOP domain in MUC4/Y-promoted tumour angiogenesis and metastasis in pancreatic cancer.

BackgroundMUC4 is a high molecular weight membrane protein that is overexpressed in pancreatic cancer (PC) and is associated with the development and progression of this disease. However, the exact mechanisms through which MUC4 domains promote these biological processes have rarely been studied, partly because of its high molecular weight, difficulty to overexpress it. Here, we use MUC4/Y, one of the MUC4 transcript variants, as a model molecule to investigate the AMOP-domain of MUC4(MUC/Y).MethodsWe used cell proliferation, migration, invasion and tube formation assays in vitro to explore the abilities of AMOP domain in PC. In vivo, the matrigel plug assay, orthotopic implantation and Kaplan-Meier survival curves were used to check the results we observed in vitro. Finally, we discovered the underlying mechanism through western blot and immunofluorescence.ResultsWe found that MUC4/Y overexpression could enhance the angiogenic and metastatic properties of PC cells, both in vitro and in vivo. However, the deletion of AMOP domain could cutback these phenomena. Additionally, Kaplan-Meier survival curves showed that mice injected with MUC4/Y overexpressed cells had shorter survival time, compared with empty-vector-transfected cells (MUC4/Y-EV), or cells expressing MUC4/Y without the AMOP domain (MUC4/Y-AMOP△). Our data also showed that overexpression of MUC4/Y could activate NOTCH3 signaling, increasing the expression of downstream genes: VEGF-A, MMP-9 and ANG-2.ConclusionsThe AMOP domain had an important role in MUC4/Y (MUC4)-mediated tumour angiogenesis and metastasis of PC cells; and the NOTCH3 signaling was involved. These findings provided new insights into PC therapies. Our study also supplies a new method to study other high molecular membrane proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0369-0) contains supplementary material, which is available to authorized users.

Synthesis of membrane proteins in eukaryotic cell‐free systems

Cell‐free protein synthesis (CFPS) is a valuable method for the fast expression of difficult‐to‐express proteins as well as posttranslationally modified proteins. Since cell‐free systems circumvent possible cytotoxic effects caused by protein overexpression in living cells, they significantly enlarge the scale and variety of proteins that can be characterized. We demonstrate the high potential of eukaryotic CFPS to express various types of membrane proteins covering a broad range of structurally and functionally diverse proteins. Our eukaryotic cell‐free translation systems are capable to provide high molecular weight membrane proteins, fluorescent‐labeled membrane proteins, as well as posttranslationally modified proteins for further downstream analysis.

The effect of pds gene silencing on chloroplast pigment composition, thylakoid membrane structure and photosynthesis efficiency in tobacco plants

Nicotiana tabacum plants that are silenced in pds gene have been constructed by RNAi technology. Transgenics showed different degrees of albinism and PSII efficiency was decreased significantly compared to wild type plants. The total carotenoid contents decreased. The ratio of chlorophyll (Chl) a to b was unchanged while the content of both declined. The grana stacking of thylakoid in variegation part was as normal as in the control and etioplast occurred in the white sector of leaf. The concentration of thylakoid membrane protein of molecular weight about 20 kDa was significantly increased compared to wild type plants according to the same contents of Chl. These observations suggest the exact matching of pigments and proteins is important for energy absorption and transfer in photosynthesis and mismatching of them could cause malfunction of PSII.

Reflections

CNS white matter from higher vertebrates and cultured differentiated oligodendrocytes are nonpermissive substrates for neurite growth and fibroblast spreading. Membrane proteins of 35 kd and 250 kd with highly nonpermissive substrate properties could be extracted from CNS myelin fractions. Monoclonal antibodies were raised against these proteins: IN-1 and IN-2 bound both to the 35 kd and 250 kd inhibitors and to the surface to differentiated cultured oligodendrocytes. Adsorption of nonpermissive CNS myelin or nonpermissive oligodendrocytes with either antibody markedly improved their substrate properties. Optic nerve explants injected with IN-1 or IN-2 allowed axon ingrowth of cocultured sensory and sympathetic neurons. We conclude that the nonpermissive substrate properties of CNS white matter are due to these membrane proteins on the surface of differentiated oligodendrocytes and to their in vivo product, myelin.

Progress of Drug Transport Study Based on Absolute Quantitative Method for Membrane Transporter Proteins

Knowing the amount of membrane transporter expression in human tissue is one of the key issues in the rational and reliable prediction of pharmacokinetic profiles in humans. Recently, we have developed a simultaneous and highly sensitive method for the absolute quantification of multiple membrane transporter proteins in mammalian tissues. To develop quantitative analysis of high molecular-weight membrane proteins, we have solved problems using proteomics technology as follows: 1) The target proteins are detected via tryptic peptides that can be dissolved and analyzed with LC-MS/MS, while membrane protein is difficult to dissolve. 2) LC-MS/MS in multiple reaction monitoring (MRM) mode produce a highly sensitive and selective response for transporter proteins with low expression by separation from highly abundant molecules. 3) Analyte specificity for each peptide was demonstrated in amino acid sequences using multiple MRM detection. Selection of the peptide probe was very important for highly sensitive analysis with LC-MS/MS. We set criteria for peptide selection using an informatics approach. Peptides without unstable residue, double basic residues, and integral membrane domain were selected as useful probes. The developed method will provide an inclusive assay platform for all transporter proteins expressed in both animal/human tissues and will contribute to progress in drug discovery and development.

At the frontline of Alzheimer’s disease treatment: γ-secretase inhibitor/modulator mechanism

Genetic and biological studies provide evidence that the production and deposition of amyloid-beta peptides (Abeta) contribute to the etiology of Alzheimer's disease. beta- and gamma-secretases, which are responsible for the generation of Abeta, are plausible molecular targets for Alzheimer's disease treatment. gamma-Secretase is an unusual aspartic protease that cleaves the scissile bond within the transmembrane domain. This unusual enzyme is composed of a high molecular weight membrane protein complex containing presenilin, nicastrin, Aph-1 and Pen-2. Drugs that regulate the production of Abeta by inhibiting or modulating gamma-secretase activity could provide a disease-modifying effect on Alzheimer's disease, although recent studies suggest that gamma-secretase plays important roles in cellular signaling including Notch. Thus, understanding the molecular mechanism whereby gamma-secretase recognizes and cleaves its substrate is a critical issue for the development of compounds that specifically regulate Abeta-generating gamma-secretase activity. This review focuses on the structure and function relationship of gamma-secretase complex and the mode of action of the gamma-secretase inhibitors.

Interaction of Bartonella henselae with Endothelial Cells Promotes Monocyte/Macrophage Chemoattractant Protein 1 Gene Expression and Protein Production and Triggers Monocyte Migration

Bacillary angiomatosis (BA), one of the many clinical manifestations resulting from infection with the facultative intracellular bacterium Bartonella henselae, is characterized by angiogenic lesions. Macrophages have been identified as important effector cells contributing to the angiogenic process during B. henselae infection by infiltrating BA lesions and secreting vascular endothelial growth factor. Monocyte-macrophage chemoattractant protein 1 (MCP-1) recruits macrophages to sites of inflammation. In this study, we investigated the ability of B. henselae to upregulate MCP-1 gene expression and protein production in the human microvascular endothelial cell line HMEC-1. MCP-1 mRNA was induced at 6 and 24 h after treatment with bacteria, whereas protein production was elevated at 6, 24, and 48 h. This induction was not dependent on the presence of bacterial lipopolysaccharide or endothelial cell toll-like receptor 4. However, MCP-1 production was dependent on NF-kappaB activity. Outer membrane proteins of low molecular weight were able to upregulate MCP-1 production. Furthermore, supernatants from B. henselae-infected HMEC-1 were able to induce chemotaxis of THP-1 monocytes. These data suggest a mechanism by which the macrophage effector cell is recruited to the endothelium during B. henselae infection and then contributes to bacterial-induced angiogenesis.

Intracellular Membrane Localization of Pseudomonas ExoS and Yersinia YopE in Mammalian Cells

ExoS (453 amino acids) is a bi-functional type-III cytotoxin of Pseudomonas aeruginosa. Residues 96-233 comprise the Rho GTPase-activating protein (Rho GAP) domain, while residues 234-453 comprise the 14-3-3-dependent ADP-ribosyltransferase domain. Residues 51-72 represent a membrane localization domain (MLD), which targets ExoS to perinuclear vesicles within mammalian cells. YopE (219 amino acids) is a type-III cytotoxin of Yersinia that is also a Rho GAP. Residues 96-219 comprise the YopE Rho GAP domain. While the Rho GAP domains of ExoS and YopE share structural homology, unlike ExoS, the intracellular localization of YopE within mammalian cells has not been resolved and is the subject of this investigation. Deletion mapping showed that the N terminus of YopE was required for intracellular membrane localization of YopE in CHO cells. A fusion protein containing the N-terminal 84 amino acids of YopE localized to a punctate-perinuclear region in mammalian cells and co-localized with a fusion protein containing the MLD of ExoS. Residues 54-75 of YopE (termed YopE-MLD) were necessary and sufficient for intracellular localization in mammalian cells. The YopE-MLD localized ExoS to intracellular membranes and targeted ExoS to ADP-ribosylate small molecular weight membrane proteins as observed for native type-III delivered ExoS. These data indicate that the YopE MLD functionally complements the ExoS MLD for intracellular targeting in mammalian cells.

Overexpression and purification of recombinant membrane PsbH protein in Escherichia coli

In this work, we featured an expression system that enables the production of sufficient quantities (∼mg) of low molecular weight membrane protein of photosystem II, PsbH protein, for solid-state NMR as well as other biophysical studies. PsbH gene from cyanobacterium Synechocystis sp. PCC 6803 was cloned into a plasmid expression vector, which allowed expression of the PsbH protein as a glutathione- S transferase (GST) fusion protein in Escherichia coli BL21(DE3) cells. A relatively large GST anchor overcomes foreseeable problems with the low solubility of membrane proteins and the toxicity caused by protein incorporation into the membrane of the host organism. As a result, the majority of fusion protein was obtained in a soluble state and could be purified from crude bacterial lysate by affinity chromatography on immobilized glutathione under non-denaturing conditions. The PsbH protein was cleaved from the carrier protein with Factor Xa protease and purified on DEAE–cellulose column with yields of up to 2.1 μg protein/ml of bacterial culture. The procedure as we optimized is applicable for isolation of small membrane proteins for structural studies.

Identification of Salmonella enteritidis outer membrane proteins expressed during attachment to human intestinal epithelial cells.

To express and identify the Salmonella enterica ser. Enteritidis outer membrane proteins that are involved in the attachment to human intestinal epithelial cells. Salmonella Enteritidis attachment proteins were expressed by infection of human intestinal epithelial cells (int-407) with Salm. Enteritidis strain SE28, followed by pulse labelling with [35S]-methionine. Outer membrane proteins were separated by TritonX-114 phase partitioning, and were detected by Salm. Enteritidis-specific polyclonal antibody. Outer membrane proteins of molecular weights 82.3, 75.6, 49.3, 35.5 and 19.3 kDa were newly synthesized. Expression of 31.2 and 16 kDa proteins was up-regulated, whereas the expression of 40.7 kDa protein was down-regulated. Polyclonal antibodies against the 82.3 and 75.6 kDa proteins significantly (P < 0.05) reduced the binding of Salm. Enteritidis to int-407 cells in vitro. Outer membrane proteins 82.3 and 75.6 kDa are potentially involved in the attachment of Salm. Enteritidis to the intestinal mucosa. Outer membrane proteins 82.3 and 75.6 kDa identified in this study could be used as potential vaccines to block or reduce Salm. Enteritidis colonization in chickens.

Isolation of monoclonal antibodies that inhibit the binding of infectious bursal disease virus to LSCC-BK3 cells.

Three hybridoma cell lines producing monoclonal antibodies (MAbs) against LSCC-BK3 cells which are susceptible to infectious bursal disease virus (IBDV) infection were produced and characterized. The MAbs, designated T7, Q11 and Q13, inhibited the attachment of IBDV to LSCC-BK3 cells. Furthermore, these MAbs bound to LSCC-BK3 but not to nonpermissive cells in flow cytometry. MAb T7 detected a 110-kDa membrane protein of LSCC-BK3 cells, whereas Q11 and Q13 reacted with membrane proteins of molecular weights 58-, 85-, 90- and 110-kDa. These observations imply that the 110-kDa protein recognized by all the MAbs is associated with IBDV binding. The MAbs established in this study are useful for studying the interaction between IBDV and its target cell.

Flagellar Membrane Proteins of Tetraselmis striata Butcher (Chlorophyta)

Highly purified flagella of the green alga Tetraselmis striata (Chlorophyta) were extracted by Triton X-114 phase partitioning. SDS-PAGE analysis revealed that most proteins were present in the aqueous phase, only two prominent flagellar membrane proteins (fmp) of apparent molecular weight 145 and 57 kDa (fmp145 and fmp57) were enriched in the detergent phase. Fmp145 was purified by gel permeation chromatography. Glycosidase treatment in combination with lectin blot analysis showed that fmp145 is a glycoprotein containing 3-5 N-glycans of the high mannose and/or hybrid type. A polyclonal antibody (anti-fmp136) was raised against the deglycosylated form of fmp145 and used to localize fmp145 by immunofluorescence and immunoelectron microscopy. Immunogold labeling showed fmp145 to be present between the scale layers and the flagellar membrane. During flagellar regeneration fmp145 is incorporated evenly and rapidly into the newly developing flagella. Anti-fmp136 specifically cross-reacted with flagella of only a subgroup of Tetraselmis strains characterized by a specific flagellar hair type (type II according to Marin et al. 1993) and thus could be a useful immunomarker for the identification of Tetraselmis strains by fluorescence microscopy.

An immunological strategy To monitor In situ the phosphate starvation state in thiobacillus ferrooxidans

Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. During the process of ore bioleaching, the microorganisms are subjected to several stressing conditions, including the lack of some essential nutrients, which can affect the rates and yields of bioleaching. When T. ferrooxidans is starved for phosphate, the cells respond by inducing the synthesis of several proteins, some of which are outer membrane proteins of high molecular weight (70,000 to 80,000). These proteins were considered to be potential markers of the phosphate starvation state of these microorganisms. We developed a single-cell immunofluorescence assay that allowed monitoring of the phosphate starvation condition of this biomining microorganism by measuring the increased expression of the surface proteins. In the presence of low levels of arsenate (2 mM), the growth of phosphate-starved T. ferrooxidans cells was greatly inhibited compared to that of control nonstarved cells. Therefore, the determination of the phosphorus nutritional state is particularly relevant when arsenic compounds are solubilized during the bioleaching of different ores.

Molecules inhibiting neurite growth: a minireview.

Molecules and activities which repulse growing neurites or induce growth cone collapse and long-lasting growth inhibition have been defined over the last 10 years. Recently, specific guidance roles for developing axons and pathways could be associated with such repulsive effects. A high molecular weight membrane protein located in CNS myelin acts as potent neurite growth inhibitor and may play a role as a negative control element for sprouting, neurite growth and regeneration, and for the plasticity of the adult CNS. Interestingly, some guidance molecules can have positive, growth-promoting as well as negative, repulsive effects for specific types of neurons. These results underline the complex mechanisms involved in neurite guidance which depends on the interpretation of combinations of incoming signals by particular growth cones.

Iron-regulated outer membrane proteins of Aeromonas salmonicida are important protective antigens in Atlantic salmon against furunculosis

Four iron-regulated outer membrane proteins (IROMPs) of molecular weight 82, 77, 72 and 70 kilodaltons (kDa) were identified in 18 isolates of Aeromonas salmonicida grown in the presence of the synthetic iron chelators ethylene diamine di ( o-hydroxyphenylacetic acid) (EDDA) or 2,2′-dipyridyl. The IROMPs were shown to be immunogenic in Atlantic salmon by immunoblotting techniques. Reaction of immunoblots with a monospecific rabbit anti-IROMP antiserum indicated that the IROMPs were present and immunologically cross-reactive in each of 18 isolates of A. salmonicida most of which were obtained from Atlantic salmon with furunculosis. Outer membrane proteins (OMPs) of A. salmonicida grown in the presence of 2,2′-dipyridyl to induce IROMPs were evaluated for their ability to actively immunise Atlantic salmon. Compared with OMPs prepared from A. salmonicida grown in iron-replete media, IROMP preparations conferred significant protection against natural and experimental heterologous A. salmonicida challenge. Passive immunisation of Atlantic salmon with Atlantic salmon antisera containing antibodies to IROMPs and monospecific rabbit anti-IROMP antiserum or affinity purified immunoglobulin G (IgG), conferred significant protection against a heterologous A. salmonicida bath challenge. Both salmon antisera raised to IROMP preparations and the monospecific rabbit anti-IROMP antiserum were bactericidal for avirulent (A-layer negative) and virulent (A-layer positive) strains of A. salmonicida in vitro. The results indicate that IROMPs of A. salmonicida represent important protective antigens against furunculosis.

36] Peroxidative activities of hemoglobin and hemoglobin derivatives

Publisher Summary This chapter discusses the peroxidative activities of hemoglobin and hemoglobin derivatives. Methemoglobin and metmyoglobin can react with preexisting lipid hydroperoxides to yield lipid-associated free radicals, which resulted in the propagation of free radical-mediated reactions that produced extensive lipid peroxidation. Methemoglobin and metmyoglobin can initiate the peroxidation of polyunsaturated fatty acids. Substantial membrane lipid peroxidation for model cells composed of hemoglobin entrapped in chemically defined liposomes. Human erythrocytes are also shown to be susceptible to hemoglobin-catalyzed, H 2 O 2 -dependent lipid peroxidation. Additionally, lipids, proteins, and carbohydrates are also susceptible to oxidative degradation by hemoglobin. Thus, the addition of H 2 O 2 to erythrocytes or erythrocyte membranes produces high molecular weight membrane protein aggregates, whereas deoxyribose is readily degraded by H 2 O 2 in the presence of hemoglobin. The mechanism by which hemoglobin exerts its peroxidative activity is similar to that of the peroxidases, in which hydrogen peroxide first reacts with the native enzyme and withdraws two electrons.

A Novel Antigen (H47 Ag) on Human Lymphocytes Involved in T Cell Activation

Surface molecules involved in human T cell activation were investigated using a newly developed monoclonal antibody (H47 mAb). H47 antigen (Ag) recognized by H47 mAb was expressed on approximately 10% of resting T cells (mostly CD4 -CD8 +), 30% of phorbol 12-myristate 13-acetate (PMA)-activated T cells (both CD4 +CD8 - and CD4 -CD8 +), and most NK, B cells, and monocytes in the peripheral blood mononuclear cells (PBMC). H47 mAb respectively immunoprecipdated a 100 or 120-kD molecular weight (MW) membrane protein of T cells and monocytes under nonreducing or reducing conditions, suggesting that H47 Ag consists of a single polypeptide that has intramolecular disulfide bonds. H47 mAb significantly enhanced PMA-induced proliferation of PBMC in a monocyte-independent fashion. H47 mAb, however, failed to enhance T cell proliferation induced by anti-CD3 mAb, anti-CD2 mAb, or phytohemagglutinin (PHA). H47 mAb also enhanced PMA-induced interleukin-2 receptor (IL-2R) expression and IL-2 synthesis, but did not induce a change in intracellular free calcium ([Ca 2+] i) of T cells. These results suggest that H47 Ag is a new membrane molecule involved in PMA-induced T cell activation.

A Monoclonal Antibody (H227) Recognizing a New Epitope of 4F2 Molecular Complex Associated with T Cell Activation

We developed and characterized a monoclonal antibody (mAb), H227, recognizing a new epitope of human 4F2 molecular complex whose roles remain to be identified. Both staining patterns and molecular sizes recognized by H227 mAb were the same to those of 4F2 mAb. Thus, both H227 and 4F2 mAbs were reactive to monocytes, thymocytes, and activated lymphocytes and immunoprecipitated the 125-kDa molecular weight membrane protein under nonreducing conditions and 85-kDa heavy-chain and 40-kDa light-chain proteins under reducing conditions. These two mAbs immunoprecipitated only the 85-kDa heavy-chain protein when the cell lysate was initially treated with dithiothreitol. Sequential immunoprecipitation proved their cross-reactivity. Both the mAbs inhibited phytohemagglutinin- or concanavalin A-induced proliferation of peripheral blood mononuclear cells (PBMC). In contrast to these similarities, the pretreatment of cells with one mAb failed to block the reactivity to the other, suggesting that their epitopes were different from each other. Furthermore, only H227 mAb augmented phorbol myrislate acetate (PMA)-induced PBMC proliferation in association with increase in interleukin 2 receptor expression. In summary, H227 mAb recognizes a new epitope of 4F2 heavy chain that might be involved in the PMA-induced T cell activation pathway and therefore shall be a useful tool for understanding the roles of the 4F2 molecular complex.

Control of the calcium pump of cardiac sarcoplasmic reticulum. A specific role for the pentameric structure of phospholamban?

Intracellular sources of calcium provide most of that required for cardiac muscle contraction. Regulation of the filling of these calcium stores by control of the enzyme responsible for calcium uptake, the sarcoplasmic reticular calcium pump, provides a mechanism by which the contractile properties of the heart can be adapted. In fact, this single biochemical event accounts for most of the changes in calcium handling associated with beta adrenergic stimulation. The calcium pump of cardiac sarcoplasmic reticulum is regulated by the phosphorylation status of a second sarcoplasmic reticular component, phospholamban. This is a low molecular weight membrane protein which assembles as an oligomer (homopentamer) and inhibits pump function by physical interaction with a "regulatory" motif on the pump. This interaction (and the state of inhibition) is broken following phosphorylation of phospholamban by cAMP-, cGMP, or Ca2+/calmodulin dependent protein kinases. This model of pump control shares a number of features associated with control of the plasma membrane calcium pump; however, the oligomeric arrangement of the regulator is specific for the sarcoplasmic reticular ATPase. The functional significance of the oligomer is currently unknown, but this format may provide functional advantages such as (1) offering an effective substrate structure for kinase/phosphatase, promoting rapid changes in phosphate status; (2) facilitating intrapentamer cooperation, translating low stoichiometric phosphorylation into large changes in pump function; or (3) coordinating multiple pump units around a single pentamer thus restricting conformational freedom and suppressing enzyme activity.