The IA-2 interactome

Aims/hypothesis Islet antigen-2 (IA-2), a major autoantigen in type 1 diabetes, is an enzymatically inactive member of the transmembrane protein tyrosine phosphatase (PTP) family. IA-2 is located in dense-core secretory vesicles and is involved in the regulation of insulin secretion. The present experiments were initiated to identify those proteins that interact with IA-2 (i.e. the IA-2 interactome) as a first step towards elucidating the mechanism(s) by which IA-2 influences insulin secretion and serves as an autoantigen. Materials and methods To determine the proteins with which IA-2 interacts, a yeast two-hybrid system was used to screen a human foetal library, and deletion mutants were used to determine the binding sites. Positive interactions were confirmed by immunoprecipitation pull-down experiments using cell lysate from transfected mammalian cell lines. Results Six new interacting proteins were identified by this approach: mitogen-activated protein kinase-activating death domain (MADD), the MADD isoform IG20, PTPρ, PTPσ, sorting nexin 19 (SNX19) and cyclophilin A. Using a series of IA-2 deletion mutants, we identified the regions on the IA-2 molecule to which five of the interacting proteins bound. Amino acids 744–979 of IA-2 were required for the maximum binding of MADD, IG20 and SNX19, whereas amino acids 602–907 of IA-2 were required for the maximum binding of PTPρ and PTPσ. Pull-down experiments with cell lysate from transfected mammalian cells confirmed the binding of the interacting proteins to IA-2. Conclusions/interpretation The IA-2 interactome based on, pull-down experiments, currently consists of 12 proteins. The identification of these interacting proteins provides clues as to how IA-2 exerts its biological functions.     

Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator.

The yeast two-hybrid system was used to identify proteins that interact with Ras. The H-Ras protein was found to interact with a guanine nucleotide dissociation stimulator (GDS) that has been previously shown to regulate guanine nucleotide exchange on another member of the Ras protein family, Ral. The interaction is mediated by the C-terminal, noncatalytic segment of the RalGDS and can be detected both in vivo, using the two-hybrid system, and in vitro, with purified recombinant proteins. The interaction of the RalGDS C-terminal segment with Ras is specific, dependent on activation of Ras by GTP, and blocked by a mutation that affects Ras effector function. These characteristics are similar to those previously demonstrated for the interaction between Ras and its putative effector, Raf, suggesting that the RalGDS may also be a Ras effector. Consistent with this idea, the RalGDS was found to inhibit the binding of Raf to Ras.     

Expression of tight and adherens junction proteins in ulcerative colitis associated colorectal carcinoma: upregulation of claudin-1, claudin-3, claudin-4, and β-catenin

Background  Tight junction (TJ) proteins play a critical role in cellular adhesion, glandular differentiation, and cellular proliferation. The function of these proteins is compromised in a number of intestinal diseases, including ulcerative colitis that has an increased incidence for colorectal carcinoma (CAC). The aim of this study was to determine the expression of TJ proteins, claudin-1–4, occludin, ZO-1, and the adherens junction (AJ) protein β-catenin in CAC. Methods  Sixteen colectomy specimens with CAC, adjoining intraepithelial neoplasia, and normal mucosa were studied by immunofluorescence. A semiquantitative evaluation of all investigated proteins was performed by scoring the staining intensity, and the TJ and AJ protein expression in neoplastic cells was compared to normal and intraepithelial neoplastic colonic mucosa. Results  Using an intensity scoring system, mucosa of crypts and surfaces of CAC exhibited significantly elevated expression levels of claudin-1, claudin-3, claudin-4, and β-catenin compared to intraepithelial neoplasia and normal mucosa (p < 0.05). These data were confirmed by a comparative score. The expression of claudin-2, occludin, and ZO-1 showed no differences between the groups. Conclusion  TJ proteins claudin-1, claudin-3, claudin-4, and the AJ protein β-catenin are overexpressed in CAC. This suggests that these proteins may become potential markers and targets in CAC. Grant:  Supported by a grant from the Deutsche Forschungsgemeinschaft (BR 2093/4-1).     

NACA as a Potential Cellular Target of Hepatitis B Virus PreS1 Protein

The mechanisms of the attachment and penetration of hepatitis B virus remain obscure. It has been demonstrated that the preS1 region is essential for viral assembly and infectivity, however, as its cellular receptor has still not been identified unequivocally, we used a yeast two-hybrid system to screen the cellular proteins that can interact with preS1 protein. The protein recovered from a human liver cDNA library was nascent polypeptide-associated complex α polypeptide. The interaction between preS1 and nascent polypeptide-associated complex α polypeptide was verified by mating experiment and coimmunoprecipitation of COS7 cell lysates expressing both proteins. Based on these results, we speculate that nascent polypeptide-associated complex α polypeptide is a functional target of hepatitis B virus preS1 protein in cells.This work was supported by Grant C0310018 from the Natural Science Foundation of Fujian Province.Dan Li and Xiao Zhong Wang contributed equally to this work.     

Lactogen enhances Nb2 cell GTPase activity after 4 hours incubation

The lactogen receptor has been suggested to associate with one or more G proteins despite the absence of a 7-transmembrane spanning sequence. These studies were designed to determine whether lactogens acutely increase GTP binding to or GTPase activity in Nb2 cell membrane. Incubation of Nb2 cell membrane with either ovine PRL (10 ng/ml) or diluent for 0–1 h resulted in a decrease in total³⁵S-GTP binding to both with no difference in GTP binding between PRL- and diluent-treated membranes. There was also no change in³⁵S-GTP binding to Nb2 cell membrane incubated with increasing oPRL concentrations (0.001–100 ng/ml) for 60 min. α-³²P-GTP photoaffinity labelling was used to evaluate changes in GTP binding to specific G proteins. Photoaffinity labelling of α-³²P-GTP to no G protein was changed after preincubation with oPRL (10 ng/ml) for 0–60 min or with oPRL (0.01–10 ng/ml) for 60 min. Finally, it was determined whether oPRL had any acute effect on GTPase activity, as determined by release of³²Pi from γ-³²P-GTP. When Nb2 cell membrane was preincubated for 0–60 min with oPRL (10 ng/ml) or a range of oPRL concentrations (0–10 ng/ml), no change in GTPase activity was observed. However, when Nb2 cells were incubated with lactogen for 0–7 h, GTPase activity in equal quantities of Nb2 cell membrane prepared from those cells increased over time. Increased GTPase activity (64.9–74.4%;P<0.03 compared to 0 h) was observed after 4–7 h incubation with lactogen. In summary, addition of lactogen to Nb2 cell membrane did not acutely increase either GTP binding or GTPase activity. Yet when Nb2 cells were incubated with lactogen for 4 h prior to preparation of membrane, GTPase activity was significantly increased. This evidence, in addition to our previous results showing that 4 h incubation with lactogen increased G protein β subunit concentration and pertussis toxin-stimulated ADP-ribosylation of Gi, support a role for delayed lactogen modulation of one or more G proteins in the Nb2 cell, requiring at least 4 h for maximal effect.     

酵母双杂交系统在痘病毒与宿主相互作用中的研究进展

痘病毒全基因组序列分析为痘病毒的研究提供了丰富的信息。近年来,一些痘病毒编码的蛋白之间,以及病毒蛋白与宿主蛋白之间的相互作用的研究证明其在痘病毒复制、感染、致病及传播过程中起关键作用,因此受到了广泛的关注。酵母双杂交系统是研究蛋白质之间相互作用的重要工具,该技术可以发现新蛋白之间的相互作用,确定蛋白相互作用的区域,从而为研究各蛋白质的生物学功能以及蛋白质之间相互作用的分子机制奠定基础。本文综述了酵母双杂交系统在痘病毒蛋白之间及病毒-宿主相互作用的研究现状及优缺点。     

Protein homeostasis and molecular chaperones in aging

Molecular chaperones are ubiquitous, highly conserved proteins responsible for the maintenance of protein folding homeostasis in cells. Environmental stress causes proteotoxic damage, which triggers chaperone induction and the subsequent reparation of cellular damage by chaperones, including disposing irreparable protein ensembles. We summarize the current view of protein damage, turnover, the stress response and chaperone function in aging, and review novel data showing that accumulation of misfolded proteins outcompete and overload the limited resources of the protein folding, maintenance and turnover system, compromising general protein homeastasis and cellular function. Possible involvement of chaperones and proteolysis in immunosenescence is highlighted. Defects in zinc metabolism are also addressed in relation to aging and changes in chaperone levels.Presented at the ZincAge Conference, Madrid, February 10–13, 2006     

Tight binding of proteins to membranes from older human cells

The lens is an ideal model system for the study of macromolecular aging and its consequences for cellular function, since there is no turnover of lens fibre cells. To examine biochemical processes that take place in the lens and that may also occur in other long-lived cells, membranes were isolated from defined regions of human lenses that are synthesised at different times during life, and assayed for the presence of tightly bound cytosolic proteins using quantitative iTRAQ proteomics technology. A majority of lens beta crystallins and all gamma crystallins became increasingly membrane bound with age, however, the chaperone proteins alpha A and alpha B crystallin, as well as the thermally-stable protein, βB2 crystallin, did not. Other proteins such as brain-associated signal protein 1 and paralemmin 1 became less tightly bound in the older regions of the lens. It is evident that protein–membrane interactions change significantly with age. Selected proteins that were formerly cytosolic become increasingly tightly bound to cell membranes with age and are not removed even by treatment with 7 M urea. It is likely that such processes reflect polypeptide denaturation over time and the untoward binding of proteins to membranes may alter membrane properties and contribute to impairment of communication between older cells.     

Protein Content in Diabetes Nutrition Plan

Medical nutrition therapy plays a major role in diabetes management. Macronutrient composition has been debated for a long time. However, there is increasing evidence that a modest increase in dietary protein intake above the current recommendation is a valid option toward better diabetes control, weight reduction, and improvement in blood pressure, lipid profile, and markers of inflammation. Increasing the absolute protein intake to 1.5–2 g/kg (or 20–30% of total caloric intake) during weight reduction has been suggested for overweight and obese patients with type 2 diabetes and normal kidney function. Increased protein intake does not increase plasma glucose, but increases the insulin response and results in a significant reduction in hemoglobin A_1c. In addition, a higher dietary protein intake reduces hunger, improves satiety, increases thermogenesis, and limits lean muscle mass loss during weight reduction using a reduced calorie diet and increased physical activity. It is preferable to calculate protein intake for patients with diabetes as grams per kilogram of body weight and not as a fixed percentage of total energy intake to avoid protein malnutrition when a hypocaloric diet is used. The relationship between protein intake as grams per kilogram of body weight and albumin excretion rate is very weak, except in hypertensive patients and particularly in those with uncontrolled diabetes. A protein intake of 0.8–1 g/kg should be recommended only for patients with diabetes and chronic kidney disease. Other patients with diabetes should not reduce protein intake to less than 1 g/kg of body weight. This review discusses the effects of different amounts of protein intake in a diabetes meal plan. It particular, it discusses the effects of protein intake on renal function, the effects of protein content on diabetes control, and the effects of increased dietary protein on body weight.     

Upregulated Expression of hITF in Crohn’s Disease and Screening of hITF Interactant by a Yeast Two-Hybrid System

Aims  

To study the expression of human intestinal trefoil factor (hITF) mRNA in Crohn’s disease and to screen the cellular proteins that can interact with the hITF protein by a yeast two-hybrid system in order to explore the mechanism of hITF in protecting intestinal mucosa from injury.     

A data-mining approach to rank candidate protein-binding partners—The case of biogenesis of lysosome-related organelles complex-1 (BLOC-1)

Summary  The study of protein–protein interactions is a powerful approach to uncovering the molecular function of gene products associated with human disease. Protein–protein interaction data are accumulating at an unprecedented pace owing to interactomics projects, although it has been recognized that a significant fraction of these data likely represents false positives. During our studies of biogenesis of lysosome-related organelles complex-1 (BLOC-1), a protein complex involved in protein trafficking and containing the products of genes mutated in Hermansky–Pudlak syndrome, we faced the problem of having too many candidate binding partners to pursue experimentally. In this work, we have explored ways of efficiently gathering high-quality information about candidate binding partners and presenting the information in a visually friendly manner. We applied the approach to rank 70 candidate binding partners of human BLOC-1 and 102 candidates of its counterpart from Drosophila melanogaster. The top candidate for human BLOC-1 was the small GTPase encoded by the RAB11A gene, which is a paralogue of the Rab38 and Rab32 proteins in mammals and the lightoid gene product in flies. Interestingly, genetic analyses in D. melanogaster uncovered a synthetic sick/lethal interaction between Rab11 and lightoid. The data-mining approach described herein can be customized to study candidate binding partners for other proteins or possibly candidates derived from other types of ‘omics’ data. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Competing interests: None declared References to electronic databases: Hermansky–Pudlak syndrome: OMIM #203300; Dystrobrevin-binding protein 1 (DTNBP1): OMIM #607145. Biogenesis of lysosome-related organelles complex 1 subunit 3 (BLOC1S3): OMIM #609762. Alliance for Cell Signaling: . Drosophila Interactions Database: . Entrez Gene (NCBI): . Saccharomyces Genome Database: . Human Protein Reference Database (HPRD): . BLAST (NCBI): . Network Protein Sequence Analysis Tools server: . Presented at the Annual Symposium of the SSIEM, Lisbon, Portugal, 2–5 September 2008     

Thick Filament Proteins and Performance in Human Heart Failure

Modifications in thick filament protein content and performance are thought to underlie contraction-relaxation dysfunction in human heart failure. It has been found that myofibrillar Mg.ATPase is reduced in failing myocardium, which may be due in part to the reduction in α-myosin heavy chain (MHC) isoform content from ∼5–10% in normal myocardium to <2% in failing myocardium. The physiological importance of this seemingly small amount of α-MHC appears substantiated by the development of cardiopathologies in humans with mutated α-MHC at normal abundance. Therefore, the replacement of α-MHC by β-MHC (possessing slower actomyosin enzymatic kinetics) may underlie to a significant degree the reduced myocardial shortening velocity and reduced relaxation function in human heart failure. The atrial isoform of myosin essential light chain (ELC) may replace up to 25% of the ventricular isoform in failing ventricles and in so doing promotes myocardial shortening velocity. An elevated accumulation of the higher performing atrial-ELC, unlike the reduced content of the higher performing α-MHC, is therefore considered a compensatory response in heart failure. Phosphorylation of the myofilament proteins myosin regulatory light chain and troponin-I are both reduced in heart failure and collectively result in an elevated myofilament sensitivity to calcium activation, which inhibits relaxation function. These and other modifications in thick filament proteins, as discussed in this review, directly affect mechanical power output and relaxation function of the myocardium and thereby may be considered to cause or in some cases to compensate for the otherwise ineffective myocardial performance in heart failure.     

Use of two-hybrid methodology for identifying proteins of interest in endocrinology.

During the last 10 years, much progress has been made in understanding signal transduction. However, the function of many newly identified proteins remains unknown. The protein/protein interactions have emerged as a major biochemical mechanism of signal transduction. They are of major interest to elucidate the role of a protein in one or another cellular process. The two-hybrid system is especially well designed for such investigation. Here we show that the contribution of this technique already is and will be essential in dissecting the molecular mechanism of transduction pathways in many cell types.     

Is It Time to Screen for the Haptoglobin Genotype to Assess the Cardiovascular Risk Profile and Vitamin E Therapy Responsiveness in Patients with Diabetes?

Diabetes mellitus (DM) carries an increased risk for cardiovascular complications. Haptoglobin (Hp) is an abundant plasma protein with an antioxidant function by virtue of its ability to block the oxidative activity of extracorpuscular hemoglobin. There exist two common functional alleles at the Hp genetic locus, denoted 1 and 2, with three Hp genotypes (Hp 1–1, 2–1, and 2–2). The Hp protein expressed in Hp 2–2 individuals is markedly inferior in protecting against hemoglobin-induced oxidative stress. Hp 2–2 DM individuals have been shown to be at increased risk for the development of diabetes complications, particularly diabetic cardiovascular disease (CVD). We review the biological mechanisms underlying the interaction between the Hp genotype and DM on CVD and the accumulating evidence in favor of Hp genotyping all individuals with DM and providing antioxidant vitamin E supplementation specifically to Hp 2–2 DM individuals to reduce their CVD morbidity and mortality.     

The Na–K–Cl Co-transporter in astrocyte swelling

Ion channels, exchangers and transporters are known to be involved in cell volume regulation. A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. In particular, activation of the Na⁺–K⁺–Cl⁻ cotransporters has been shown to regulate cell volume in many conditions. The Na⁺–K⁺–Cl− cotransporters (NKCC) are a class of membrane proteins that transport Na, K, and Cl ions into and out of a wide variety of epithelial and nonepithelial cells. Studies have established the role of NKCC1 in astrocyte swelling/brain edema in ischemia and trauma. Our recent studies suggest that NKCC1 activation is also involved in astrocyte swelling induced by ammonia and in the brain edema in the thioacetamide model of acute liver failure. This review will focus on mechanisms of NKCC1 activation and its contribution to astrocyte swelling/brain edema in neurological disorders, with particular emphasis on ammonia neurotoxicity and acute liver failure.     

HEV-ORF3 Encoding Phosphoprotein Interacts With Hepsin

Background:

Hepatitis E virus (HEV) is a major causative agent of acute clinical hepatitis in adults through much of Asia, the Middle East and Africa. Open reading frame 3 (ORF3) encodes around 120 amino acids of phosphorylation protein that associates with the cytoskeleton, while its precise biological function is still unknown.

Objectives:

In order to understand the function of ORF3 protein (pORF3) in depth, HEV ORF3 interacting proteins were screened in human hepatocytes cDNA library using two-hybrid system techniques and further verification of the interactions were carried out through co-immunoprecipitation (Co-IP).

Materials and Methods:

The Cyto-Trap two-hybrid system technology, a classical method for analyzing protein interactions, was used to screen the pORF3 interacting proteins from human hepatocytes cDNA library.

Results:

Through the Cyto-Trap two-hybrid system, eight proteins interacting with pORF3 were winnowed. The Co-IP results confirmed that hepsin which is reported to function as the inhibitor of several tumors reacted with pORF3.

Conclusions:

Out of eight screened proteins interacting with pORF3, hepsin was confirmed to have specific interactions with pORF3.