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1.
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. 相似文献
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3.
Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator. 总被引:24,自引:2,他引:22
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F Hofer S Fields C Schneider G S Martin 《Proceedings of the National Academy of Sciences of the United States of America》1994,91(23):11089-11093
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. 相似文献
4.
S. T. Mees R. Mennigen T. Spieker E. Rijcken N. Senninger J. Haier M. Bruewer 《International journal of colorectal disease》2009,24(4):361-368
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). 相似文献
5.
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. 相似文献
6.
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. 相似文献
7.
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。 相似文献
8.
Protein homeostasis and molecular chaperones in aging 总被引:1,自引:1,他引:0
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 相似文献
9.
Truscott RJ Comte-Walters S Ablonczy Z Schwacke JH Berry Y Korlimbinis A Friedrich MG Schey KL 《Age (Dordrecht, Netherlands)》2011,33(4):543-554
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. 相似文献
10.
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 A1c. 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. 相似文献
11.
Huaxiu Shi Jianlin Ren Hongzhi Xu Jinshui Pan Xiaoyan Hao Lydia Leyland Barlow Weiguo Dong 《Digestive diseases and sciences》2010,55(10):2929-2939
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. 相似文献12.
I. A. Rodriguez-Fernandez E. C. Dell’Angelica 《Journal of inherited metabolic disease》2009,32(2):190-203
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 相似文献
13.
Thick Filament Proteins and Performance in Human Heart Failure 总被引:2,自引:0,他引:2
Palmer BM 《Heart failure reviews》2005,10(3):187-197
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. 相似文献
14.
I Pirson C Jacobs I Vandenbroere H El Housni J E Dumont D Perez-Morga 《Molecular and cellular endocrinology》1999,151(1-2):137-141
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. 相似文献
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16.
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. 相似文献
17.
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. 相似文献
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Chunyan Wang Liang Guo Dayi Yu Xiuguo Hua Zhibiao Yang Congli Yuan Li Cui 《Hepatitis monthly》2014,14(1)