Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand protein family. ALG-2 forms a homodimer, a heterodimer with another penta-EF-hand protein, peflin, and a complex with its interacting protein, named AIP1 or Alix. By yeast two-hybrid screening using human ALG-2 as bait, we isolated a cDNA of a novel ALG-2-interacting protein, which turned out to be annexin XI. Deletion analysis revealed that ALG-2 interacted with the N-terminal domain of annexin XI (AnxN), which has an amino acid sequence similar to that of the C-terminal region of AIP1/Alix. Using recombinant biotin-tagged ALG-2 and the glutathione S-transferase (GST) fusion protein of AnxN, the direct interaction was analyzed by an ALG-2 overlay assay and by real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. The dissociation constant (K(d)) was estimated to be approximately 70 nM. The Ca(2+)-dependent fluorescence change of ALG-2 in the presence of the hydrophobicity fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was inhibited by mixing with GST-AnxN, suggesting that the Pro/Gly/Tyr/Ala-rich hydrophobic region in AnxN masked the Ca(2+)-dependently exposed hydrophobic surface of ALG-2.
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PMID:ALG-2 interacts with the amino-terminal domain of annexin XI in a Ca(2+)-dependent manner. 1188 39

The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand (PEF) protein family. ALG-2 forms a homodimer, a heterodimer with another PEF protein, peflin, and a complex with its interacting protein, named Alix or AIP1. We previously identified annexin XI as a novel ALG-2-binding partner. Both the N-terminal regulatory domain of annexin XI (Anx11N) and the ALG-2-binding domain of Alix/AIP1 are rich in Pro, Gly, Ala, Tyr and Gln. This PGAYQ-biased amino acid composition is also found in the N-terminal extension of annexin VII (Anx7N). Using recombinant ALG-2 proteins and the glutathione S-transferase (GST) fusion proteins of Anx7N and Anx11N, the direct Ca(2+)-dependent interaction was analyzed by a biotin-tagged ALG-2 overlay assay and by a real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. Both GST-Anx7N and GST-Anx11N showed similar binding kinetics against ALG-2 as well as ALG-2-DeltaN23, which lacked the hydrophobic N-terminal region. Two binding sites were predicted in both Anx7N and Anx11N, and the dissociation constants (K(d)) were estimated to be approximately 40-60 nM for the high-affinity site and 500-700 nM for the low-affinity site.
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PMID:The penta-EF-hand domain of ALG-2 interacts with amino-terminal domains of both annexin VII and annexin XI in a Ca2+-dependent manner. 1244 60

The penta-EF hand (PEF) family of calcium binding proteins includes grancalcin, peflin, sorcin, calpain large and small subunits as well as ALG-2. Systematic testing of the heterodimerization abilities of the PEF proteins using the yeast two-hybrid and glutathione S-transferase pull-down assays revealed the new finding that grancalcin interacts strongly with sorcin. In addition, sorcin and grancalcin can be co-immunoprecipitated from lysates of human umbilical vein endothelial cells. Our results indicate that heterodimerization, in addition to differential interactions with target proteins, might be a way to regulate and fine tune processes mediated by calcium binding proteins of the penta-EF hand type.
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PMID:The PEF family proteins sorcin and grancalcin interact in vivo and in vitro. 1280 66

Alix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca(2+)-binding protein, ALG-2 (apoptosis-linked gene 2), through its C-terminal proline-rich region. In this study, we searched for proteins that interact with human AlixDeltaC (a truncated form not containing the C-terminal region) by using a yeast two-hybrid screen, and we identified two similar human proteins, CHMP4a and CHMP4b (chromatin-modifying protein; charged multivesicular body protein), as novel binding partners of Alix. The interaction of Alix with CHMP4b was confirmed by a glutathione S-transferase pull-down assay and by co-immunoprecipitation experiments. Fluorescence microscopic analysis revealed that CHMP4b transiently expressed in HeLa cells mainly exhibited a punctate distribution in the perinuclear area and co-localized with co-expressed Alix. The distribution of CHMP4b partly overlapped the distributions of early and late endosomal marker proteins, EEA1 (early endosome antigen 1) and Lamp-1 (lysosomal membrane protein-1), respectively. Transient overexpression of CHMP4b induced the accumulation of ubiquitinated proteins as punctate patterns that were partly overlapped with the distribution of CHMP4b and inhibited the disappearance of endocytosed epidermal growth factor. In contrast, stably expressed CHMP4b in HEK293 cells was observed diffusely in the cytoplasm. Transient overexpression of AlixDeltaC in stably CHMP4b-expressing cells, however, induced formation of vesicle-like structures in which CHMP4b and AlixDeltaC were co-localized. SKD1(E235Q), a dominant negative form of the AAA type ATPase SKD1 that plays critical roles in the endocytic pathway, was co-immunoprecipitated with CHMP4b. Furthermore, CHMP4b co-localized with SKD1(E235Q) as punctate patterns in the perinuclear area, and Alix was induced to exhibit dot-like distributions overlapped with SKD1(E235Q) in HeLa cells. These results suggest that CHMP4b and Alix participate in formation of multivesicular bodies by cooperating with SKD1.
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PMID:The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting. 1286 Sep 94

Snf7p (sucrose non-fermenting) and Vps20p (vacuolar protein-sorting) are small coil-coiled proteins involved in yeast MVB (multivesicular body) structure, formation and function. In the present study, we report the identification of three human homologues of yeast Snf7p, designated hSnf7-1, hSnf7-2 and hSnf7-3, and a single human Vps20p homologue, designated hVps20, that may have similar roles in humans. Immunofluorescence studies showed that hSnf7-1 and hSnf7-3 localized in large vesicular structures that also co-localized with late endosomal/lysosomal structures induced by overexpressing an ATPase-defective Vps4-A mutant. In contrast, overexpressed hVps20 showed a typical endosomal membrane-staining pattern, and co-expression of hVps20 with Snf7-1 dispersed the large Snf7-staining vesicles. Interestingly, overexpression of both hSnf7 and hVps20 proteins induced a post-endosomal defect in cholesterol sorting. To explore possible protein-protein interactions involving hSnf7 proteins, we used information from yeast genomic studies showing that yeast Snf7p can interact with proteins involved in MVB function. Using a glutathione S-transferase-capture approach with several mammalian homologues of such yeast Snf7p-interacting proteins, we found that all three hSnf7s interacted with mouse AIP1 [ALG-2 (apoptosis-linked gene 2) interacting protein 1], a mammalian Bro1p [BCK1 (bypass of C kinase)-like resistance to osmotic shock]-containing protein involved in cellular vacuolization and apoptosis. Whereas mapping experiments showed that the N-terminus of AIP1 containing both a Bro1 and an alpha-helical domain were required for interaction with hSnf7-1, Snf7-1 did not interact with another human Bro1-containing molecule, rhophilin-2. Co-immunoprecipitation experiments confirmed the in vivo interaction of hSnf7-1 and AIP1. Additional immunofluorescence experiments showed that hSnf7-1 recruited cytosolic AIP1 to the Snf7-induced vacuolar-like structures. Together these results suggest that mammalian Vps20, AIP1 and Snf7 proteins, like their yeast counterparts, play roles in MVB function.
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PMID:Structure and function of human Vps20 and Snf7 proteins. 1458 93

The ALG-2-interacting protein Alix has recently been demonstrated to associate with CHMP4b that is a human homologue of yeast Snf7p (also named Vps32p) and a member of the family of small coiled-coil proteins named CHMP implicated in playing roles in multivesicular body sorting. In addition to the previously isolated cDNAs for two CHMP4 proteins (CHMP4a and CHMP4b), we isolated a cDNA for a new member of the CHMP4 subfamily (designated CHMP4c). Northern blot analyses revealed different expression patterns of the mRNAs for the three CHMP4 isoforms in human tissues. CHMP4b messages were expressed at higher levels in all 12 tissues tested in comparison with the CHMP4a and CHMP4c transcripts, particularly in heart and skeletal muscle. The interaction with Alix was detected for each CHMP4 isoform by co-immunoprecipitation experiments using lysates of HEK293 cells expressing each epitope-tagged CHMP4 protein and Alix fused with green fluorescent protein. Further, using recombinant glutathione S-transferase (GST) fusion protein of truncated Alix (amino acids 1-423) and thioredoxin-tagged CHMP4 proteins, the direct interactions were detected by a GST pull-down assay, where CHMP4b showed a stronger interaction than other CHMP4 isoforms. These results suggest that CHMP4b is a major binding partner of Alix among the three CHMP4 isoforms.
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PMID:CHMP4b is a major binding partner of the ALG-2-interacting protein Alix among the three CHMP4 isoforms. 1467 97

ALG-2 (apoptosis-linked gene 2) is a Ca2+-binding protein that belongs to the PEF (penta-EF-hand) protein family. Alix (ALG-2-interacting protein X)/AIP1 (ALG-2-interacting protein 1), one of its binding partners, interacts with TSG101 and CHMP4 (charged multivesicular body protein 4), which are components of ESCRT-I (endosomal sorting complex required for transport I) and ESCRT-III respectively. In the present study, we investigated the association between ALG-2 and ESCRT-I. By a GST (glutathione S-transferase) pull-down assay using HEK-293T (human embryonic kidney 293T) cell lysates, endogenous TSG101 and two other exogenously expressed ESCRT-I components [hVps28 (human vacuolar protein sorting 28) and hVps37A] were shown to associate with GST-ALG-2 in the presence of Ca2+. By the yeast two-hybrid assay, however, a positive interaction was observed with only TSG101 among the three ESCRT-I components, suggesting that ALG-2 associates with hVps28 and hVps37A indirectly through TSG101. Using various deletion mutants of TSG101, the central PRR (proline-rich region) was found to be sufficient for interaction with ALG-2 by the GST-pull-down assay. Direct binding of ALG-2 to the TSG101 PRR was demonstrated by an overlay assay using biotin-labelled ALG-2 as a probe. In immunofluorescence microscopic analysis of HeLa cells that overexpressed a GFP (green fluorescent protein)-fused ATPase-defective dominant-negative form of SKD1/Vps4B (GFP-SKD1(E235Q)), ALG-2 exhibited a punctate distribution at the perinuclear area and co-localized with GFP-SKD1(E235Q) to aberrant endosomes. This punctate distribution of ALG-2 was markedly diminished by treatment of HeLa cells with a membrane-permeant Ca2+ chelator. Moreover, a Ca2+-binding-defective mutant of ALG-2 did not co-localize with GFP-SKD1(E235Q). Our findings suggest that ALG-2 may function as a Ca2+-dependent accessory protein of the endosomal sorting machinery by interacting directly with TSG101 as well as with Alix.
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PMID:The penta-EF-hand protein ALG-2 interacts directly with the ESCRT-I component TSG101, and Ca2+-dependently co-localizes to aberrant endosomes with dominant-negative AAA ATPase SKD1/Vps4B. 1600 3

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.
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PMID:Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins. 1697 57

Intracellular localization of the penta-EF-hand Ca2+-binding protein ALG-2 in HeLa cells was investigated by immunofluorescent confocal microscopy using a polyclonal antibody. In addition to its presence in the nucleus, ALG-2 was found to be distributed in a punctate pattern in the cytoplasm, where it was partly co-stained with an endoplasmic reticulum (ER) exit site marker p125. In vitro GST pull down analysis demonstrated that ALG-2 and its alternatively spliced isoform interact with the COPII component Sec31A in a Ca2+-dependent manner, and a biotin-labeled ALG-2 overlay assay revealed direct binding of ALG-2 to Sec31A. Biochemical and immunofluorescent microscopic analyses showed that ALG-2 was enriched at the Sec31A-localizing membrane compartments upon stimulation with the Ca2+ ionophore A23187. In contrast, treatment of cells with the membrane-permeant Ca2+ chelator BAPTA-AM led to a dispersion of ALG-2 throughout the cells and to a significant loss of Sec31A in the perinuclear region. These findings establish Sec31A as a novel target for ALG-2 and provide a framework for studies on the roles of ALG-2 in ER-Golgi transport.
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PMID:ALG-2 directly binds Sec31A and localizes at endoplasmic reticulum exit sites in a Ca2+-dependent manner. 1719 69

The vasopressin type 2 receptor (V2R) is a G protein-coupled receptor that plays a central role in renal water reabsorption. Termination of ligand (vasopressin) stimulation is an important physiological regulatory event, but few proteins that interact with the V2R during downregulation after vasopressin (VP) binding have been identified. Using yeast two-hybrid screening of a human kidney cDNA library, we show that a 100-kDa protein called ALG-2-interacting protein X (Alix) interacts with the last 29 amino acids of the V2R COOH terminus. This was confirmed by pull-down assays using a GST-V2R-COOH-tail fusion protein. Alix was immunolocalized in principal cells of the kidney, which also express the V2R. The function of the Alix-V2R interaction was studied by transfecting Alix into LLC-PK(1) epithelial cells expressing V2R-green fluorescent protein (GFP). Under basal conditions, V2R-GFP localized mainly at the plasma membrane. On VP treatment, V2R-GFP was internalized into perinuclear vesicles in the nontransfected cells. In contrast, V2R-GFP fluorescence was virtually undetectable 2 h after exposure to VP in cells that coexpressed Alix. Western blotting using an anti-GFP antibody showed marked degradation of the V2R after 2 h in the presence of VP and Alix, a time point at which little or no degradation was detected in the absence of Alix. In contrast, little or no degradation of the parathyroid hormone receptor was detectable in the presence or absence of Alix and/or the PTH ligand. The VP-induced disappearance of V2R-GFP was abolished by chloroquine, a lysosomal degradation inhibitor, but not by MG132, a proteosome inhibitor. These data suggest that Alix increases the rate of lysosomal degradation of V2R and may play an important regulatory role in the VP response by modulating V2R downregulation.
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PMID:Alix (AIP1) is a vasopressin receptor (V2R)-interacting protein that increases lysosomal degradation of the V2R. 1728


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