Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recipient IgG immunity against leukoreduced donor platelets is dependent on indirect T-cell allorecognition and is suppressed in vivo by inhibitors (aminoguanidine, AMG) of inducible nitric oxide synthase (iNOS). To examine recipient processing pathways of donor platelet antigens, enriched macrophages (antigen-presenting cells [APC]) from BALB/c (H-2(d)) mice were pulsed with allogeneic C57BL/6 (H-2(b)) platelets and transfused weekly into naive BALB/c mice. Platelet-pulsed APC stimulated IgG antidonor antibody production in 45% of recipients by the second transfusion and in 100% by the sixth transfusion; this response was enhanced by pulsing in the presence of interferon-gamma. By the sixth transfusion, high-titer IgG1 (mean titer 4990) and IgG2a (1933) isotypes specific for donor major histocompatibility complex (MHC) class I antigens were detected. Platelet pulsing in the presence of AMG or colchicine significantly inhibited the ability of APC to stimulate IgG alloantibodies; only 50% (P <.005) and 20% (P <.0001) of recipients, respectively, produced antibodies by the sixth transfusion. AMG inhibition was reversed by the addition of L-arginine, the substrate for iNOS. In contrast, pulsing in the presence of chloroquine, the proteasome inhibitory peptide MG115, or Brefeldin A enhanced APC immunity (70-100% of recipients antibody positive by the second transfusion [P <.05]); these agents allowed the pulsed APC to stimulate IgG2a but inhibited IgG1 production and this correlated with a reduction in serum interleukin (IL)-4 levels. The results suggest that for donor platelet antigens to stimulate IgG alloantibodies, recipient APC use the essential generation of nitric oxide and a noncytosolic, pH-independent processing pathway, which can be exploited as an effective immunotherapy target to further inhibit alloimmunization against leukoreduced platelets. (Blood. 2000;95:1735-1742)
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PMID:Unique processing pathways within recipient antigen-presenting cells determine IgG immunity against donor platelet MHC antigens. 1068 32

The basic helix-loop-helix/leucine zipper (bHLH/ZIP) microphthalmia-associated transcription factor (MITF) regulates transcription of genes encoding enzymes essential for melanin biosynthesis in melanocytes and retinal pigmented epithelial cells. To determine how MITF activity is regulated, we used the yeast two-hybrid system to identify proteins expressed by human melanoma cells that interact with MITF. The majority of clones that showed positive interaction with a 158-amino-acid region of MITF containing the bHLH/ZIP domain (aa 168-325) encoded the ubiquitin conjugating enzyme hUBC9. The association of MITF with hUBC9 was further confirmed by an in vitro GST pull-down assay. Although hUBC9 is known to interact preferentially with SENTRIN/SUMO1, in vitro transcription/translation analysis demonstrated greater association of MITF with ubiquitin than with SENTRIN. Importantly, cotransfection of MITF and hUBC9 expression vectors resulted in MITF protein degradation. MITF protein was stabilized by the proteasome inhibitor MG132, indicating the role of the ubiquitin-proteasome system in MITF degradation. Serine 73, which is located in a region rich in proline, glutamic acid, serine, and threonine (PEST), regulates MITF protein stability, since a serine to alanine mutation prevented hUBC9-mediated MITF (S73A) degradation. Furthermore, we identified lysine 201 as a potential ubiquitination site. A lysine to arginine mutation abolished MITF (K201R) degradation by hUBC9 in vivo. Our experiments indicate that by targeting MITF for proteasome degradation, hUBC9 is a critical regulator of melanocyte differentiation.
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PMID:Regulation of microphthalmia-associated transcription factor MITF protein levels by association with the ubiquitin-conjugating enzyme hUBC9. 1069 30

Hepatitis B virus (HBV) has a unique fourth open reading frame coding for a 16.5-kDa protein known as hepatitis B virus X protein (HBX). The importance of HBX in the life cycle of HBV has been well established, but the underlying molecular function of HBX remains controversial. We previously identified a proteasome subunit PSMA7 that interacts specifically with HBX in the Saccharomyces cerevisiae two-hybrid system. Here we demonstrate that PSMC1, an ATPase-like subunit of the 19 S proteasome component, also interacts with HBX and PSMA7. Analysis of the interacting domains among PSMA7, PSMC1, and HBX by deletion and site-directed mutagenesis suggested a mutually competitive structural relationship among these polypeptides. The competitive nature of these interactions is further demonstrated using a modified yeast two-hybrid dissociator system. The crucial HBX sequences involved in interaction with PSMA7 and PSMC1 are important for its function as a transcriptional coactivator. HBX, while functioning as a coactivator of AP-1 and acidic activator VP-16 in mammalian cells, had no effect on the transactivation function of their functional orthologs GCN4 and Gal4 in yeast. Overexpression of PSMC1 seemed to suppress the expression of various reporters in mammalian cells; this effect, however, was overcome by coexpression of HBX. In addition, HBX expression inhibited the cellular turnover of c-Jun and ubiquitin-Arg-beta-galactosidase, two well known substrates of the ubiquitin-proteasome pathway. Thus, interaction of HBX with the proteasome complex in metazoan cells may underlie the functional basis of proteasome as a cellular target of HBX.
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PMID:Structural and functional characterization of interaction between hepatitis B virus X protein and the proteasome complex. 1074 18

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. We used an expression-cloning screen to search for mouse proteins that are degraded by the ubiquitin/proteasome-dependent N-end rule pathway in a reticulocyte lysate. One substrate thus identified was RGS4, a member of the RGS family of GTPase-activating proteins that down-regulate specific G proteins. A determinant of the RGS4 degradation signal (degron) was located at the N terminus of RGS4, because converting cysteine 2 to either glycine, alanine, or valine completely stabilized RGS4. Radiochemical sequencing indicated that the N-terminal methionine of the lysate-produced RGS4 was replaced with arginine. Since N-terminal arginine is a destabilizing residue not encoded by RGS4 mRNA, we conclude that the degron of RGS4 is generated through the removal of N-terminal methionine and enzymatic arginylation of the resulting N-terminal cysteine. RGS16, another member of the RGS family, was also found to be an N-end rule substrate. RGS4 that was transiently expressed in mouse L cells was short-lived in these cells. However, the targeting of RGS4 for degradation in this in vivo setting involved primarily another degron, because N-terminal variants of RGS4 that were stable in reticulocyte lysate remained unstable in L cells.
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PMID:RGS4 is arginylated and degraded by the N-end rule pathway in vitro. 1078 90

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. N-terminal asparagine and glutamine are tertiary destabilizing residues, in that they are enzymatically deamidated to yield secondary destabilizing residues aspartate and glutamate, which are conjugated to arginine, a primary destabilizing residue. N-terminal arginine of a substrate protein is bound by the Ubr1-encoded E3alpha, the E3 component of the ubiquitin-proteasome-dependent N-end rule pathway. We describe the construction and analysis of mouse strains lacking the asparagine-specific N-terminal amidase (Nt(N)-amidase), encoded by the Ntan1 gene. In wild-type embryos, Ntan1 was strongly expressed in the branchial arches and in the tail and limb buds. The Ntan1(-/-) mouse strains lacked the Nt(N)-amidase activity but retained glutamine-specific Nt(Q)-amidase, indicating that the two enzymes are encoded by different genes. Among the normally short-lived N-end rule substrates, only those bearing N-terminal asparagine became long-lived in Ntan1(-/-) fibroblasts. The Ntan1(-/-) mice were fertile and outwardly normal but differed from their congenic wild-type counterparts in spontaneous activity, spatial memory, and a socially conditioned exploratory phenotype that has not been previously described with other mouse strains.
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PMID:Altered activity, social behavior, and spatial memory in mice lacking the NTAN1p amidase and the asparagine branch of the N-end rule pathway. 1080 55

We reported the first case of a congenital histidine-rich glycoprotein deficiency (HRG Tokushima) in which substitution of Gly85 with Glu (G85E) in the first cystatin domain resulted in intracellular degradation and a low plasma level of HRG [Shigekiyo, T. et al. (1998) Blood 91, 128-133]. Recently, we identified the gene mutation of a second case of HRG deficiency as a Cys223 to Arg (C223R) mutation in the second cystatin domain. To investigate the molecular and cellular bases of these deficiencies, we expressed these HRG mutants in baby hamster kidney (BHK) cells. Pulse-chase experiments in the absence and presence of various proteinase inhibitors revealed that, while wild-type HRG was completely secreted during 4-h chase periods, both the G85E and C223R mutants were only partially secreted and primarily degraded within the cells. The intracellular degradation of the C223R mutant was almost completely inhibited in the presence of a proteasome inhibitor, lactacystin, carbobenzoxy-leucyl-leucyl-leucinal or N-acetyl-leucyl-leucyl-norleucinal, resulting in increased secretion of the C223R mutant, and thus implicating the proteasome system in this degradation process. In contrast, the sum of the amounts of the G85E mutant inside and outside the cells decreased during the chase periods even in the presence of the proteasome inhibitor, carbobenzoxy-leucyl-leucyl-leucinal or N-acetyl-leucyl-leucyl-norleucinal, although proteasome-specific inhibitor lactacystin and one of the cysteine protease inhibitors, E-64-d, prevented the intracellular degradation. These results suggested that intracellular degradation of G85E HRG occurred to some extent through a hitherto unknown mechanism. Similar studies involving recombinant mutants in which Gly85 or Cys223 was replaced with several other amino acids revealed that proteins with mutations leading to the destruction of the predicted b-sheet structure of the cystatin domains were eliminated by the intracellular quality control system.
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PMID:Intracellular degradation of histidine-rich glycoprotein mutants: tokushima-1 and 2 mutants are degraded by different proteolytic systems. 1092 Feb 55

It has been demonstrated from studies using NF-kappaB inhibitors that NF-kappaB may be involved in the iNOS induction stimulated by cytokines and/or lipopolysaccharide (LPS) in various cell types and tissues. However, the actions of the inhibitors are less selective and highly cytotoxic. We constructed stable clones of C6 cells transfected with two types of IkappaBalpha mutant genes (IkappaBalpha(SS --> AA); Ser-32/36 to Ala-32/36, IkappaBalpha(KK --> RR); Lys-21/22 to Arg-21/22). IkappaBalpha(SS --> AA) strongly inhibited (1) LPS-, IL-1beta-, and TNF-alpha-induced nuclear translocation and DNA binding of NF-kappaB to the kappaB site; and (2) iNOS induction stimulated by LPS or IL-1beta plus IFN-gamma. These results indicate that NF-kappaB plays a critical role in cytokines and/or LPS-induced iNOS induction. Surprisingly, similar to the endogenous IkappaBalpha, IkappaBalpha(KK --> RR) was degraded by various stimuli, and proteasome inhibitors blocked this event. These results suggest that another Lys residue(s), other than Lys-21/22, may be required for the ligand-induced IkappaBalpha degradation by the ubiquitin-proteasome pathway.
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PMID:Involvement of nuclear factor-kappaB (NF-kappaB) signaling in the expression of inducible nitric oxide synthase (iNOS) gene in rat C6 glioma cells. 1096 56

In normal cells, p53 is maintained at a low level by ubiquitin-mediated proteolysis, but after genotoxic insult this process is inhibited and p53 levels rise dramatically. Ubiquitination of p53 requires the ubiquitin-activating enzyme Ubc5 as a ubiquitin conjugation enzyme and Mdm2, which acts as a ubiquitin protein ligase. In addition to the N-terminal region, which is required for interaction with Mdm2, the C-terminal domain of p53 modulates the susceptibility of p53 to Mdm2-mediated degradation. To analyze the role of the C-terminal domain in p53 ubiquitination, we have generated p53 molecules containing single and multiple lysine-to-arginine changes between residues 370 and 386. Although wild-type (WT) and mutant molecules show similar subcellular distributions, the mutants display a higher transcriptional activity than WT p53. Simultaneous mutation of lysine residues 370, 372, 373, 381, 382, and 386 to arginine residues (6KR p53 mutant) generates a p53 molecule with potent transcriptional activity that is resistant to Mdm2-induced degradation and is refractory to Mdm2-mediated ubiquitination. In contrast to WT p53, transcriptional activity directed by the 6KR p53 mutant fails to be negatively regulated by Mdm2. Those differences are also manifest in HeLa cells which express the human papillomavirus E6 protein, suggesting that p53 C-terminal lysine residues are also implicated in E6-AP-mediated ubiquitination. These data suggest that p53 C-terminal lysine residues are the main sites of ubiquitin ligation, which target p53 for proteasome-mediated degradation.
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PMID:Multiple C-terminal lysine residues target p53 for ubiquitin-proteasome-mediated degradation. 1104 42

Our previous biochemical studies of HIV-1 and MuLV virions isolated and identified mature Gag products, HIV-1 p6(Gag) and MuLV p12(Gag), that were conjugated to a single ubiquitin. To study the importance of the monoubiquitination of Gag, a series of lysine to arginine mutants were constructed that eliminated ubiquitination at one or both of the lysines in HIV-1(NL4-3) p6(Gag) and both lysines in Moloney MuLV p12(Gag). HPLC and immunoblot analysis of the HIV-1 mutants demonstrated that either of the lysines in p6(Gag), K27 or K33, could be monoubiquitinated. However, infectivity assays showed that monoubiquitination of HIV-1 p6(Gag) or MuLV p12(Gag) is not required for viral replication in vitro. Pulse-chase radiolabeling of HIV-1-producing cells revealed that monoubiquitination of p6(Gag) does not affect the short-term release of virus from the cell, the maturation of Pr55(Gag), or the sensitivity of these processes to proteasome inhibitors. Experiments with protease-deficient HIV-1 showed that Pr55(Gag) can be monoubiquitinated, suggesting that p6(Gag) is first modified as a domain within Gag. Examination of the proteins inside an HIV-1 mutant found that free ubiquitin was incorporated into the virions in the absence of the lysines in p6(Gag), showing that the ubiquitin inside the virus is not initially brought in as a p6(Gag) conjugate. Although our results establish that monoubiquitination of p6(Gag) and p12(Gag) is not required for viral replication in vitro, this modification may be a by-product of interactions between Gag and cellular proteins during assembly and budding.
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PMID:Ubiquitination of HIV-1 and MuLV Gag. 1111 87

The E7 oncoprotein of the high risk human papillomavirus type 16 (HPV-16), which is etiologically associated with uterine cervical cancer, is a potent immortalizing and transforming agent. It probably exerts its oncogenic functions by interacting and altering the normal activity of cell cycle control proteins such as p21WAF1, p27KIP1 and pRb, transcriptional activators such as TBP and AP-1, and metabolic regulators such as M2-pyruvate kinase (M2-PK). Here we show that E7 is a short-lived protein and its degradation both in vitro and in vivo is mediated by the ubiquitin-proteasome pathway. Interestingly, ubiquitin does not attach to any of the two internal Lysine residues of E7. Substitution of these residues with Arg does not affect the ability of the protein to be conjugated and degraded; in contrast, addition of a Myc tag to the N-terminal but not to the C-terminal residue, stabilizes the protein. Also, deletion of the first 11 amino acid residues stabilizes the protein in cells. Taken together, these findings strongly suggest that, like MyoD and the Epstein Barr Virus (EBV) transforming Latent Membrane Protein 1 (LMPI), the first ubiquitin moiety is attached linearly to the free N-terminal residue of E7. Additional ubiquitin moieties are then attached to an internal Lys residue of the previously conjugated molecule. The involvement of E7 in many diverse and apparently unrelated processes requires tight regulation of its function and cellular level, which is controlled in this case by ubiquitin-mediated proteolysis.
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PMID:Degradation of the E7 human papillomavirus oncoprotein by the ubiquitin-proteasome system: targeting via ubiquitination of the N-terminal residue. 1112 26


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