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)

MAP kinase activation by growth factors depends on cell adhesion to the extracellular matrix. Disrupting the cell adhesion process in NIH 3T3 fibroblasts induced an almost complete inhibition of MAP kinase, which was impaired by proteasome inhibitors. In the absence of cell anchorage, c-Raf-1 expression was dramatically decreased after 24 h. This down-regulation was suppressed by proteasome inhibitors, suggesting that a proteasome-dependent degradation of Raf occurred in the absence of cell adhesion. Proteasome inhibitors did not affect Raf-1 levels in adherent cells, indicating that this degradation only occurred in the absence of cell adhesion. Finally, ectopic coexpression of Raf-1 and ubiquitin in HEK-293 and NIH 3T3 cells generated ubiquitylated forms of Raf-1, both in adherent and suspended cells, suggesting a possible ubiquitin-dependent degradation of the protein.
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PMID:Cell adhesion protects c-Raf-1 against ubiquitin-dependent degradation by the proteasome. 1207 72

Mutations in the alpha-synuclein gene (A30P and A53T) are reported to cause familial Parkinson's disease (PD), but it is not known how they result in selective dopaminergic cell death. Here we report on effects of mutant alpha-synucleins on dopamine transporter (DAT)-mediated toxicity of the selective dopaminergic neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+) in vitro. We established human embryonic kidney HEK-293 cell lines stably co-expressing each alpha-synuclein isoform and the human DAT. We demonstrate that expression of all alpha-synuclein isoforms enhances toxicity of general complex I inhibition (rotenone), but only the expression of mutant alpha-synucleins induces significant increased DAT-dependent toxicity of very low concentrations of MPP+ compared to wild-type protein. Proteasomal inhibition by lactacystin does not alter MPP+-toxicity in all cell lines. Our data suggest a new mechanism of MPP+-induced dopaminergic toxicity by an interaction between mutant alpha-synucleins and the DAT, which is independent of the function of the proteasome.
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PMID:Expression of mutant alpha-synucleins enhances dopamine transporter-mediated MPP+ toxicity in vitro. 1215 87

The early response gene IEX-1 is involved in the regulation of cellular growth and survival, and its expression is related to stress-, growth- and death-inducing signals. Addressing the role of IEX-1 in the promotion of apoptosis, we investigated the effect of IEX-1 on nuclear factor-kappaB (NF-kappaB) activation. Stably transfected HEK-293 cells conditionally overexpressing IEX-1 exhibit decreased levels of NF-kappaB activity, either basal or TNFalpha induced, as shown by gel-shift and luciferase reporter gene assay. Furthermore, activated p65 accumulated in the nuclei of 293 cells to a lower degree, if IEX-1 expression was increased. This inhibited NF-kappaB activation was preceded by an altered turnover of IkappaBalpha and phospho-IkappaBalpha. In addition, IEX-1 expression also inhibited the activity of the 26S-proteasome, as shown by a fluorometric proteasome assay. Conversely, disruption of IEX-1 expression in 293 cells by stable transfection with specific anti-IEX-1 hammerhead ribozymes increased NF-kappaB activity, and accelerated the degradation of IkappaBalpha. Along with these opposite effects of IEX-1 expression and IEX-1 disruption on NF-kappaB activation, the sensitivity of 293 cells towards various apoptotic stimuli also changed. In contrast to ribozyme-transduced 293 cells that were significantly less sensitive to apoptosis, this sensitivity was enhanced if IEX-1 expression was increased. Our data suggest that IEX-1 - itself an NF-kappaB target gene - inhibits the activation of this transcription factor, and hereby may counteract the antiapoptotic potential of NF-kappaB.
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PMID:The early response gene IEX-1 attenuates NF-kappaB activation in 293 cells, a possible counter-regulatory process leading to enhanced cell death. 1276 4

Expansion of the CAG trinucleotide repeat encoding glutamine in the androgen receptor gene leads to spinobulbar muscular atrophy (SBMA), a neurodegenerative disorder in a family of polyglutamine diseases with enigmatic pathogenic mechanisms. One established property of glutamine residues is their ability to act as an amine accepter in a transglutaminase-catalyzed reaction, resulting in a proteolytically resistant glutamyl-lysine cross-link. To examine underlying disease mechanisms we investigated the relationship between polyglutamine-expanded androgen receptor and transglutaminase. We found androgen receptor N-terminal fragments are a substrate for transglutaminase. Western blots of the proteins following incubation with transglutaminase show that several different epitopes of the AR appear to be lost. We propose that this is due to the transglutaminase cross-linking of the AR, which interferes with antibody recognition. Furthermore, HEK GFP(u)-1 cells expressing polyglutamine-expanded androgen receptor and transglutaminase exhibit ligand-dependent proteasome dysfunction; this effect was not observed in the presence of cystamine, a transglutaminase inhibitor. In addition, transglutaminase-mediated isopeptide bonds were detected in brains of SBMA transgenic mice, but not in controls, suggesting involvement of transglutaminase-catalyzed reactions in polyglutamine disease pathogenesis. Our hypothesis is that cross-linked AR cannot to be degraded by the proteasome and obstructs the proteasome pore, preventing normal function. Because of the central role the ubiquitin-proteasome degradation system plays in fundamental cellular processes, any alteration in its function could cause cell death, ultimately contributing to SBMA pathogenesis.
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PMID:Transglutaminase potentiates ligand-dependent proteasome dysfunction induced by polyglutamine-expanded androgen receptor. 1281 78

The stability of the endoplasmic reticulum (ER) glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the key enzyme in cholesterol biosynthesis, is negatively regulated by sterols. HMGR is anchored in the ER via its N-terminal region, which spans the membrane eight times and contains a sterol-sensing domain. We have previously established that degradation of mammalian HMGR is mediated by the ubiquitin-proteasome system (Ravid, T., Doolman, R., Avner, R., Harats, D., and Roitelman, J. (2000) J. Biol. Chem. 275, 35840-35847). Here we expressed in HEK-293 cells an HA-tagged-truncated version of HMGR that encompasses all eight transmembrane spans (350 N-terminal residues). Similar to endogenous HMGR, degradation of this HMG(350)-3HA protein was accelerated by sterols, validating it as a model to study HMGR turnover. The degradation of HMG(240)-3HA, which lacks the last two transmembrane spans yet retains an intact sterol-sensing domain, was no longer accelerated by sterols. Using HMG(350)-3HA, we demonstrate that transmembrane region of HMGR is ubiquitinated in a sterol-regulated fashion. Through site-directed Lys --> Arg mutagenesis, we pinpoint Lys(248) and Lys(89) as the internal lysines for ubiquitin attachment, with Lys(248) serving as the major acceptor site for polyubiquitination. Moreover, the data indicate that the N terminus is also ubiquitinated. The degradation rates of the Lys --> Arg mutants correlates with their level of ubiquitination. Notably, lysine-less HMG(350)-3HA is degraded faster than wild-type protein, suggesting that lysines other than Lys(89) and Lys(248) attenuate ubiquitination at the latter residues. The ATP-dependent ubiquitination of HMGR in isolated microsomes requires E1 as the sole cytosolic protein, indicating that ER-bound E2 and E3 enzymes catalyze this modification. Polyubiquitination of HMGR is correlated with its extraction from the ER membrane, a process likely to be assisted by cytosolic p97/VCP/Cdc48p-Ufd1-Npl4 complex, as only ubiquitinated HMGR pulls down p97.
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PMID:Ubiquitin is conjugated by membrane ubiquitin ligase to three sites, including the N terminus, in transmembrane region of mammalian 3-hydroxy-3-methylglutaryl coenzyme A reductase: implications for sterol-regulated enzyme degradation. 1524 8

Autosomal dominant and recessive distal renal tubular acidosis (dRTA) can be caused by mutations in the anion exchanger 1 (AE1 or SLC4A1) gene, which encodes the erythroid chloride/bicarbonate anion exchanger membrane glycoprotein (eAE1) and a truncated kidney isoform (kAE1). The biosynthesis and trafficking of kAE1 containing a novel recessive missense dRTA mutation (kAE1 S773P) was studied in transiently transfected HEK-293 cells, expressing the mutant alone or in combination with wild-type kAE1 or another recessive mutant, kAE1 G701D. The kAE1 S773P mutant was expressed at a three times lower level than wild-type, had a 2-fold decrease in its half-life, and was targeted for degradation by the proteasome. It could not be detected at the plasma membrane in human embryonic kidney cells and showed predominant endoplasmic reticulum immunolocalization in both human embryonic kidney and LLC-PK1 cells. The oligosaccharide on a kAE1 S773P N-glycosylation mutant (N555) was not processed to the complex form indicating impaired exit from the endoplasmic reticulum. The kAE1 S773P mutant showed decreased binding to an inhibitor affinity resin and increased sensitivity to proteases, suggesting that it was not properly folded. The other recessive dRTA mutant, kAE1 G701D, also exhibited defective trafficking to the plasma membrane. The recessive kAE1 mutants formed dimers like wild-type AE1 and could hetero-oligomerize with wild-type kAE1 or with each other. Hetero-oligomers of wild-type kAE1 with recessive kAE1 S773P or G701D, in contrast to the dominant kAE1 R589H mutant, were delivered to the plasma membrane.
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PMID:Trafficking defects of a novel autosomal recessive distal renal tubular acidosis mutant (S773P) of the human kidney anion exchanger (kAE1). 1525 44

A widely expressed protein containing UBA (ubiquitin-associated) and UBX (ubiquitin-like) domains was identified as a substrate of SAPKs (stress-activated protein kinases). Termed SAKS1 (SAPK substrate-1), it was phosphorylated efficiently at Ser200 in vitro by SAPK3/p38gamma, SAPK4/p38delta and JNK (c-Jun N-terminal kinase), but weakly by SAPK2a/p38alpha, SAPK2b/p38beta2 or ERK (extracellular-signal-regulated kinase) 2. Ser200, situated immediately N-terminal to the UBX domain, became phosphorylated in HEK-293 (human embryonic kidney) cells in response to stressors. Phosphorylation was not prevented by SB 203580 (an inhibitor of SAPK2a/p38alpha and SAPK2b/p38beta2) and/or PD 184352 (which inhibits the activation of ERK1 and ERK2), and was similar in fibroblasts lacking both SAPK3/p38gamma and SAPK4/p38delta or JNK1 and JNK2. SAKS1 bound ubiquitin tetramers and VCP (valosin-containing protein) in vitro via the UBA and UBX domains respectively. The amount of VCP in cell extracts that bound to immobilized GST (glutathione S-transferase)-SAKS1 was enhanced by elevating the level of polyubiquitinated proteins, while SAKS1 and VCP in extracts were coimmunoprecipitated with an antibody raised against S5a, a component of the 19 S proteasomal subunit that binds polyubiquitinated proteins. PNGase (peptide N-glycanase) formed a 1:1 complex with VCP and, for this reason, also bound to immobilized GST-SAKS1. We suggest that SAKS1 may be an adaptor that directs VCP to polyubiquitinated proteins, and PNGase to misfolded glycoproteins, facilitating their destruction by the proteasome.
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PMID:A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs. 1536 74

Topological studies of multi-spanning membrane proteins commonly use sequentially truncated proteins fused to a C-terminal translocation reporter to deduce transmembrane (TM) segment orientation and key biogenesis events. Because these truncated proteins represent an incomplete stage of synthesis, they transiently populate intermediate folding states that may or may not reflect topology of the mature protein. For example, in Xenopus oocytes, the aquaporin-1 (AQP1) water channel is cotranslationally directed into a four membrane-spanning intermediate, which matures into the six membrane-spanning topology at a late stage of synthesis (Skach, W. R., Shi, L. B., Calayag, M. C., Frigeri, A., Lingappa, V. R., and Verkman, A. S. (1994) J. Cell Biol. 125, 803-815 and Lu, Y., Turnbull, I. R., Bragin, A., Carveth, K., Verkman, A. S., and Skach, W. R. (2000) Mol. Biol. Cell 11, 2973-2985). The hallmark of this process is that TM3 initially acquires an Nexo/Ccyto (Type I) topology and must rotate 180 degrees to acquire its mature orientation. In contrast, recent studies in HEK-293 cells have suggested that TM3 acquires its mature topology cotranslationally without the need for reorientation (Dohke, Y., and Turner, R. J. (2002) J. Biol. Chem. 277, 15215-15219). Here we re-examine AQP1 biogenesis and show that irrespective of the reporter or fusion site used, oocytes and mammalian cells yielded similar topologic results. AQP1 intermediates containing the first three TM segments generated two distinct cohorts of polypeptides in which TM3 spanned the ER membrane in either an Ncyto/Cexo (mature) or Nexo/Ccyto (immature) topology. Pulse-chase analyses revealed that the immature form was predominant immediately after synthesis but that it was rapidly degraded via the proteasome-mediated endoplasmic reticulum associated degradation (ERAD) pathway with a half-life of less than 25 min in HEK cells. As a result, the mature topology predominated at later time points. We conclude that (i) differential stability of biogenesis intermediates is an important factor for in vivo topological analysis of truncated chimeric proteins and (ii) cotranslational events of AQP1 biogenesis reflect a common AQP1 folding pathway in diverse expression systems.
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PMID:Differential stability of biogenesis intermediates reveals a common pathway for aquaporin-1 topological maturation. 1551 32

Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder caused by expansion of the polyglutamine tract in the SCA1 gene product, ataxin-1. Using d2EGFP, a short-lived enhanced green fluorescent protein, we investigated whether polyglutamine-expanded ataxin-1 affects the function of the proteasome, a cellular multicatalytic protease that degrades most misfolded proteins and regulatory proteins. In Western blot analysis and immunofluorescence experiments, d2EGFP was less degraded in HEK 293T cells transfected with ataxin-1(82Q) than in cells transfected with lacZ or empty vector controls. To test whether the stability of the d2EGFP protein was due to aggregation of ataxin-1, we constructed a plasmid carrying ataxin-1-Delta114, lacking the self-association region (SAR), and examined degradation of the d2EGFP. Both the level of ataxin-1-Delta114 aggregates and the amount of d2EGFP were drastically reduced in cells containing ataxin-1-Delta114. Furthermore, d2EGFP localization experiments showed that polyglutamine-expanded ataxin-1 inhibited the general function of the proteasome activity. Taken together, these results demonstrate that polyglutamine-expanded ataxin-1 decreases the activity of the proteasome, implying that a disturbance in the ubiquitin-proteasome pathway is directly involved in the development of spinocerebellar ataxia type1.
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PMID:Proteasome function is inhibited by polyglutamine-expanded ataxin-1, the SCA1 gene product. 1575 Mar 36

Components of the ubiquitin-proteasome system function on the surface of the endoplasmic reticulum (ER) to select misfolded proteins for degradation. Herein we describe methods that allow for the study of the pathway for proteasomal degradation of the cystic fibrosis transmembrane conductance regulator (CFTR). The experimental system described employs transiently transfected HEK-293 cells and is utilized to monitor the biogenesis of CFTR by Western blot and pulse-chase analysis.
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PMID:Cystic fibrosis transmembrane conductance regulator as a model substrate to study endoplasmic reticulum protein quality control in mammalian cells. 1591 41


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