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)

The cytochrome P-450 family of enzymes performs an incredibly diverse range of detoxification and oxidation reactions within the cell and constitutes between 5 and 10% of protein in hepatic endoplasmic reticulum. In this report it is demonstrated that constitutively expressed membranous P-450s are targeted for destruction by the proteasome, in a process which is ubiquitin-independent and is demonstrated in vitro to require prior labilization of the enzyme. This process was specific for P-450s CYP1A2, CYP2E1, CYP3A, and CYP4A and was not demonstrated to be involved in the turnover of CYP1A1, CYP2B1/2, or NADPH reductase. In reconstitution experiments using purified proteasomes and microsomal fractions, labilized P-450 conformations are protected from 20 S proteasome degradation by substrate addition, with proteolysis occurring while P-450s are still attached to the endoplasmic reticulum.
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PMID:Evidence of proteasome-mediated cytochrome P-450 degradation. 909 10

Cytochrome P450, CYP3A4, is the dominant human liver endoplasmic reticulum (ER) hemoprotein enzyme, responsible for the metabolism of over 60% of clinically relevant drugs. We have previously shown that mechanism-based suicide inactivation of CYP3A4 and its rat liver ER orthologs, CYPs 3A, via heme-modification of their protein moieties, results in their ubiquitin (Ub)-dependent 26S proteasomal degradation (Korsmeyer et al. (1999) Arch. Biochem. Biophys. 365, 31; Wang et al. (1999) Arch. Biochem. Biophys. 365, 45). This is not surprising given that the heme-modified CYP3A proteins are structurally damaged. To determine whether the turnover of the native enzyme similarly recruited this pathway, we heterologously expressed this protein in wild-type Saccharomyces cerevisiae and mutant strains (hrd1Delta, hrd2-1, and hrd3Delta) previously shown to be deficient in the Ub-dependent 26S proteasomal degradation of the polytopic ER protein 3-hydroxy-3-methylglutaryl-CoA reductase (isoform Hmg2p), the rate-limiting enzyme in sterol biosynthesis, as well as in strains deficient in ER-associated Ub-conjugating enzymes, Ubc6p and/or Ubc7p (Hampton et al. (1996) Mol. Biol. Cell 7, 2029; Hampton and Bhakta (1997) Proc. Natl. Acad. Sci. USA 94, 12,944). Our findings reveal that in common with the degradation of Hmg2p, that of native CYP3A4 also requires Hrd2p (a subunit of the 19S cap complex of the 26S proteasome) and Ubc7p, and to a much lesser extent Hrd3p, a component of the ER-associated Ub-ligase complex. In contrast to Hmg2p-degradation, that of native CYP3A4 does not appear to absolutely require Hrd1p, another component of the ER-associated Ub-ligase complex. Furthermore, studies in a S. cerevisiae pep4Delta strain proven to be deficient in the vacuolar degradation of carboxypeptidase Y indicated that CYP3A4 degradation is also largely independent of vacuolar (lysosomal) proteolytic function. The degradation of two other native ER proteins, Sec61p and Sec63p, normal components of the ER translocon, were also examined in parallel and found to be stabilized to some extent in HRD2- and UBC7-deficient strains. Together these findings attest to the remarkable mechanistic diversity in the normal degradation of ER proteins.
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PMID:Ubiquitin-dependent 26S proteasomal pathway: a role in the degradation of native human liver CYP3A4 expressed in Saccharomyces cerevisiae? 1151 67

We characterize a novel microsome system that forms high-molecular-mass (HMM) CYP3A, CYP2E1, and ubiquitin conjugates, but does not alter CYP4A or most other microsomal proteins. The formation of the HMM bands was observed in hepatic microsomes isolated from rats treated 1 week or more with high doses (50 mg/kg/day) of nicardipine, clotrimazole, or pregnenolone 16alpha-carbonitrile, but not microsomes from control, dexamethasone-, nifedipine-, or diltiazem-treated rats. Extensive washing of the microsomes to remove loosely attached proteins or cytosolic contaminants did not prevent the conjugation reaction. In contrast to prototypical ubiquitination pathways, this reaction did not require addition of ubiquitin, ATP, Mg(2+), or cytosol. Addition of cytosol did result in the degradation of the HMM CYP3A bands in a process that was not blocked by proteasome inhibitors. Immunoprecipitated CYP3A contained HMM ubiquitin. Even so, mass spectrometric analysis of tryptic peptides indicated that the HMM CYP3A was in molar excess to ubiquitin, suggesting that the formation of the HMM CYP3A may have resulted from conjugation to itself or a diffuse pool of ubiquitinated proteins already present in the microsomes. Addition of CYP3A substrates inhibited the formation of the HMM CYP3A and the cytosol-dependent degradation of HMM CYP3A. These results suggest that after extended periods of elevated CYP3A expression, microsomal factors are induced that catalyze the formation of HMM CYP3A conjugates that contain ubiquitin. This conjugation reaction, however, seems to be distinct from the classical ubiquitination pathway but may be related to the substrate-dependent stabilization of CYP3A observed in vivo.
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PMID:Cytochrome P450 3A conjugation to ubiquitin in a process distinct from classical ubiquitination pathway. 1190 Dec 29

We have previously reported that CYP3A cross-links with polyubiquitinated proteins in microsomes from nicardipine-treated rats in a process that is distinct from classical polyubiquitination. To further examine the role of the proteasome in CYP3A degradation, we investigated the effects of proteasome inhibitors lactacystin, MG132, proteasome inhibitor 1, and hemin in primary cultures of rat and human hepatocytes. With the exception of hemin, these agents increased the total pool of ubiquitinated proteins in microsomes isolated from rat hepatocytes, indicating that lactacystin, MG132, and proteasome inhibitor 1 effectively inhibited the proteasome in these cells. All four agents caused a reduction in the amount of the major approximately 55-kDa CYP3A band, opposite to what would be expected if the ubiquitin-proteasome pathway degraded CYP3A. Only hemin treatment caused an increase in high molecular mass (HMM) CYP3A bands. Because hemin treatment did not alter levels of ubiquitin in CYP3A immunoprecipitates, the HMM CYP3A bands formed in response to hemin treatment clearly were not due to proteasome inhibition. Rather, because hemin treatment also caused an increase in HMM CYP3A in the detergent-insoluble fraction of the 10,000g pellet, the HMM CYP3A seems to represent a large protein complex that is unlikely to primarily represent ubiquitination.
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PMID:Suppression of cytochrome P450 3A protein levels by proteasome inhibitors. 1262 52

The hepatic cytochromes P450 (P450s) are monotopic endoplasmic reticulum (ER)-anchored hemoproteins engaged in the enzymatic oxidation of a wide variety of endo- and xenobiotics. In the course of these reactions, the enzymes generate reactive O(2) species and/or reactive metabolic products that can attack the P450 heme and/or protein moiety and structurally and functionally damage the enzyme. The in vivo conformational unraveling of such a structurally damaged P450 signals its rapid removal via the cellular sanitation system responsible for the proteolytic disposal of structurally aberrant, abnormal, and/or otherwise malformed proteins. A key player in this process is the ubiquitin (Ub)-dependent 26S proteasome system. Accordingly, the structurally deformed P450 protein is first branded for recognition and proteolytic removal by the 26S proteasome with an enzymatically incorporated polyUb tag. P450s of the 3A subfamily such as the major human liver enzyme CYP3A4 are notorious targets for this process, and they represent excellent prototypes for the understanding of integral ER protein ubiquitination. Not all the participants in hepatic CYP3A ubiquitination and subsequent proteolytic degradation have been identified. The following discussion thus addresses the various known and plausible events and/or cellular participants involved in this multienzymatic P450 ubiquitination cascade, on the basis of our current knowledge of other eukaryotic models. In addition, because the detection of ubiquitinated P450s is technically challenging, the critical importance of appropriate methodology is also discussed.
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PMID:Cytochrome P450 ubiquitination: branding for the proteolytic slaughter? 1582 84

Bortezomib (Velcade, PS-341), a dipeptidyl boronic acid, is a first-in-class proteasome inhibitor approved in 2003 for the treatment of multiple myeloma. In a preclinical toxicology study, bortezomib-treated rats resulted in liver enlargement (35%). Ex vivo analyses of the liver samples showed an 18% decrease in cytochrome P450 (P450) content, a 60% increase in palmitoyl coenzyme A beta-oxidation activity, and a 41 and 23% decrease in CYP3A protein expression and activity, respectively. Furthermore, liver samples of bortezomib-treated rats had little change in CYP2B and CYP4A protein levels and activities. To address the likelihood of clinical drug-drug interactions, the P450 inhibition potential of bortezomib and its major deboronated metabolites M1 and M2 and their dealkylated metabolites M3 and M4 was evaluated in human liver microsomes for the major P450 isoforms 1A2, 2C9, 2C19, 2D6, and 3A4/5. Bortezomib, M1, and M2 were found to be mild inhibitors of CYP2C19 (IC(50) approximately 18.0, 10.0, and 13.2 microM, respectively), and M1 was also a mild inhibitor of CYP2C9 (IC(50) approximately 11.5 microM). However, bortezomib, M1, M2, M3, and M4 did not inhibit other P450s (IC(50) values > 30 microM). There also was no time-dependent inhibition of CYP3A4/5 by bortezomib or its major metabolites. Based on these results, no major P450-mediated clinical drug-drug interactions are anticipated for bortezomib or its major metabolites. To our knowledge, this is the first report on P450-mediated drug-drug interaction potential of proteasome inhibitors or boronic acid containing therapeutics.
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PMID:Investigation of drug-drug interaction potential of bortezomib in vivo in female Sprague-Dawley rats and in vitro in human liver microsomes. 1644 66

Whereas many cytochrome P450 enzymes are transcriptionally suppressed by inflammatory stimuli, down-regulation of CYP2B protein by the inflammatory cytokine interleukin (IL)-1beta is nitric oxide (NO)-dependent and occurs via polyubiquitination and proteasomal degradation. Here, we used iTRAQ proteomic analysis to search for other proteins that are potentially down-regulated by cellular NO in cultured rat hepatocytes, and we identified CYP3A1 as one such protein. Therefore, we examined whether CYP3A proteins, like CYP2B, undergo NO- and proteasome-dependent degradation in response to cytokine treatment of rat hepatocytes. In cultured rat hepatocytes treated with phenobarbital, IL-1beta stimulation failed to down-regulate CYP3A1 mRNA within 24 h of treatment, whereas CYP3A protein was down-regulated to 40% of control within 6 h, showing the post-transcriptional down-regulation of CYP3A1 protein. The down-regulation of CYP3A after 9 h of stimulation by IL-1beta was attenuated by inhibitors of NO synthase (NOS) and of the proteasome, showing NO- and proteasome-dependent down-regulation at earlier time points. However, the down-regulation of CYP3A evoked by IL-1beta measured 24 h after stimulation was not affected by the inhibition of NOS or by proteasomal inhibitors, showing that CYP3A1 down-regulation at later time points is NO- and proteasome-independent. IL-6, which did not evoke NO production nor affect CYP3A1 mRNA within 24 h, produced a delayed proteasome-independent down-regulation as well. Taken together, these observations show a novel dual mode of post-transcriptional CYP3A down-regulation by cytokines: NO- and proteasome-dependent at earlier time points and NO- and proteasome-independent at later times.
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PMID:Dual mechanisms of CYP3A protein regulation by proinflammatory cytokine stimulation in primary hepatocyte cultures. 1917 75

Hepatic cytochromes P450 3A (P450s 3A) are endoplasmic reticulum (ER)-proteins, responsible for xenobiotic metabolism. They are degraded by the ubiquitin-dependent 26S proteasome. Consistent with this, we have shown that proteasomal inhibitors N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) and N-benzoyloxycarbonyl-Leu-Leu-Leu-B(OH)(2) (MG262) stabilize CYP3A proteins. However, MG132 has been reported to suppress P450s 3A as a result of impaired nuclear factor-kappaB activation and consequently reduced CYP3A protein stability. Because the MG132 concentration used in those studies was 10-fold higher than that required for CYP3A stabilization, we examined the effect of MG132 (0-300 microM) concentration-dependent proteasomal inhibition on CYP3A turnover in cultured primary rat hepatocytes. We found a biphasic MG132 concentration effect on CYP3A turnover: Stabilization at 5 to 10 muM with marked suppression at >100 microM. Proteasomal inhibitors reportedly induce ER stress, heat shock, and apoptotic response. At these high MG132 concentrations, such CYP3A suppression could be due to ER stress induction, so we monitored the activity of PERK [PKR (RNA-dependent protein kinase)-like ER kinase (EIF2AK3)], the ER stress-activated eukaryotic initiation factor 2alpha (eIF2alpha) kinase. Indeed, we found a marked (approximately 4-fold) MG132 concentration-dependent PERK autophosphorylation, along with an 8-fold increase in eIF2alpha-phosphorylation. In parallel, MG132 also activated GCN2 [general control nonderepressible-2 (EIF2AK4)] eIF2alpha kinase in a concentration-dependent manner, but not the heme-regulated inhibitor eIF2alpha kinase [(EIF2AK1)]. Pulse-chase, immunoprecipitation/immunoblotting analyses documented the consequently dramatic translational shutoff of total hepatic protein, including but not limited to CYP3A and tryptophan 2,3-dioxygenase protein syntheses. These findings reveal that at high concentrations, MG132 is indeed cytotoxic and can suppress CYP3A synthesis, a result confirmed by confocal immunofluorescence analyses of MG132-treated hepatocytes.
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PMID:Hepatic CYP3A suppression by high concentrations of proteasomal inhibitors: a consequence of endoplasmic reticulum (ER) stress induction, activation of RNA-dependent protein kinase-like ER-bound eukaryotic initiation factor 2alpha (eIF2alpha)-kinase (PERK) and general control nonderepressible-2 eIF2alpha kinase (GCN2), and global translational shutoff. 1952 Aug 99

Proteasome inhibitors are important tools for studying the roles of the proteasome in cellular processes. In this study, we observed that the proteasome inhibitors N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132), epoxomicin, and lactacystin were ineffective and bortezomib was completely effective in inhibiting cytokine-stimulated nitric oxide production in primary cultures of human hepatocytes that had been treated with the cytochrome P450 inducer phenobarbital. The inefficacy of MG132 was due to its metabolism by CYP3A enzymes, as deduced from its rapid, ketoconazole-sensitive clearance by pooled human liver microsomes and cultured hepatocytes. The efficacy of MG132 was increased by inclusion of ketoconazole in the hepatocyte incubations and decreased by prior treatment of the cultures with the CYP3A inducers phenobarbital or rifampicin. Epoxomicin was also rapidly metabolized by CYP3A, whereas bortezomib and lactacystin were much more stable metabolically in human liver microsomes or hepatocyte cultures. Thus, bortezomib is a better choice than MG132, epoxomicin, or lactacystin in cells with high activities of CYP3A enzymes. The reason for the lack of efficacy of lactacystin in human hepatocytes has yet to be determined, but it too should not be used for studies of proteasome function in human hepatocytes.
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PMID:Metabolism and action of proteasome inhibitors in primary human hepatocytes. 2083 60

The CYP3A subfamily of hepatic cytochromes P450, being engaged in the metabolism and clearance of >50% of clinically relevant drugs, can significantly influence therapeutics and drug-drug interactions. Our characterization of CYP3A degradation has indicated that CYPs 3A incur ubiquitin-dependent proteasomal degradation (UPD) in an endoplasmic reticulum (ER)-associated degradation (ERAD) process. Cytochromes P450 are monotopic hemoproteins N-terminally anchored to the ER membrane with their protein bulk readily accessible to the cytosolic proteasome. Given this topology, it was unclear whether they would require the AAA-ATPase p97 chaperone complex that retrotranslocates/dislocates ubiquitinated ER-integral and luminal proteins into the cytosol for proteasomal delivery. To assess the in vivo relevance of this p97-CYP3A association, we used lentiviral shRNAs to silence p97 (80% mRNA and 90% protein knockdown relative to controls) in sandwich-cultured rat hepatocytes. This extensive hepatic p97 knockdown remarkably had no effect on cellular morphology, ER stress, and/or apoptosis, despite the well recognized strategic p97 roles in multiple important cellular processes. However, such hepatic p97 knockdown almost completely abrogated CYP3A extraction into the cytosol, resulting in a significant accumulation of parent and ubiquitinated CYP3A species that were firmly ER-tethered. Little detectable CYP3A accumulated in the cytosol, even after concomitant inhibition of proteasomal degradation, thereby documenting a major role of p97 in CYP3A extraction and delivery to the 26 S proteasome during its UPD/ERAD. Intriguingly, the accumulated parent CYP3A was functionally active, indicating that p97 can regulate physiological CYP3A content and thus influence its clinically relevant function.
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PMID:Liver cytochrome P450 3A endoplasmic reticulum-associated degradation: a major role for the p97 AAA ATPase in cytochrome P450 3A extraction into the cytosol. 2110 9


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