CYP2E1 Degradation by in Vitro Reconstituted Systems: Role of the Molecular Chaperone hsp90

doi:10.1006/abbi.2000.1870

Archives of Biochemistry and Biophysics

Volume 379, Issue 2, 15 July 2000, Pages 321-330

https://doi.org/10.1006/abbi.2000.1870 Get rights and content

Abstract

One major mode of regulation of cytochrome P450 2E1 (CYP2E1) is at the posttranscriptional level, since many low-molecular-weight compounds stabilize the enzyme against proteolysis by the proteasome complex. In an in vitro system containing human liver microsomes, degradation of CYP2E1 in the microsomes required addition of the human liver cytosol fraction in a reaction sensitive to inhibitors of the proteasome complex. It is not clear how CYP2E1 in the microsomal membrane becomes accessible to the cytosolic proteasome. Since molecular chaperones play a role in protein folding and degradation, the possible role of heat shock proteins in CYP2E1 degradation by this reconstituted system was evaluated. Degradation of CYP2E1 required ATP; ATP-γS, a nonhydrolyzable analogue of ATP, did not catalyze CYP2E1 degradation by the cytosol fraction, indicating that ATP hydrolysis is required. Geldanamycin, a specific inhibitor of hsp90, inhibited the degradation of microsomal CYP2E1 by the cytosol fraction. Control experiments indicated that geldanamycin was not a substrate/ligand of CYP2E1 nor did it prevent microsomal lipid peroxidation, a process which increases CYP2E1 turnover. Inhibition by geldanamycin was prevented by molybdate. Both of these compounds have been shown to promote alterations in hsp90 structure and to modulate hsp90–protein interactions. The proteasome activity in the cytosol, as assayed by the cleavage of a fluorogenic peptide, was enhanced when ATP was added and inhibited by 30–40% by geldanamycin, effects that are similar, although less pronounced, to the degradation of CYP2E1 by the cytosol. Purified 20S proteasome could catalyze degradation of CYP2E1; however, in an assay using equal peptidase activity, the cytosol fraction was much more effective than the 20S proteasome in promoting CYP2E1 degradation. Immunodepletion of hsp90 from the cytosol resulted in prevention of the degradation of CYP2E1, a reaction that was reversed by the addition of pure hsp90 to this cytosol. These results suggest that in addition to the proteasome, the cytosol fraction contains other factors that modulate the efficiency of CYP2E1 degradation. The sensitivity to geldanamycin and molybdate and the immunodepletion experiments suggest that hsp90 is one of these factors that interact with CYP2E1 and/or with the proteasome to promote the degradation of this microsomal P450.

References (49)

E. Eliasson et al.
Biochem. Biophys. Res. Commun.
(1988)
B.J. Song et al.
J. Biol. Chem.
(1989)
B.J. Roberts et al.
J. Biol. Chem.
(1995)
D.J. Tierney et al.
Arch. Biochem. Biophys.
(1992)
B.J. Roberts
J. Biol. Chem.
(1997)
M.X. Yang et al.
Biochem. Biophys. Res. Commun.
(1996)
T. Goasduff et al.
Arch. Biochem. Biophys.
(1999)
K.K. Korsmeyer et al.
Arch. Biochem. Biophys.
(1999)
H.F. Wang et al.
Arch. Biochem. Biophys.
(1999)
J.L. Johnson et al.
J. Biol. Chem.
(1994)

W. Sullivan et al.

J. Biol. Chem.

(1997)

J.P. Grenert et al.

J. Biol. Chem.

(1997)

C. Prodromou et al.

Cell

(1997)

M. Sakagami et al.

Cell Stress Chaperones

(1999)

E. Eliasson et al.

J. Biol. Chem.

(1992)

A. Zhukov et al.

Biochem. Biophys. Res. Commun.

(1993)

C. Realini et al.

J. Biol. Chem.

(1995)

S. Tsubuki et al.

FEBS Lett.

(1994)

M. Chu-Ping et al.

J. Biol. Chem.

(1994)

W. Dubiel et al.

J. Biol. Chem.

(1992)

K. Tanaka et al.

J. Biol. Chem.

(1986)

T. Tokumoto et al.

Biochem. Biophys. Res. Commun.

(1993)

M.J.M. McGuire et al.

Biochim. Biophys. Acta

(1989)

L.D. Gorsky et al.

J. Biol. Chem.

(1984)

Cited by (54)

Oncogenic acidic nuclear phosphoproteins ANP32C/D are novel clients of heat shock protein 90
2015, Biochimica et Biophysica Acta - Molecular Cell Research
Citation Excerpt :
Indeed recent studies have shown that Hsp90-dependent folding and degradation are executed by distinct molecular complexes containing variable levels of Hsp70, CHIP and HOP activity that make folding predominant under normal conditions [49]. Hsp90 also actively participates in the proteasomal degradation of selected clients [35], including membrane proteins CYP2E1 [50], delta F508 CFTR mutant [51], apoprotein B [52], mutated forms of lysosomal enzyme glucocerebrosidase [53], misfolded or mutated cytosolic protein VHL [54], and chaperone-mediated autophagy for selective degradation of cytosolic proteins in lysosomes [55]. Interestingly, there are some reports of Hsp90 clients that do not undergo degradation when Hsp90 is inhibited with geldanamycin or PU-H71 but demonstrate subtle changes in their phosphorylation status (e.g. STAT3/5) [26,47].
The acidic nuclear phosphoproteins (ANP32A-H) are an evolutionarily conserved family of proteins with diverse and sometimes opposing cellular functions. Here we show that the oncogenic family members ANP32C and ANP32D are associated in complexes containing the molecular chaperone Hsp90. The oncogenic ANP32C protein appears to be highly unstable with a rapid degradation (t_1/2 > 30 min) occurring upon treatment of cells with cycloheximide. ANP32C was also found to be associated with oncogenic Hsp90 complexes by virtue of its ability to interact and be immunoprecipitated by the Hsp90 inhibitor PU-H71. Further studies treating cells with the Hsp90 inhibitors PU-H71 and 17-AAG showed atypical increased protein stability and prevention of ANP32C degradation compared to the Hsp90 client AKT. Cells overexpressing ANP32C or its mutant ANP32CY140H showed enhanced sensitivity to treatment with PU-H71 as demonstrated by CCK-8 and colony formation assays. Our results highlight that certain malignancies with ANP32C/D overexpression or mutation might be specifically targeted using Hsp90 inhibitors.
Effect of maternal folic acid supplementation on hepatic proteome in newborn piglets
2013, Nutrition
Citation Excerpt :
Specifically, HSPs maintain the structural and functional integrity of several client proteins to promote cell survival, proliferation, and regulate cell apoptosis [21]. The HSP90 occupies nearly 2% of total protein in most non-stressed cells and is involved in essential physiologic processes, such as protein trafficking and signal transduction and protein degradation, regulation, and stabilization in a wide range of client proteins [22–24]. Previous research has indicated that dietary folic acid deficiency results in a sharp decrease of hepatic HSP70 [17].
The objective of this study was to investigate the changes in the hepatic proteome of newborn piglets after maternal folic acid supplementation (FS).
Pregnant dams were fed a control diet (folic acid 1.3 mg/kg) or an FS diet (folic acid 30 mg/kg) during gestation. The liver samples of newborn piglets from each group were collected at birth for the analysis of the proteome using two-dimensional difference gel electrophoresis.
The results indicated that the expression levels of 11 proteins were changed dramatically in the newborn piglets after maternal FS. FS during gestation increased the content of proteins that regulate the immune response (90-kDa heat shock protein), energy metabolism (aconitase and succinate dehydrogenase), and intermediary metabolism (formiminotransferase cyclodeaminase and abhydrolase). In addition, maternal FS downregulated the expression of proteins associated with cellular signal transduction (heterogeneous nuclear ribonucleoprotein and exportin), proteolysis (ubiquitin-conjugating enzyme and porcine ε-trypsin), and cell migration regulation (actin-related protein-3).
These findings suggest that maternal FS alters the expression abundance of several hepatic proteins that are involved in metabolic regulation, oxidative responses, and cancer-related processes.
Quality control and fate determination of Hsp90 client proteins
2012, Biochimica et Biophysica Acta - Molecular Cell Research
Citation Excerpt :
These include cytochrome p450 2E1, mutant CFTR∆F508 and Apolipoprotein B. In each of these cases, geldanamycin was found to inhibit degradation in cell free systems when the substrate was added to cytosolic extracts in the form of microsomes. [44–46]. The degradation of a mutant form of the insulin receptor was also reduced upon injection of Hsp90 antibodies into cells [47].
Quality control processes regulate the proteome by determining whether a protein is to be folded or degraded. Hsp90 is a hub in the network of molecular chaperones that maintain this process because it promotes both folding and degradation, in addition to regulating expression of other quality control components. The significance of Hsp90's role in quality control is enhanced by the function of its clients, which include protein kinases and transcription factors, in cellular signaling. The inhibition of Hsp90 with small molecules results in the rapid degradation of such clients via the ubiquitin/proteasome pathway, and also in the induction of the Hsp70 molecular chaperone. These two events result in markedly different outcomes depending on cell type. For tumor cells there is a profound loss of signaling in growth promoting pathways. By contrast, increased amounts of Hsp70 in neuronal cells ameliorate the toxicity that is associated with the formation of aggregates observed in neurodegenerative conditions. In this review we discuss the mechanisms underlying these differential effects of Hsp90 inhibition on the quality control of distinct client proteins. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).
Ubiquitin-dependent proteasomal degradation of human liver cytochrome P450 2E1: Identification of sites targeted for phosphorylation and ubiquitination
2011, Journal of Biological Chemistry
Citation Excerpt :
Such substrate-mediated P450 induction and/or enhanced turnover can influence the severity and the time course of certain pharmacokinetic/pharmacodynamic drug-drug interactions and is an important therapeutic consideration (24–27). P450 turnover has been proposed to involve various proteolytic mechanisms (6–9, 28–38). However, it is now increasingly evident that in common with other type I monotopic ER proteins, P450s such as CYPs 3A (both native and structurally inactivated) undergo ER-associated degradation (ERAD) involving the ubiquitin (Ub)-dependent 26 S proteasomal system (UPS) (6–9, 12–16).
Human liver CYP2E1 is a monotopic, endoplasmic reticulum-anchored cytochrome P450 responsible for the biotransformation of clinically relevant drugs, low molecular weight xenobiotics, carcinogens, and endogenous ketones. CYP2E1 substrate complexation converts it into a stable slow-turnover species degraded largely via autophagic lysosomal degradation. Substrate decomplexation/withdrawal results in a fast turnover CYP2E1 species, putatively generated through its futile oxidative cycling, that incurs endoplasmic reticulum-associated ubiquitin-dependent proteasomal degradation (UPD). CYP2E1 thus exhibits biphasic turnover in the mammalian liver. We now show upon heterologous expression of human CYP2E1 in Saccharomyces cerevisiae that its autophagic lysosomal degradation and UPD pathways are evolutionarily conserved, even though its potential for futile catalytic cycling is low due to its sluggish catalytic activity in yeast. This suggested that other factors (i.e. post-translational modifications or “degrons”) contribute to its UPD. Indeed, in cultured human hepatocytes, CYP2E1 is detectably ubiquitinated, and this is enhanced on its mechanism-based inactivation. Studies in Ubc7p and Ubc5p genetically deficient yeast strains versus corresponding isogenic wild types identified these ubiquitin-conjugating E2 enzymes as relevant to CYP2E1 UPD. Consistent with this, in vitro functional reconstitution analyses revealed that mammalian UBC7/gp78 and UbcH5a/CHIP E2-E3 ubiquitin ligases were capable of ubiquitinating CYP2E1, a process enhanced by protein kinase (PK) A and/or PKC inclusion. Inhibition of PKA or PKC blocked intracellular CYP2E1 ubiquitination and turnover. Here, through mass spectrometric analyses, we identify some CYP2E1 phosphorylation/ubiquitination sites in spatially associated clusters. We propose that these CYP2E1 phosphorylation clusters may serve to engage each E2-E3 ubiquitination complex in vitro and intracellularly.
Impaired methylation as a novel mechanism for proteasome suppression in liver cells
2010, Biochemical and Biophysical Research Communications
The proteasome is a multi-catalytic protein degradation enzyme that is regulated by ethanol-induced oxidative stress; such suppression is attributed to CYP2E1-generated metabolites. However, under certain conditions, it appears that in addition to oxidative stress, other mechanisms are also involved in proteasome regulation. This study investigated whether impaired protein methylation that occurs during exposure of liver cells to ethanol, may contribute to suppression of proteasome activity. We measured the chymotrypsin-like proteasome activity in Huh7CYP cells, hepatocytes, liver cytosols and nuclear extracts or purified 20S proteasome under conditions that maintain or prevent protein methylation. Reduction of proteasome activity of hepatoma cell and hepatocytes by ethanol or tubercidin was prevented by simultaneous treatment with S-adenosylmethionine (SAM). Moreover, the tubercidin-induced decline in proteasome activity occurred in both nuclear and cytosolic fractions. In vitro exposure of cell cytosolic fractions or highly purified 20S proteasome to low SAM:S-adenosylhomocysteine (SAH) ratios in the buffer also suppressed proteasome function, indicating that one or more methyltransferase(s) may be associated with proteasomal subunits. Immunoblotting a purified 20S rabbit red cell proteasome preparation using methyl lysine-specific antibodies revealed a 25 kDa proteasome subunit that showed positive reactivity with anti-methyl lysine. This reactivity was modified when 20S proteasome was exposed to differential SAM:SAH ratios. We conclude that impaired methylation of proteasome subunits suppressed proteasome activity in liver cells indicating an additional, yet novel mechanism of proteasome activity regulation by ethanol.
Proteasome Activation by Hepatitis C Core Protein Is Reversed by Ethanol-Induced Oxidative Stress
2008, Gastroenterology
Citation Excerpt :
The nature of the putative proteasome-interacting protein(s) in mitochondria and ER is not clear. Heat shock proteins (HSP) are potential candidates for providing communication between mitochondria, microsomes, and the proteasome because the role of HSP in interactions between the microsomal enzyme CYP2E1 and the proteasome has been established.33 Mitochondria may also affect the activity of 20S-PA28 complex via interactions between mitochondrial HSP70 and PA28 because mitochondria express HSP7034 and the association between HSP70 and PA28 during substrate refolding has been reported.35
Background & Aims: The proteasome is a major cellular proteinase. Its activity is modulated by cellular oxidants. Hepatitis C core protein and ethanol exposure both cause enhanced oxidant generation. The aim was to investigate whether core protein, by its ability to generate oxidants, alters proteasome activity and whether these alterations are further affected by ethanol exposure. Methods: These interactions were examined in Huh-7 cell lines that expressed inducible HCV core protein and/or constitutive cytochrome P450 2E1 (CYP2E1) and as purified components in a cell-free system. Chymotrypsin-like proteasome activity was measured fluorometrically. Results: Proteasome activity in core-positive 191-20 cells was 20% higher than that in core-negative cells and was enhanced 3-fold in CYP2E1-expressing L14 cells. Exposure of core-positive cells to glutathione ethyl ester, catalase, or the CYP2E1 inhibitor diallyl sulfide partially reversed the elevation of proteasome activity in core-positive cells, whereas ethanol exposure suppressed proteasome activity. The results indicate that proteasome activity was up-regulated by low levels of core-induced oxidative stress but down-regulated by high levels of ethanol-elicited stress. These findings were partially mimicked in a cell-free system. Addition of core protein enhanced the peptidase activity of purified 20S proteasome containing the proteasome activator PA28 and was further potentiated by addition of liver mitochondrial and/or microsome fractions. However, proteasome activation was significantly attenuated when fractions were obtained from ethanol-fed animals. Conclusions: HCV core protein interacts with PA28, mitochondrial, and endoplasmic reticulum proteins to cause low levels of oxidant stress and proteasome activation, which is dampened during ethanol metabolism when oxidant generation is higher.

View all citing articles on Scopus

¹: To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, Box 1020, Mount Sinai School of Medicine, New York, NY 10029. Fax: (212) 996-7214.

View full text

Regular ArticleCYP2E1 Degradation by in Vitro Reconstituted Systems: Role of the Molecular Chaperone hsp90

Abstract

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell

Cell Stress Chaperones

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

J. Biol. Chem.

Regular Article
CYP2E1 Degradation by in Vitro Reconstituted Systems: Role of the Molecular Chaperone hsp90