CYP3A4 ubiquitination by gp78 (the tumor autocrine motility factor receptor, AMFR) and CHIP E3 ligases

Human liver CYP3A4 is an endoplasmic reticulum (ER)-anchored hemoprotein responsible for the metabolism of >50% of clinically prescribed drugs. After heterologous expression in Saccharomyces cerevisiae, it is degraded via the ubiquitin (Ub)-dependent 26S proteasomal pathway that utilizes Ubc7p/Cue1p, but none of the canonical Ub-ligases (E3s) Hrd1p/Hrd3p, Doa10p, and Rsp5p involved in ER-associated degradation (ERAD). To identify an Ub-ligase capable of ubiquitinating CYP3A4, we examined various in vitro reconstituted mammalian E3 systems, using purified and functionally characterized recombinant components. Of these, the cytosolic domain of the ER-protein gp78, also known as the tumor autocrine motility factor receptor (AMFR), an UBC7-dependent polytopic RING-finger E3, effectively ubiquitinated CYP3A4 in vitro, as did the UbcH5a-dependent cytosolic E3 CHIP. CYP3A4 immunoprecipitation coupled with anti-Ub immunoblotting analyses confirmed its ubiquitination in these reconstituted systems. Thus, both UBC7/gp78 and UbcH5a/CHIP may be involved in CYP3A4 ERAD, although their relative physiological contribution remains to be established.     

Liver Cytochrome P450 3A Ubiquitination in Vivo by gp78/Autocrine Motility Factor Receptor and C Terminus of Hsp70-interacting Protein (CHIP) E3 Ubiquitin Ligases: PHYSIOLOGICAL AND PHARMACOLOGICAL RELEVANCE*

CYP3A4 is a dominant human liver cytochrome P450 enzyme engaged in the metabolism and disposition of >50% of clinically relevant drugs and held responsible for many adverse drug-drug interactions. CYP3A4 and its mammalian liver CYP3A orthologs are endoplasmic reticulum (ER)-anchored monotopic proteins that undergo ubiquitin (Ub)-dependent proteasomal degradation (UPD) in an ER-associated degradation (ERAD) process. These integral ER proteins are ubiquitinated in vivo, and in vitro studies have identified the ER-integral gp78 and the cytosolic co-chaperone, CHIP (C terminus of Hsp70-interacting protein), as the relevant E3 Ub-ligases, along with their cognate E2 Ub-conjugating enzymes UBC7 and UbcH5a, respectively. Using lentiviral shRNA templates targeted against each of these Ub-ligases, we now document that both E3s are indeed physiologically involved in CYP3A ERAD/UPD in cultured rat hepatocytes. Accordingly, specific RNAi resulted in ≈80% knockdown of each hepatic Ub-ligase, with a corresponding ≈2.5-fold CYP3A stabilization. Surprisingly, however, such stabilization resulted in increased levels of functionally active CYP3A, thereby challenging the previous notion that E3 recognition and subsequent ERAD of CYP3A proteins required ab initio their structural and/or functional inactivation. Furthermore, coexpression in HepG2 cells of both CYP3A4 and gp78, but not its functionally inactive RING-finger mutant, resulted in enhanced CYP3A4 loss greater than that in corresponding cells expressing only CYP3A4. Stabilization of a functionally active CYP3A after RNAi knockdown of either of the E3s, coupled with the increased CYP3A4 loss on gp78 or CHIP coexpression, suggests that ERAD-associated E3 Ub-ligases can influence clinically relevant drug metabolism by effectively regulating the physiological CYP3A content and consequently its function.     

Ubiquitin ligase gp78 increases solubility and facilitates degradation of the Z variant of alpha-1-antitrypsin

Deficiency of circulating alpha-1-antitrypsin (AAT) is the most widely recognized abnormality of a proteinase inhibitor that causes lung disease. AAT-deficiency is caused by mutations of the AAT gene that lead to AAT protein retention in the endoplasmic reticulum (ER). Moreover, the mutant AAT accumulated in the ER predisposes the homozygote to severe liver injuries, such as neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. Despite the fact that mutant AAT protein is subject to ER-associated degradation (ERAD), yeast genetic studies have determined that the ubiquitination machinery, Hrd1/Der3p-cue1p-Ubc7/6p, which plays a prominent role in ERAD, is not involved in degradation of mutant AAT. Here we report that gp78, a ubiquitin ligase (E3) pairing with mammalian Ubc7 for ERAD, ubiquitinates and facilitates degradation of ATZ, the classic deficiency variant of AAT having a Z mutation (Glu 342 Lys). Unexpectedly, gp78 over-expression also significantly increases ATZ solubility. p97/VCP, an AAA ATPase essential for retrotranslocation of misfolded proteins from the ER during ERAD, is involved in gp78-mediated degradation of ATZ. Surprisingly, unlike other ERAD substrates that cause ER stress leading to apoptosis when accumulated in the ER, ATZ, in fact, increases cell proliferation when over-expressed in cells. This effect can be partially inhibited by gp78 over-expression. These data indicate that gp78 assumes multiple unique quality control roles over ATZ, including the facilitation of degradation and inhibition of aggregation of ATZ.     

Ubiquitin-dependent proteasomal degradation of human liver cytochrome P450 2E1: identification of sites targeted for phosphorylation and ubiquitination

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.     

The E3 Ubiquitin Ligase gp78 Protects against ER Stress in Zebrafish Liver

Enhanced endoplasmic reticulum （ER）-associated protein degradation （ERAD） activity by the unfolded protein response （UPR） represents one of the mechanisms for restoring ER homeostasis. In vitro evidence indicates that the mammalian gp78 protein is an E3 ubiquitin ligase that facilitates ERAD by polyubiquitinating and targeting proteins for proteasomal degradation under both physiologic and stress conditions. However, the in vivo function of gp78 in maintaining ER protein homeostasis remains untested. Here we show that like its mammalian counterpart, the zebrafish gp78 is also an E3 ubiquitin ligase as revealed by in vitro ubiquitination assays. Expression analysis uncovered that gp78 is highly expressed in several organs, including liver and brain, of both larval and adult fish. Treatment of larvae or adult fish with tunicamycin induces ER stress and upregulates the expression of several key components of the gp78 ERAD complex in the liver. Moreover, liver-specific overexpression of the dominant-negative form of gp78 （gp78-R2M） renders liver more sensitive to tunicamycin-induced ER stress and enhances the expression of sterol response element binding protein （Srebp）-target genes, which was largely suppressed in fish overexpressing wild-type gp78. Together, these data indicate that gp78 plays a critical role in protecting against ER stress in liver.     

A Role for Protein Phosphorylation in Cytochrome P450 3A4 Ubiquitin-dependent Proteasomal Degradation

Cytochromes P450 (P450s) incur phosphorylation. Although the precise role of this post-translational modification is unclear, marking P450s for degradation is plausible. Indeed, we have found that after structural inactivation, CYP3A4, the major human liver P450, and its rat orthologs are phosphorylated during their ubiquitin-dependent proteasomal degradation. Peptide mapping coupled with mass spectrometric analyses of CYP3A4 phosphorylated in vitro by protein kinase C (PKC) previously identified two target sites, Thr²⁶⁴ and Ser⁴²⁰. We now document that liver cytosolic kinases additionally target Ser⁴⁷⁸ as a major site. To determine whether such phosphorylation is relevant to in vivo CYP3A4 degradation, wild type and CYP3A4 with single, double, or triple Ala mutations of these residues were heterologously expressed in Saccharomyces cerevisiae pep4Δ strains. We found that relative to CYP3A4wt, its S478A mutant was significantly stabilized in these yeast, and this was greatly to markedly enhanced for its S478A/T264A, S478A/S420A, and S478A/T264A/S420A double and triple mutants. Similar relative S478A/T264A/S420A mutant stabilization was also observed in HEK293T cells. To determine whether phosphorylation enhances CYP3A4 degradation by enhancing its ubiquitination, CYP3A4 ubiquitination was examined in an in vitro UBC7/gp78-reconstituted system with and without cAMP-dependent protein kinase A and PKC, two liver cytosolic kinases involved in CYP3A4 phosphorylation. cAMP-dependent protein kinase A/PKC-mediated phosphorylation of CYP3A4wt but not its S478A/T264A/S420A mutant enhanced its ubiquitination in this system. Together, these findings indicate that phosphorylation of CYP3A4 Ser⁴⁷⁸, Thr²⁶⁴, and Ser⁴²⁰ residues by cytosolic kinases is important both for its ubiquitination and proteasomal degradation and suggest a direct link between P450 phosphorylation, ubiquitination, and degradation.Hepatic cytochromes P450 (P450s)³ are integral endoplasmic reticulum (ER)-anchored hemoproteins engaged in the oxidative biotransformation of various endo- and xenobiotics. Of these, human CYP3A4 is the most dominant liver enzyme, accounting for >30% of the hepatic microsomal P450 complement, and responsible for the oxidative metabolism of over 50% of clinically relevant drugs (1). In common with all the other ER-bound P450s, CYP3A4 is a monotopic protein with its N-terminal ≈33-residue domain embedded in the ER membrane with the bulk of its structure in the cytosol. Our in vivo studies of the heterologously expressed CYP3A4 in the yeast Saccharomyces cerevisiae as well as of its rat liver CYP3A2/3A23 orthologs in primary hepatocytes have revealed that human and rat liver CYPs 3A are turned over via ubiquitin (Ub)-dependent proteasomal degradation (UPD) (2–8). Thus, CYPs 3A represent excellent prototypic substrates of ER-associated degradation (ERAD), specifically of the ERAD-C pathway (6–11). Consistent with this CYP3A ERAD process, our studies of in vivo and/or in vitro reconstituted systems have led us to conclude that CYPs 3A are ubiquitinated by the UBC7/gp78 Ub-ligase complex and recruited by the p97-Npl4-Ufd1 complex before their degradation by the 26 S proteasome (4–8, 12). Because all these processes are energy-dependent, it is not surprising that in vitro reconstitution of CYP3A4 UPD requires ATP. However, inclusion of γ-S-[³²P]ATP in an in vitro reconstituted CYP3A4 ubiquitination system catalyzed by rat liver cytosolic fraction II (FII) resulted in CYP3A4 protein phosphorylation, i.e. γ-[³²P]phosphoryl transfer onto CYP3A4 target residues (13, 14). This phosphorylation was enhanced after cumene hydroperoxide (CuOOH)-mediated CYP3A4 inactivation. The physiological role, if any, of this CYP3A4 post-translational modification is unclear.CYP3A4 is not the only P450 that is phosphorylated. Since the in vitro phosphorylation of a hepatic P450 (CYP2B4) by cAMP-dependent protein kinase A (PKA) was first described (15), various P450s, particularly those belonging to the subfamily 2, were documented to be phosphorylated in cell-free systems, hepatocyte incubations, and intact animals (16–32). Common features of such P450 phosphorylation were the presence of a cytosolically exposed PKA recognition sequence (RRXS) with the Ser residue as the exclusive kinase target, and the ensuing loss of prosthetic heme, conversion to the inactive P420 species, and consequent dramatic functional inactivation (15–20). Studies in intact rats also identified CYPs 3A and 2C6 as kinase targets (21). Although both these P450s lack the hallmark PKA recognition sequence, apparently they possess secondary PKA targeting sequences or are phosphorylated by other protein kinases such as PKC. Indeed, in vitro studies revealed that P450s were phosphorylated in an isoform-dependent manner by either PKA or PKC, except for CYP2B1, which was heavily phosphorylated by both (20). Over the years since this particular post-translational P450 modification was recognized, it has been assigned various functional roles (17, 29–33). Among these, as first proposed by Taniguchi et al. (16) and later explored both by Eliasson et al. (23–26) and us (13, 14), P450 phosphorylation served as a marker for its degradation. Accordingly, the phosphorylation of CYP2E1Ser¹²⁹ and CYP3A1Ser³⁹³ by a microsomal cAMP-dependent protein kinase has been proposed to predispose these P450s but not the similarly phosphorylated CYP2B1 to proteolytic degradation by an integral ER Mg²⁺-ATP-activated serine protease (23–27). However, heterologous expression of CYP2E1S129A/S129G site-directed mutants in COS7 cells apparently had no effect on its relative stability thereby revealing that if CYP2E1 phosphorylation is important for its degradation (34, 35), then alternate Ser/Thr residues (i.e. in plausible secondary PKA recognition sites, Lys-Lys-Ser²⁰⁹-Lys and Lys-Lys-Ser⁴⁴⁹-Ala) may be recruited.On the other hand, on the basis of rapid phosphorylation of CuOOH-inactivated CYP3A4 that precedes its ubiquitination and 26 S proteasomal degradation in an in vitro liver cytosolic FII-catalyzed system, we have proposed that CYP3A4 phosphorylation was essential for targeting it to proteins participating in its UPD/ERAD (13). Indeed, several examples of similar phosphorylation for targeting proteins to UPD exist, of which IκBα phosphorylation is the most notable and perhaps the best documented (36–47; see “Discussion”).Our in vitro studies with specific kinase inhibitors as probes identified both PKC and PKA as the major FII kinases responsible for CYP3A4 phosphorylation (14). Indeed, in vitro model studies of CYP3A4 with PKC as the kinase, coupled with lysylendopeptidase C (Lys-C) digestion of the phosphorylated protein and liquid chromatography-tandem mass spectrometric (LC-MS/MS) analyses of the Lys-C digests, identified two PKC-phosphorylated CYP3A4 peptides ²⁵⁸ESRLEDpTQK²⁶⁶ and ⁴¹⁴FLPERFpSK⁴²¹ unambiguously phosphorylated at Thr²⁶⁴ and Ser⁴²⁰ (14). These same residues were also phosphorylated in corresponding studies with PKA.⁴ Furthermore, although both native and CuOOH-inactivated CYP3A4 were phosphorylated at Thr²⁶⁴, Ser⁴²⁰ phosphorylation was particularly enhanced after CuOOH-mediated CYP3A4 inactivation (14). Corresponding studies of CuOOH-inactivated CYP3A4 using rat liver cytosolic FII as the source of the kinase(s), revealed ³²P phosphorylation of both these peptides as well as that of an additional CYP3A4 peptide ⁴⁷⁷LS(p)LGGLLQPEKPVVLK⁴⁹². Unlike the unambiguous mass spectrometric identification of Thr²⁶⁴ and Ser⁴²⁰ as the phosphorylated CYP3A4 residues, the phosphorylation of Ser⁴⁷⁸, the only plausible phosphorylatable residue in this ³²P-labeled peptide, was not similarly established. Nevertheless, the predominant phosphorylation of Thr²⁶⁴ in native CYP3A4 (14), but of two additional residues in the CuOOH-inactivated enzyme, is consistent with the inactivation-induced structural unraveling of this enzyme with exposure of otherwise concealed and/or kinase-inaccessible domains (48). Such unraveling of CYP3A4 protein stems from the irreversible modification of its active site by fragments generated from CuOOH-mediated oxidative destruction of its prosthetic heme (49). In this study, using mass spectrometric analyses of Lys-C digests of FII-phosphorylated CYP3A4, we have provided unambiguous evidence that in addition to Thr²⁶⁴ and Ser⁴²⁰, Ser⁴⁷⁸ is indeed phosphorylated. More importantly, through alanine-scanning mutagenesis of these three residues, we now document that although neither the structural conformation nor the catalytic function of this triple CYP3A4T264A/S420A/S478A mutant is altered, its degradation after heterologous expression in S. cerevisiae is significantly impaired. This is also true of CYP3A4T264A/S420A/S478A mutant degradation in human embryonic kidney (HEK293T) cells. Furthermore, using an in vitro reconstituted CYP3A4 ubiquitination system, catalyzed by human Ub-conjugating E2 enzyme UBC7 and integral ER protein gp78 as the E3 Ub ligase (12), we document that PKA/PKC-mediated phosphorylation of the wild type CYP3A4 (CYP3A4wt) considerably enhanced its UBC7/gp78-mediated ubiquitination. Together these findings reveal the critical importance of CYP3A4 phosphorylation at these residues for its UPD and suggest a direct link between phosphorylation and its ubiquitination and degradation.     

Membrane-associated ubiquitin ligase complex containing gp78 mediates sterol-accelerated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase

The endoplasmic reticulum (ER)-associated degradation (ERAD) pathway in the yeast Saccharomyces cerevisiae is mediated by two membrane-bound ubiquitin ligases, Doa10 and Hrd1. These enzymes are found in distinct multiprotein complexes that allow them to recognize and target a variety of substrates for proteasomal degradation. Although multiprotein complexes containing mammalian ERAD ubiquitin ligases likely exist, they have yet to be identified and characterized in detail. Here, we identify two ER membrane proteins, SPFH2 and TMUB1, as associated proteins of mammalian gp78, a membrane-bound ubiquitin ligase that bears significant sequence homology with mammalian Hrd1 and mediates sterol-accelerated ERAD of the cholesterol biosynthetic enzyme HMG-CoA reductase. Co-immunoprecipitation studies indicate that TMUB1 bridges SPFH2 to gp78 in ER membranes. The functional significance of these interactions is revealed by the observation that RNA interference (RNAi)-mediated knockdown of SPFH2 and TMUB1 blunts both the sterol-induced ubiquitination and degradation of endogenous reductase in HEK-293 cells. These studies mark the initial steps in the characterization of the mammalian gp78 ubiquitin ligase complex, the further elucidation of which may yield important insights into mechanisms underlying gp78-mediated ERAD.     

Huntingtin Interacts with the Cue Domain of gp78 and Inhibits gp78 Binding to Ubiquitin and p97/VCP

Huntington''s disease (HD) is caused by polyglutamine expansion in huntingtin (htt) protein, but the exact mechanism of HD pathogenesis remains uncertain. Recent evidence suggests that htt proteins with expanded polyglutamine tracts induce endoplasmic reticulum (ER) stress, probably by interfering with ER-associated degradation (ERAD). Here we report that mutant htt interacts and interferes with the function of gp78, an ER membrane-anchored ubiquitin ligase (E3) involved in ERAD. Mapping studies showed that the HEAT repeats 2&3 of htt interact with the cue domain of gp78. The interaction competitively reduces polyubiquitinated protein binding to gp78 and also sterically blocks gp78 interaction of p97/VCP, a molecular chaperone that is essential for ERAD. These effects of htt negatively regulate the function of gp78 in ERAD and are aggravated by polyglutamine expansion. Paradoxically, gp78 is still able to ubiquitinate and facilitate degradation of htt proteins with expanded polyglutamine. The impairment of ERAD by mutant htt proteins is associated with induction of ER stress. Our studies provide a novel molecular mechanism that supports the involvement of ER stress in HD pathogenesis.     

AAA ATPase p97/valosin-containing protein interacts with gp78, a ubiquitin ligase for endoplasmic reticulum-associated degradation

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control mechanism that eliminates unwanted proteins from the endoplasmic reticulum (ER) through a ubiquitin-dependent proteasomal degradation pathway. gp78 is a previously described ER membrane-anchored ubiquitin ligase (E3) involved in ubiquitination of ER proteins. AAA ATPase (ATPase associated with various cellular activities) p97/valosin-containing protein (VCP) subsequently dislodges the ubiquitinated proteins from the ER and chaperones them to the cytosol, where they undergo proteasomal degradation. We now report that gp78 physically interacts with p97/VCP and enhances p97/VCP-polyubiquitin association. The enhanced association correlates with decreases in ER stress-induced accumulation of polyubiquitinated proteins. This effect is abolished when the p97/VCP-interacting domain of gp78 is removed. Further, using ERAD substrate CD3delta, gp78 consistently enhances p97/VCP-CD3delta binding and facilitates CD3delta degradation. Moreover, inhibition of endogenous gp78 expression by RNA interference markedly increases the levels of total polyubiquitinated proteins, including CD3delta, and abrogates VCP-CD3delta interactions. The gp78 mutant with deletion of its p97/VCP-interacting domain fails to increase CD3delta degradation and leads to accumulation of polyubiquitinated CD3delta, suggesting a failure in delivering ubiquitinated CD3delta for degradation. These data suggest that gp78-p97/VCP interaction may represent one way of coupling ubiquitination with retrotranslocation and degradation of ERAD substrates.     

ER stress differentially regulates the stabilities of ERAD ubiquitin ligases and their substrates

Endoplasmic reticulum (ER) stress-induced accumulation of misfolded proteins in the ER stimulates the ER-associated degradation (ERAD) process. ERAD in turn eliminates those misfolded proteins. Upregulation of ubiquitination enzymes is an essential mechanism by which ER stress enhances ERAD. However, ectopic overexpression of ubiquitination enzymes often fails to increase, and sometimes, inhibits ERAD. To further understand how ER stress regulates ERAD, we studied the effects of ER stress on ubiquitin ligase (E3) gp78-mediated ERAD and on the stabilities of gp78 and another ERAD E3 Hrd1. The results showed that ER stress-inducing agent tunicamycin significantly enhanced ERAD in cells that either express endogenous or overexpress gp78. Importantly, ER stress could increase ERAD even when new protein synthesis was inhibited by cycloheximide. Surprisingly, tunicamycin treatment stabilized gp78, an established ERAD E3 and an ERAD substrate as well, for up to 8h. By contrast, ER stress had little effects on the stability of another E3 Hrd1 except that it reduced the total ubiquitination level of Hrd1. Our data suggest that ER stress differentially regulates the stabilities of ERAD E3s and their substrates, which may represent a novel mechanism by which ER stress increases ERAD.     

Ubiquitination regulates the assembly of VLDL in HepG2 cells and is the committing step of the apoB-100 ERAD pathway

Apolipoprotein B-100 (apoB-100) is degraded by endoplasmic reticulum-associated degradation (ERAD) when lipid availability limits assembly of VLDLs. The ubiquitin ligase gp78 and the AAA-ATPase p97 have been implicated in the proteasomal degradation of apoB-100. To study the relationship between ERAD and VLDL assembly, we used small interfering RNA (siRNA) to reduce gp78 expression in HepG2 cells. Reduction of gp78 decreased apoB-100 ubiquitination and cytosolic apoB-ubiquitin conjugates. Radiolabeling studies revealed that gp78 knockdown increased secretion of newly synthesized apoB-100 and, unexpectedly, enhanced VLDL assembly, as the shift in apoB-100 density in gp78-reduced cells was accompanied by increased triacylglycerol (TG) secretion. To explore the mechanisms by which gp78 reduction might enhance VLDL assembly, we compared the effects of gp78 knockdown with those of U0126, a mitogen-activated protein kinase/ERK kinase1/2 inhibitor that enhances apoB-100 secretion in HepG2 cells. U0126 treatment increased secretion of both apoB100 and TG and decreased the ubiquitination and cellular accumu-lation of apoB-100. Furthermore, p97 knockdown caused apoB-100 to accumulate in the cell, but if gp78 was concomitantly reduced or assembly was enhanced by U0126 treatment, cellular apoB-100 returned toward baseline. This indicates that ubiquitination commits apoB-100 to p97-mediated retrotranslocation during ERAD. Thus, decreasing ubiquitination of apoB-100 enhances VLDL assembly, whereas improving apoB-100 lipidation decreases its ubiquitination, suggesting that ubiquitination has a regulatory role in VLDL assembly.     

Gp78, a membrane-anchored ubiquitin ligase, associates with Insig-1 and couples sterol-regulated ubiquitination to degradation of HMG CoA reductase

Sterol-regulated ubiquitination is an obligatory step in ER-associated degradation (ERAD) of HMG CoA reductase, a rate-limiting enzyme in cholesterol synthesis. Accelerated degradation of reductase, one of several strategies animal cells use to limit production of cholesterol, requires sterol-induced binding of the enzyme to ER membrane proteins called Insigs. Once formed, the reductase-Insig complex is recognized by a putative membrane-associated ubiquitin ligase (E3) that mediates the reductase ubiquitination reaction. Here, we show that gp78, a membrane bound E3, binds to Insig-1 and is required for sterol-regulated ubiquitination of reductase. In addition, gp78 couples regulated ubiquitination to degradation of reductase by binding to VCP, an ATPase that plays a key role in recognition and degradation of ERAD substrates. The current results identify gp78 as the E3 that initiates sterol-accelerated degradation of reductase, and Insig-1 as a bridge between gp78/VCP and the reductase substrate.     

Identification of SVIP as an endogenous inhibitor of endoplasmic reticulum-associated degradation

Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a process known as ER-associated degradation (ERAD), which starts with misfolded protein recognition, followed by ubiquitination, retrotranslocation to the cytosol, deglycosylation, and targeting to the proteasome for degradation. Actions of multisubunit protein machineries in the ER membrane integrate these steps. We hypothesized that regulation of the multisubunit machinery assembly is a mechanism by which ERAD activity is regulated. To test this hypothesis, we investigated the potential regulatory role of the small p97/VCP-interacting protein (SVIP) on the formation of the ERAD machinery that includes ubiquitin ligase gp78, AAA ATPase p97/VCP, and the putative channel Derlin1. We found that SVIP is anchored to microsomal membrane via myristoylation and co-fractionated with gp78, Derlin1, p97/VCP, and calnexin to the ER. Like gp78, SVIP also physically interacts with p97/VCP and Derlin1. Overexpression of SVIP blocks unassembled CD3delta from association with gp78 and p97/VCP, which is accompanied by decreases in CD3delta ubiquitination and degradation. Silencing SVIP expression markedly enhances the formation of gp78-p97/VCP-Derlin1 complex, which correlates with increased degradation of CD3delta and misfolded Z variant of alpha-1-antitrypsin, established substrates of gp78. These results suggest that SVIP is an endogenous inhibitor of ERAD that acts through regulating the assembly of the gp78-p97/VCP-Derlin1 complex.     

Differential regulation of CFTRΔF508 degradation by ubiquitin ligases gp78 and Hrd1

The most common mutation associated with cystic fibrosis is the deletion of phenylalanine 508 of cystic fibrosis transmembrane conductance regulator (CFTRΔF508). This mutation renders otherwise functional protein susceptible to ER-associated degradation (ERAD) and prevents CFTR from exiting the ER and trafficking to the plasma membrane. In this study, we demonstrate that RNAi-mediated silencing of gp78, an established ubiquitin ligase (E3) involved in ERAD, leads to accumulation of CFTRΔF508 protein in cells. gp78 facilitates the degradation of CFTRΔF508 by enhancing both its ubiquitination and interaction with p97/VCP. SVIP, which is the inhibitor of gp78, causes accumulation of CFTRΔF508. We showed that endogenous gp78 co-immunoprecipitates with Hrd1. Furthermore, the results indicate that silencing the expression of another ERAD E3, Hrd1, leads to stabilization of gp78 and decline in gp78 ubiquitination; thereby enhancing CFTRΔF508 degradation. The results support that gp78 is an E3 targeting CFTRΔF508 for degradation and Hrd1 inhibits CFTRΔF508 degradation by acting as an E3 for gp78.     

Different p97/VCP complexes function in retrotranslocation step of mammalian ER-associated degradation (ERAD)

Studies in yeast indicate that three specialized endoplasmic reticulum-associated degradation (ERAD) pathways, namely ERAD-L, -M, or -C, dispose substrates with structural lesions in the lumenal, transmembrane, or cytosolic domains, respectively. The ubiquitin ligase (E3) Hrd1p and its cooperating partners are required for ERAD-L and -M pathways, whereas Doa10p complex is required for the ERAD-C pathway. We investigated these pathways in mammalian cells by assessing the requirements of the mammalian ERAD E3s, gp78 and Hrd1, in degradation of four substrates each with different type of structural lesions: CD3δ, Z-variant α1-antitrypsin, tyrosinase (C89R) and mutant cystic fibrosis transmembrane conductance regulator (CFTRΔF508). We demonstrated that tyrosinase (C89R) is a substrate for Hrd1 while all others are gp78 substrates. Knockdown of Hrd1 diminished gp78 substrate levels, but silencing of gp78 had no effect on Hrd1's substrate, suggesting that the functional interaction between Hrd1 and gp78 is unidirectional. Furthermore, while Ufd1 is dispensable for gp78-mediated ERAD, it is essential for Hrd1-mediated ERAD. Interestingly, Npl4 was found to be a key component for both pathways. These results suggest that the Hrd1-mediated ERAD requires a well-established retrotranslocation machinery, the p97/VCP-Ufd1-Npl4 complex, whereas the gp78 pathway needs only p97/VCP and Npl4. In addition, the three distinct ERAD pathways described in yeast may not be strictly conserved in mammalian cells as gp78 can function on three substrates with different structural lesions.     

Gp78 cooperates with RMA1 in endoplasmic reticulum-associated degradation of CFTRDeltaF508

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin–proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTRΔF508, by specifically promoting ubiquitylation of CFTRΔF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRΔF508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTRΔF508 and catalyzes further polyubiquitylation of CFTRΔF508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTRΔF508.     

The endoplasmic reticulum (ER)-associated degradation system regulates aggregation and degradation of mutant neuroserpin

Familial encephalopathy with neuroserpin inclusion bodies is a neurodegenerative disorder characterized by the accumulation of neuroserpin polymers in the endoplasmic reticulum (ER) of cortical and subcortical neurons in the CNS because of neuroserpin point mutations. ER-associated degradation (ERAD) is involved in mutant neuroserpin degradation. In this study, we demonstrate that two ER-associated E3 ligases, Hrd1 and gp78, are involved in the ubiquitination and degradation of mutant neuroserpin. Overexpression of Hrd1 and gp78 decreases the mutant neuroserpin protein level, whereas Hrd1 and gp78 knockdown increases mutant neuroserpin stability. Moreover, ERAD impairment by mutant valosin-containing protein increases the mutant neuroserpin protein level and aggregate formation. Thus, these findings identify mutant neuroserpin as an ERAD target and show that Hrd1 and gp78 mediate mutant neuroserpin turnover through the ERAD pathway.     

Targeting of gp78 for ubiquitin-mediated proteasomal degradation by Hrd1: Cross-talk between E3s in the endoplasmic reticulum

There are an increasing number of ubiquitin ligases (E3s) implicated in endoplasmic reticulum (ER)-associated degradation (ERAD) in mammals. The two for which the greatest amount of information exists are the RING finger proteins gp78 and Hrd1, which are the structural orthologs of the yeast ERAD E3 Hrd1p. We now report that Hrd1, also known as synoviolin, targets gp78 for proteasomal degradation independent of the ubiquitin ligase activity of gp78, without evidence of a reciprocal effect. This degradation is observed in mouse embryonic fibroblasts lacking Hrd1, as well as with acute manipulation of Hrd1. The significance of this is underscored by the diminished level of a gp78-specific substrate, Insig-1, when Hrd1 expression is decreased and gp78 levels are consequently increased. These finding demonstrate a previously unappreciated level of complexity of the ubiquitin system in ERAD and have potentially important ramifications for processes where gp78 is implicated including regulation of lipid metabolism, metastasis, cystic fibrosis and neurodegenerative disorders.     

Inhibitory Interactions between Phosphorylation Sites in the C Terminus of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid-type Glutamate Receptor GluA1 Subunits

The C terminus of AMPA-type glutamate receptor (AMPAR) GluA1 subunits contains several phosphorylation sites that regulate AMPAR activity and trafficking at excitatory synapses. Although many of these sites have been extensively studied, little is known about the signaling mechanisms regulating GluA1 phosphorylation at Thr-840. Here, we report that neuronal depolarization in hippocampal slices induces a calcium and protein phosphatase 1/2A-dependent dephosphorylation of GluA1 at Thr-840 and a nearby site at Ser-845. Despite these similarities, inhibitors of NMDA-type glutamate receptors and protein phosphatase 2B prevented depolarization-induced Ser-845 dephosphorylation but had no effect on Thr-840 dephosphorylation. Instead, depolarization-induced Thr-840 dephosphorylation was prevented by blocking voltage-gated calcium channels, indicating that distinct Ca²⁺ sources converge to regulate GluA1 dephosphorylation at Thr-840 and Ser-845 in separable ways. Results from immunoprecipitation/depletion assays indicate that Thr-840 phosphorylation inhibits protein kinase A (PKA)-mediated increases in Ser-845 phosphorylation. Consistent with this, PKA-mediated increases in AMPAR currents, which are dependent on Ser-845 phosphorylation, were inhibited in HEK-293 cells expressing a Thr-840 phosphomimetic version of GluA1. Conversely, mimicking Ser-845 phosphorylation inhibited protein kinase C phosphorylation of Thr-840 in vitro, and PKA activation inhibited Thr-840 phosphorylation in hippocampal slices. Together, the regulation of Thr-840 and Ser-845 phosphorylation by distinct sources of Ca²⁺ influx and the presence of inhibitory interactions between these sites highlight a novel mechanism for conditional regulation of AMPAR phosphorylation and function.