Expression of SARS-CoV-2 Nonstructural Proteins 3 and 4 Can Tune the Unfolded Protein Response in Cell Culture.

Coronaviruses (CoV), including SARS-CoV-2, modulate host proteostasis through the activation of stress-responsive signaling pathways such as the Unfolded Protein Response (UPR), which remedies misfolded protein accumulation by attenuating translation and increasing protein folding capacity. While CoV nonstructural proteins (nsps) are essential for infection, little is known about the role of nsps in modulating the UPR. We characterized the impact of overexpression of SARS-CoV-2 nsp4, a key driver of replication, on the UPR in cell culture using quantitative proteomics to sensitively detect pathway-wide upregulation of effector proteins. We find that nsp4 preferentially activates the ATF6 and PERK branches of the UPR. Previously, we found that an N-terminal truncation of nsp3 (nsp3.1) can suppress pharmacological ATF6 activation. To determine how nsp3.1 and nsp4 tune the UPR, their coexpression demonstrated that nsp3.1 suppresses nsp4-mediated PERK, but not ATF6 activation. Reanalysis of SARS-CoV-2 infection proteomics data revealed time-dependent activation of PERK targets early in infection, which subsequently fades. This temporal regulation suggests a role for nsp3 and nsp4 in tuning the PERK pathway to attenuate host translation beneficial for viral replication while avoiding later apoptotic signaling caused by chronic activation. This work furthers our understanding of CoV-host proteostasis interactions and highlights the power of proteomic methods for systems-level analysis of the UPR.

SARS-CoV-2 Nonstructural Proteins 3 and 4 tune the Unfolded Protein Response.

Coronaviruses (CoV), including SARS-CoV-2, modulate host proteostasis through activation of stress-responsive signaling pathways such as the Unfolded Protein Response (UPR), which remedies misfolded protein accumulation by attenuating translation and increasing protein folding capacity. While CoV nonstructural proteins (nsps) are essential for infection, little is known about the role of nsps in modulating the UPR. We characterized the impact of SARS-CoV-2 nsp4, a key driver of replication, on the UPR using quantitative proteomics to sensitively detect pathway-wide upregulation of effector proteins. We find nsp4 preferentially activates the ATF6 and PERK branches of the UPR. Previously, we found an N-terminal truncation of nsp3 (nsp3.1) can suppress pharmacological ATF6 activation. To determine how nsp3.1 and nsp4 tune the UPR, their co-expression demonstrated that nsp3.1 suppresses nsp4-mediated PERK, but not ATF6 activation. Re-analysis of SARS-CoV-2 infection proteomics data revealed time-dependent activation of PERK targets early in infection, which subsequently fades. This temporal regulation suggests a role for nsp3 and nsp4 in tuning the PERK pathway to attenuate host translation beneficial for viral replication while avoiding later apoptotic signaling caused by chronic activation. This work furthers our understanding of CoV-host proteostasis interactions and highlights the power of proteomic methods for systems-level analysis of the UPR.

Bok: real killer or bystander with non-apoptotic roles?

Bcl-2-related ovarian killer, Bok, was first labeled "pro-apoptotic" due to its ability to cause cell death when over-expressed. However, it has become apparent that this is not a good name, since Bok is widely expressed in tissues other than ovaries. Further, there is serious doubt as to whether Bok is a real "killer," due to disparities in the ability of over-expressed versus endogenous Bok to trigger apoptosis. In this brief review, we rationalize these disparities and argue that endogenous Bok is very different from the pro-apoptotic, mitochondrial outer membrane permeabilization mediators, Bak and Bax. Instead, Bok is a stable, endoplasmic reticulum-located protein bound to inositol 1,4,5 trisphosphate receptors. From this location, Bok plays a variety of roles, including regulation of endoplasmic reticulum/mitochondria contact sites and mitochondrial dynamics. Therefore, categorizing Bok as a "killer" may well be misleading and instead, endogenous Bok would better be considered an endoplasmic reticulum-located "bystander", with non-apoptotic roles.

Binding of the erlin1/2 complex to the third intralumenal loop of IP3R1 triggers its ubiquitin-proteasomal degradation.

Long-term activation of inositol 1,4,5-trisphosphate receptors (IP3Rs) leads to their degradation by the ubiquitin-proteasome pathway. The first and rate-limiting step in this process is thought to be the association of conformationally active IP3Rs with the erlin1/2 complex, an endoplasmic reticulum-located oligomer of erlin1 and erlin2 that recruits the E3 ubiquitin ligase RNF170, but the molecular determinants of this interaction remain unknown. Here, through mutation of IP3R1, we show that the erlin1/2 complex interacts with the IP3R1 intralumenal loop 3 (IL3), the loop between transmembrane (TM) helices 5 and 6, and in particular, with a region close to TM5, since mutation of amino acids D-2471 and R-2472 can specifically block erlin1/2 complex association. Surprisingly, we found that additional mutations in IL3 immediately adjacent to TM5 (e.g., D2465N) almost completely abolish IP3R1 Ca2+ channel activity, indicating that the integrity of this region is critical to IP3R1 function. Finally, we demonstrate that inhibition of the ubiquitin-activating enzyme UBE1 by the small-molecule inhibitor TAK-243 completely blocked IP3R1 ubiquitination and degradation without altering erlin1/2 complex association, confirming that association of the erlin1/2 complex is the primary event that initiates IP3R1 processing and that IP3R1 ubiquitination mediates IP3R1 degradation. Overall, these data localize the erlin1/2 complex-binding site on IP3R1 to IL3 and show that the region immediately adjacent to TM5 is key to the events that facilitate channel opening.

Endogenous Bok is stable at the endoplasmic reticulum membrane and does not mediate proteasome inhibitor-induced apoptosis.

Controversy surrounds the cellular role of the Bcl-2 family protein Bok. On one hand, it has been shown that all endogenous Bok is bound to inositol 1,4,5-trisphosphate receptors (IP3Rs), while other data suggest that Bok can act as a pro-apoptotic mitochondrial outer membrane permeabilization mediator, apparently kept at very low and non-apoptotic levels by efficient proteasome-mediated degradation. Here we show that 1) endogenous Bok is expressed at readily-detectable levels in key cultured cells (e.g., mouse embryonic fibroblasts and HCT116 cells) and is not constitutively degraded by the proteasome, 2) proteasome inhibitor-induced apoptosis is not mediated by Bok, 3) endogenous Bok expression level is critically dependent on the presence of IP3Rs, 4) endogenous Bok is rapidly degraded by the ubiquitin-proteasome pathway in the absence of IP3Rs at the endoplasmic reticulum membrane, and 5) charged residues in the transmembrane region of Bok affect its stability, ability to interact with Mcl-1, and pro-apoptotic activity when over-expressed. Overall, these data indicate that endogenous Bok levels are not governed by proteasomal activity (except when IP3Rs are deleted) and that while endogenous Bok plays little or no role in apoptotic signaling, exogenous Bok can mediate apoptosis in a manner dependent on its transmembrane domain.

Identification of the Bok Interactome Using Proximity Labeling.

The function of the Bcl-2 family member Bok is currently enigmatic, with various disparate roles reported, including mediation of apoptosis, regulation of mitochondrial morphology, binding to inositol 1,4,5-trisphosphate receptors, and regulation of uridine metabolism. To better define the roles of Bok, we examined its interactome using TurboID-mediated proximity labeling in HeLa cells, in which Bok knock-out leads to mitochondrial fragmentation and Bok overexpression leads to apoptosis. Labeling with TurboID-Bok revealed that Bok was proximal to a wide array of proteins, particularly those involved in mitochondrial fission (e.g., Drp1), endoplasmic reticulum-plasma membrane junctions (e.g., Stim1), and surprisingly among the Bcl-2 family members, just Mcl-1. Comparison with TurboID-Mcl-1 and TurboID-Bak revealed that the three Bcl-2 family member interactomes were largely independent, but with some overlap that likely identifies key interactors. Interestingly, when overexpressed, Mcl-1 and Bok interact physically and functionally, in a manner that depends upon the transmembrane domain of Bok. Overall, this work shows that the Bok interactome is different from those of Mcl-1 and Bak, identifies novel proximities and potential interaction points for Bcl-2 family members, and suggests that Bok may regulate mitochondrial fission via Mcl-1 and Drp1.

Bok binds to a largely disordered loop in the coupling domain of type 1 inositol 1,4,5-trisphosphate receptor.

Bcl-2-related ovarian killer (Bok) binds tightly to inositol 1,4,5-trisphosphate receptors (IP3Rs). To better understand this interaction, we sought to elucidate the Bok binding determinants in IP3R1, focusing on the ∼75 amino acid loop (residues 1882-1957) between α helices 72 and 73. Bioinformatic analysis revealed that the majority of this loop is intrinsically disordered, with two flanking regions of high disorder next to a low disorder central region (∼residues 1914-1926) that is predicted to contain two fused, disjointed transient helical elements. Experiments with IP3R1 mutants, combined with computational analysis, indicated that small deletions in this central region block Bok binding due to perturbation of the helical elements. Studies in vitro with purified Bok and IP3R1-derived peptides revealed high affinity binding to amino acids 1898-1940 of IP3R1 (Kd ∼65 nM) and that binding affinity is also dependent upon both of the high disorder flanking regions. The strength of the Bok-IP3R1 interaction was demonstrated by the ability of IP3R1 or Bok to recruit transmembrane domain-free Bok or IP3R1 mutants, respectively, to membranes in intact cells, and that these two mutants can bind in the cytosol independently of membrane association. Overall, we show that Bok binding to IP3R1 occurs within a largely disordered loop between α helices 72 and 73 and that high affinity binding is mediated by multivalent interactions.

Epsin-mediated degradation of IP3R1 fuels atherosclerosis.

The epsin family of endocytic adapter proteins are widely expressed, and interact with both proteins and lipids to regulate a variety of cell functions. However, the role of epsins in atherosclerosis is poorly understood. Here, we show that deletion of endothelial epsin proteins reduces inflammation and attenuates atherosclerosis using both cell culture and mouse models of this disease. In atherogenic cholesterol-treated murine aortic endothelial cells, epsins interact with the ubiquitinated endoplasmic reticulum protein inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), which triggers proteasomal degradation of this calcium release channel. Epsins potentiate its degradation via this interaction. Genetic reduction of endothelial IP3R1 accelerates atherosclerosis, whereas deletion of endothelial epsins stabilizes IP3R1 and mitigates inflammation. Reduction of IP3R1 in epsin-deficient mice restores atherosclerotic progression. Taken together, epsin-mediated degradation of IP3R1 represents a previously undiscovered biological role for epsin proteins and may provide new therapeutic targets for the treatment of atherosclerosis and other diseases.

The emerging link between IP3 receptor turnover and Hereditary Spastic Paraplegia.

IP3 receptor turnover is mediated by the ubiquitin ligase RNF170, which is recruited to active IP3 receptors by the erlin1/2 complex. A new study by Wagner et al (Nat Commun, 2019) links four cases of Hereditary Spastic Paraplegia to inactivating mutations in RNF170. This increases the number of examples of mutations to the erlin1/2 complex-RNF170 module underlying neurodegenerative disorders.

Bok regulates mitochondrial fusion and morphology.

Bok (Bcl-2-related ovarian killer) is a member of the Bcl-2 protein family that governs the intrinsic apoptosis pathway, but the cellular role that Bok plays is controversial. Remarkably, endogenous Bok is constitutively bound to inositol 1,4,5-trisphosphate receptors (IP3Rs) and is stabilized by this interaction. Here we report that despite the strong association with IP3Rs, deletion of Bok expression by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease)-mediated gene editing does not alter calcium mobilization via IP3Rs or calcium influx into the mitochondria. Rather, Bok deletion significantly reduces mitochondrial fusion rate, resulting in mitochondrial fragmentation. This phenotype is reversed by exogenous wild-type Bok and by an IP3R binding-deficient Bok mutant, and may result from a decrease in mitochondrial motility. Bok deletion also enhances mitochondrial spare respiratory capacity and membrane potential. Finally, Bok does not play a major role in apoptotic signaling, since Bok deletion does not alter responsiveness to various apoptotic stimuli. Overall, despite binding to IP3Rs, Bok does not alter IP3R-mediated Ca2+ signaling, but is required to maintain normal mitochondrial fusion, morphology, and bioenergetics.

The Making and Breaking of Inositol 1,4,5-Trisphosphate Receptor Tetramers.

Mammalian cells express three highly conserved inositol 1,4,5-trisphosphate (IP3) receptor types (IP3R1, IP3R2 and IP3R3), which have broadly similar characteristics, but markedly different distributions, and form homo- or heterotetrameric Ca2+ channels in endoplasmic reticulum (ER) membranes. A vast array of published work details how mature, ER membrane-located IP3 receptor tetramers are regulated, but much less attention has been paid to the intriguing questions of how the tetramers are assembled and destroyed as part of their natural life cycle. Are they assembled at the ER membrane from nascent, or completely translated polypeptides? How are they disassembled and degraded? These questions and other recently defined modes of IP3 receptor processing will be briefly reviewed.

The erlin2 T65I mutation inhibits erlin1/2 complex-mediated inositol 1,4,5-trisphosphate receptor ubiquitination and phosphatidylinositol 3-phosphate binding.

The erlin1/2 complex is a ∼2-MDa endoplasmic reticulum membrane-located ensemble of the ∼40-kDa type II membrane proteins erlin1 and erlin2. The best defined function of this complex is to mediate the ubiquitination of activated inositol 1,4,5-trisphosphate receptors (IP3Rs) and their subsequent degradation. However, it remains unclear how mutations of the erlin1/2 complex affect its cellular function and cause cellular dysfunction and diseases such as hereditary spastic paraplegia. Here, we used gene editing to ablate erlin1 or erlin2 expression to better define their individual roles in the cell and examined the functional effects of a spastic paraplegia-linked mutation to erlin2 (threonine to isoleucine at position 65; T65I). Our results revealed that erlin2 is the dominant player in mediating the interaction between the erlin1/2 complex and IP3Rs and that the T65I mutation dramatically inhibits this interaction and the ability of the erlin1/2 complex to promote IP3R ubiquitination and degradation. Remarkably, we also discovered that the erlin1/2 complex specifically binds to phosphatidylinositol 3-phosphate, that erlin2 binds this phospholipid much more strongly than does erlin1, that the binding is inhibited by T65I mutation of erlin2, and that multiple determinants within the erlin2 polypeptide comprise the phosphatidylinositol 3-phosphate-binding site. Overall, these results indicate that erlin2 is the primary mediator of the cellular roles of the erlin1/2 complex and that disease-linked mutations of erlin2 can affect both IP3R processing and lipid binding.

Multifunctional Telodendrimer Nanocarriers Restore Synergy of Bortezomib and Doxorubicin in Ovarian Cancer Treatment.

We have developed multifunctional nanoparticles for codelivery of bortezomib and doxorubicin to synchronize their pharmacokinetic profiles and synergize their activities in solid tumor treatment, a need still unmet in the clinic. Micellar nanoparticles were formed by a spatially segregated, linear-dendritic telodendrimer containing three segments: a hydrophilic polyethylene glycol (PEG), a bortezomib-conjugating intermediate, and a dendritic doxorubicin-affinitive interior. Bortezomib-conjugated telodendrimers, together with doxorubicin, self-assembled into monodispersed micelles [NP(BTZ-DOX)] with small particle sizes (20-30 nm) for dual drug delivery. NP(BTZ-DOX) displayed excellent drug-loading capacity and stability, which minimized premature drug leakage and synchronized drug release profiles. Bortezomib release was accelerated significantly by acidic pH, facilitating drug availability in the acidic tumor microenvironment. Synergistic anticancer effects of combined bortezomib and doxorubicin were observed in vitro against both multiple myeloma and ovarian cancer cells. NP(BTZ-DOX) prolonged payload circulation and targeted tumors in vivo efficiently with superior signal ratios of tumor to normal organs. In vitro and in vivo proteasome inhibition analysis and biodistribution studies revealed decreased toxicity and efficient intratumoral bortezomib and doxorubicin delivery by nanoformulation. NP(BTZ-DOX) exhibited significantly improved ovarian cancer treatment in SKOV-3 xenograft mouse models in comparison with free drugs and their combinations, including bortezomib and Doxil. In summary, tumor-targeted and synchronized delivery system elicits enhanced anticancer effects and merits further development in the clinical setting. Cancer Res; 77(12); 3293-305. ©2017 AACR.

The Stability and Expression Level of Bok Are Governed by Binding to Inositol 1,4,5-Trisphosphate Receptors.

Bok is a member of the Bcl-2 protein family that governs the intrinsic apoptosis pathway, although the role that Bok plays in this pathway is unclear. We have shown previously in cultured cell lines that Bok interacts strongly with inositol 1,4,5-trisphosphate receptors (IP3Rs), suggesting that it may contribute to the structural integrity or stability of IP3R tetramers. Here we report that Bok is similarly IP3R-assocated in mouse tissues, that essentially all cellular Bok is IP3R bound, that it is the helical nature of the Bok BH4 domain, rather than specific amino acids, that mediates binding to IP3Rs, that Bok is dramatically stabilized by binding to IP3Rs, that unbound Bok is ubiquitinated and degraded by the proteasome, and that binding to IP3Rs limits the pro-apoptotic effect of overexpressed Bok. Agents that stimulate IP3R activity, apoptosis, phosphorylation, and endoplasmic reticulum stress did not trigger the dissociation of mature Bok from IP3Rs or Bok degradation, indicating that the role of proteasome-mediated Bok degradation is to destroy newly synthesized Bok that is not IP3R associated. The existence of this unexpected proteolytic mechanism that is geared toward restricting Bok to that which is bound to IP3Rs, implies that unbound Bok is deleterious to cell viability and helps explain the current uncertainty regarding the cellular role of Bok.

A Point Mutation in the Ubiquitin Ligase RNF170 That Causes Autosomal Dominant Sensory Ataxia Destabilizes the Protein and Impairs Inositol 1,4,5-Trisphosphate Receptor-mediated Ca2+ Signaling.

RNF170 is an endoplasmic reticulum membrane ubiquitin ligase that contributes to the ubiquitination of activated inositol 1,4,5-trisphosphate (IP3) receptors, and also, when point mutated (arginine to cysteine at position 199), causes autosomal dominant sensory ataxia (ADSA), a disease characterized by neurodegeneration in the posterior columns of the spinal cord. Here we demonstrate that this point mutation inhibits RNF170 expression and signaling via IP3 receptors. Inhibited expression of mutant RNF170 was seen in cells expressing exogenous RNF170 constructs and in ADSA lymphoblasts, and appears to result from enhanced RNF170 autoubiquitination and proteasomal degradation. The basis for these effects was probed via additional point mutations, revealing that ionic interactions between charged residues in the transmembrane domains of RNF170 are required for protein stability. In ADSA lymphoblasts, platelet-activating factor-induced Ca(2+) mobilization was significantly impaired, whereas neither Ca(2+) store content, IP3 receptor levels, nor IP3 production were altered, indicative of a functional defect at the IP3 receptor locus, which may be the cause of neurodegeneration. CRISPR/Cas9-mediated genetic deletion of RNF170 showed that RNF170 mediates the addition of all of the ubiquitin conjugates known to become attached to activated IP3 receptors (monoubiquitin and Lys(48)- and Lys(63)-linked ubiquitin chains), and that wild-type and mutant RNF170 have apparently identical ubiquitin ligase activities toward IP3 receptors. Thus, the Ca(2+) mobilization defect seen in ADSA lymphoblasts is apparently not due to aberrant IP3 receptor ubiquitination. Rather, the defect likely reflects abnormal ubiquitination of other substrates, or adaptation to the chronic reduction in RNF170 levels.

The effect of M-phase stage-dependent kinase inhibitors on inositol 1,4,5-trisphosphate receptor 1 (IP3 R1) expression and localization in pig oocytes.

At fertilization, inositol 1,4,5-trisphosphate receptor type 1 (IP3 R1) has a crucial role in Ca(2+) release in mammals. Expression levels, localization and phosphorylation of IP3 R1 are important for its function, but it still remains unclear which molecule(s) regulates IP3 R1 behavior in pig oocytes. We examined whether there was a difference in localization of IP3 R1 after in vitro or in vivo maturation of pig oocytes. In mouse oocytes, large clusters of IP3 R1 were formed in the cortex of the oocyte except in a ring-shaped band of cortex adjacent to the spindle. However, no such clusters of IP3 R1 were observed in pig oocytes and there was no difference in its localization between in vitro and in vivo matured oocytes. We next tried to clarify which factor(s) regulates IP3 R1 localization, phosphorylation and expression using M-phase stage-dependent kinase inhibitors. Our results show that treatments with roscovitine (p34(cdc2) kinase inhibitor) or U0126 (mitogen-activated protein kinase inhibitor) did not affect IP3 R1 expression or localization in pig oocytes, although the latter strongly inhibited phosphorylation. However, treatment with BI-2536, an inhibitor of polo-like kinase 1 (Plk1), dramatically decreased the expression level of IP3 R1 in pig oocytes in a dose-dependent manner. From these results, it is suggested that Plk1 is involved in the regulation of IP3 R1 expression in pig oocytes.

The Bcl-2 protein family member Bok binds to the coupling domain of inositol 1,4,5-trisphosphate receptors and protects them from proteolytic cleavage.

Bok is a member of the Bcl-2 protein family that controls intrinsic apoptosis. Bok is most closely related to the pro-apoptotic proteins Bak and Bax, but in contrast to Bak and Bax, very little is known about its cellular role. Here we report that Bok binds strongly and constitutively to inositol 1,4,5-trisphosphate receptors (IP3Rs), proteins that form tetrameric calcium channels in the endoplasmic reticulum (ER) membrane and govern the release of ER calcium stores. Bok binds most strongly to IP3R1 and IP3R2, and barely to IP3R3, and essentially all cellular Bok is IP3R bound in cells that express substantial amounts of IP3Rs. Binding to IP3Rs appears to be mediated by the putative BH4 domain of Bok and the docking site localizes to a small region within the coupling domain of IP3Rs (amino acids 1895-1903 of IP3R1) that is adjacent to numerous regulatory sites, including sites for proteolysis. With regard to the possible role of Bok-IP3R binding, the following was observed: (i) Bok does not appear to control the ability of IP3Rs to release ER calcium stores, (ii) Bok regulates IP3R expression, (iii) persistent activation of inositol 1,4,5-trisphosphate-dependent cell signaling causes Bok degradation by the ubiquitin-proteasome pathway, in a manner that parallels IP3R degradation, and (iv) Bok protects IP3Rs from proteolysis, either by chymotrypsin in vitro or by caspase-3 in vivo during apoptosis. Overall, these data show that Bok binds strongly and constitutively to IP3Rs and that the most significant consequence of this binding appears to be protection of IP3Rs from proteolysis. Thus, Bok may govern IP3R cleavage and activity during apoptosis.

Vitrification procedure decreases inositol 1,4,5-trisphophate receptor expression, resulting in low fertility of pig oocytes.

Although cryopreservation of mammalian oocytes is an important technology, it is well known that unfertilized oocytes, especially in pigs, are highly sensitive to low temperature and that cryopreserved oocytes show low fertility and developmental ability. The aim of the present study was to clarify why porcine in vitro matured (IVM) oocytes at the metaphase II (MII) stage showed low fertility and developmental ability after vitrification. In vitro matured cumulus oocyte complexes (COCs) were vitrified with Cryotop and then evaluated for fertility through in vitro fertilization (IVF). Although sperm-penetrated oocytes were observed to some extent (30-40%), the rate of pronuclear formation was low (9%) and none of them progressed to the two-cell stage. The results suggest that activation ability of cryopreserved oocytes was decreased by vitrification. We examined the localization and expression level of the type 1 inositol 1,4,5 trisphosphate receptor (IP3 R1), the channel responsible for Ca(2+) release during IVF in porcine oocytes. Localization of IP3 R1 close to the plasma membrane and total expression level of IP3 R1 protein were both decreased by vitrification. In conclusion, our present study indicates that vitrified-warmed porcine COCs showed a high survival rate but low fertility after IVF. This low fertility seems to be due to the decrease in IP3 R1 by the vitrification procedure.

Differential regulation of HMG-CoA reductase and Insig-1 by enzymes of the ubiquitin-proteasome system.

The endoplasmic reticulum (ER)-resident enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyzes the rate-limiting step in sterol production and is the therapeutic target of statins. Understanding HMG-CoA reductase regulation has tremendous implications for atherosclerosis. HMG-CoA reductase levels are regulated in response to sterols both transcriptionally, through a complex regulatory loop involving the ER Insig proteins, and posttranslationally, by Insig-dependent protein degradation by the ubiquitin-proteasome system. The ubiquitin ligase (E3) gp78 has been implicated in the sterol-regulated degradation of HMG-CoA reductase and Insig-1 through ER-associated degradation (ERAD). More recently, a second ERAD E3, TRC8, has also been reported to play a role in the sterol-accelerated degradation of HMG-CoA reductase. We interrogated this network in gp78(-/-) mouse embryonic fibroblasts and also assessed two fibroblast cell lines using RNA interference. Although we consistently observe involvement of gp78 in Insig-1 degradation, we find no substantive evidence to support roles for either gp78 or TRC8 in the robust sterol-accelerated degradation of HMG-CoA reductase. We discuss factors that might lead to such discrepant findings. Our results suggest a need for additional studies before definitive mechanistic conclusions are drawn that might set the stage for development of drugs to manipulate gp78 function in metabolic disorders.

Clustered hydrophobic amino acids in amphipathic helices mediate erlin1/2 complex assembly.

Erlin1 and erlin2 are highly homologous, ∼40kDa, endoplasmic reticulum membrane proteins that assemble into a ring-shaped complex with a mass of ∼2 MDa. How this complex is formed is not understood, but appears to involve multiple interactions, including a coiled-coil region that mediates lower-order erlin assembly, and a short hydrophobic region, termed the "assembly domain", that mediates higher-order assembly into ∼2 MDa complexes. Here we have used molecular modeling, mutagenesis and cross-linking to examine the role of the assembly domain in higher-order assembly. We find (i) that the assembly domains of erlin1 and erlin2 are amphipathic helices, (ii) that erlin1 alone and erlin2 alone can assemble into ∼2 MDa complexes, (iii) that higher-order assembly is strongly inhibited by point mutations to the assembly domain, (iv) that three interacting hydrophobic residues in the assembly domain and aromaticity are essential for higher-order assembly, and (iv) that while erlins1 and 2 are equally capable of forming lower-order homo- and hetero-oligomers, hetero-oligomers are the most prevalent form when erlin1 and erlin2 are co-expressed. Overall, we conclude that the ∼2 MDa erlin1/2 complex is composed of an assemblage of lower-order hetero-oligomers, probably heterotrimers, linked together by assembly domain hydrophobic residues.