The chemokine CCL1 triggers an AMFR-SPRY1 pathway that promotes differentiation of lung fibroblasts into myofibroblasts and drives pulmonary fibrosis.

Recruitment of immune cells to the site of inflammation by the chemokine CCL1 is important in the pathology of inflammatory diseases. Here, we examined the role of CCL1 in pulmonary fibrosis (PF). Bronchoalveolar lavage fluid from PF mouse models contained high amounts of CCL1, as did lung biopsies from PF patients. Immunofluorescence analyses revealed that alveolar macrophages and CD4+ T cells were major producers of CCL1 and targeted deletion of Ccl1 in these cells blunted pathology. Deletion of the CCL1 receptor Ccr8 in fibroblasts limited migration, but not activation, in response to CCL1. Mass spectrometry analyses of CCL1 complexes identified AMFR as a CCL1 receptor, and deletion of Amfr impaired fibroblast activation. Mechanistically, CCL1 binding triggered ubiquitination of the ERK inhibitor Spry1 by AMFR, thus activating Ras-mediated profibrotic protein synthesis. Antibody blockade of CCL1 ameliorated PF pathology, supporting the therapeutic potential of targeting this pathway for treating fibroproliferative lung diseases.

Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum.

The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy1. ER-phagy receptors have a central role in this process2, but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands.

iASPP suppresses Gp78-mediated TMCO1 degradation to maintain Ca2+ homeostasis and control tumor growth and drug resistance.

Ca2+ release from the endoplasmic reticulum (ER) is an essential event in the modulation of Ca2+ homeostasis, which is coordinated by multiple biological processes, ranging from cell proliferation to apoptosis. Deregulated Ca2+ homeostasis is linked with various cancer hallmarks; thus, uncovering the mechanisms underlying Ca2+ homeostasis dynamics may lead to new anticancer treatment strategies. Here, we demonstrate that a reported Ca2+-channel protein TMCO1 (transmembrane and coiled-coil domains 1) is overexpressed in colon cancer tissues at protein levels but not at messenger RNA levels in colon cancer. Further study revealed that TMCO1 is a substrate of ER-associated degradation E3 ligase Gp78. Intriguingly, Gp78-mediated TMCO1 degradation at K186 is under the control of the iASPP (inhibitor of apoptosis-stimulating protein of p53) oncogene. Mechanistically, iASPP robustly reduces ER Ca2+ stores, mainly by competitively binding with Gp78 and interfering with Gp78-mediated TMCO1 degradation. A positive correlation between iASPP and TMCO1 proteins is further validated in human colon tissues. Inhibition of iASPP-TMCO1 axis promotes cytosolic Ca2+ overload-induced apoptotic cell death, reducing tumor growth both in vitro and in vivo. Thus, iASPP-TMCO1 represents a promising anticancer treatment target by modulating Ca2+ homeostasis.

Adding to the STING.

STING (also known as MITA) is a central component in innate immunity against DNA virus. In this issue of Immunity, Wang et al. (2014) demonstrate that K27-linked polyubiquitination of STING (MITA) by the ER-associated E3 ligase AMFR is essential for STING (MITA)-mediated signaling and innate antiviral response.

The E3 ubiquitin ligase AMFR and INSIG1 bridge the activation of TBK1 kinase by modifying the adaptor STING.

Stimulator of interferon genes (STING, also known as MITA, ERIS, or MPYS) is essential for host immune responses triggered by microbial DNAs. However, the regulatory mechanisms underlying STING-mediated signaling are not fully understood. We report here that, upon cytoplasmic DNA stimulation, the endoplasmic reticulum (ER) protein AMFR was recruited to and interacted with STING in an insulin-induced gene 1 (INSIG1)-dependent manner. AMFR and INSIG1, an E3 ubiquitin ligase complex, then catalyzed the K27-linked polyubiquitination of STING. This modification served as an anchoring platform for recruiting TANK-binding kinase 1 (TBK1) and facilitating its translocation to the perinuclear microsomes. Depletion of AMFR or INSIG1 impaired STING-mediated antiviral gene induction. Consistently, myeloid-cell-specific Insig1(-/-) mice were more susceptible to herpes simplex virus 1 (HSV-1) infection than wild-type mice. This study uncovers an essential role of the ER proteins AMFR and INSIG1 in innate immunity, revealing an important missing link in the STING signaling pathway.

Synergistic effects of autocrine motility factor and methyl jasmonate on human breast cancer cells.

Autocrine motility factor (AMF) stimulates the motility of cancer cells via an autocrine route and has been implicated in tumor progression and metastasis. Overexpression of AMF is correlated with the aggressive nature of breast cancer and is negatively associated with clinical outcomes. In contrast, AMF also has the ability to suppress cancer cells. In this study, AMFs from different cancer cells were demonstrated to have suppressive activity against MCF-7 and MDA-MB-231 breast cancer cells. In a growth and colony formation assay, AMF from AsPC-1 pancreatic cancer cells (ASPC-1:AMF) was determined to be more suppressive compared to other AMFs. It was also demonstrated that AsPC-1:AMF could arrest breast cancer cells at the G0/G1 cell cycle phase. Quantified by Western blot analysis, AsPC-1:AMF lowered levels of the AMF receptor (AMFR) and G-protein-coupled estrogen receptor (GPER), concomitantly regulating the activation of the AKT and ERK signaling pathways. JAK/STAT activation was also decreased. These results were found in estrogen receptor (ER)-positive MCF-7 cells but not in triple-negative MDA-MB-231 cells, suggesting that AsPC-1:AMF could work through multiple pathways led to apoptosis. More importantly, AsPC-1:AMF and methyl jasmonate (MJ) cooperatively and synergistically acted against breast cancer cells. Thus, AMF alone or along with MJ may be a promising breast cancer treatment option.

PINK1 Content in Mitochondria is Regulated by ER-Associated Degradation.

Maintaining a pool of functional mitochondria requires degradation of damaged ones within the cell. PINK1 is critical in this quality-control process: loss of mitochondrial membrane potential causes PINK1 to accumulate on the mitochondrial surface, triggering mitophagy. However, little is known about how PINK1 is regulated. Recently, we showed that PINK1 content is kept low in healthy mitochondria by continuous ubiquitination and proteasomal degradation of its mature form via a mechanism inconsistent with the proposed N-end rule process. Using both human female and monkey cell lines, we now demonstrate that once generated within the mitochondria, 52 kDa PINK1 adopts a mitochondrial topology most consistent with it being at the mitochondrial-endoplasmic reticulum (ER) interface. From this particular submitochondrial location, PINK1 interacts with components of the ER-associated degradation pathway, such as the E3 ligases gp78 and HRD1, which cooperate to catalyze PINK1 ubiquitination. The valosin-containing protein and its cofactor, UFD1, then target ubiquitinated PINK1 for proteasomal degradation. Our data show that PINK1 in healthy mitochondria is negatively regulated via an interplay between mitochondria and ER, and shed light on how this mitochondrial protein gains access to the proteasome.SIGNIFICANCE STATEMENT Regulation of mitochondrial content of PINK1, a contributor to mitophagy, is an important area of research. Recently, we found that PINK1 content is kept low in healthy mitochondria by continuous ubiquitination and proteasomal degradation. We now extend and refine this novel finding by showing that PINK1 localizes at the mitochondrial-endoplasmic reticulum (ER) interface, from where it interacts with the ER-associated degradation machinery, which catalyzes its ubiquitination and transfer to the proteasome. Thus, these data show that PINK1 in healthy mitochondria is negatively regulated via a mitochondria and ER interplay, and how this mitochondrial protein gains access to the proteasome.

A technique for delineating the unfolding requirements for substrate entry into retrotranslocons during endoplasmic reticulum-associated degradation.

The endoplasmic reticulum-associated degradation (ERAD) pathway mediates the endoplasmic reticulum-to-cytosol retrotranslocation of defective proteins through protein complexes called retrotranslocons. Defective proteins usually have complex conformations and topologies, and it is unclear how ERAD can thread these conformationally diverse protein substrates through the retrotranslocons. Here, we investigated the substrate conformation flexibility necessary for transport via retrotranslocons on the ERAD-L, ERAD-M, and HIV-encoded protein Vpu-hijacked ERAD branches. To this end, we appended various ERAD substrates with specific domains whose conformations were tunable in flexibility or tightness by binding to appropriate ligands. With this technique, we could define the capacity of specific retrotranslocons in disentangling very tight, less tight but well-folded, and unstructured conformations. The Hrd1 complex, the retrotranslocon on the ERAD-L branch, permitted the passage of substrates with a proteinase K-resistant tight conformation, whereas the E3 ligase gp78-mediated ERAD-M allowed passage only of nearly completely disordered but not well-folded substrates and thus may have the least unfoldase activity. Vpu-mediated ERAD, containing a potential retrotranslocon, could unfold well-folded substrates for successful retrotranslocation. However, substrate retrotranslocation in Vpu-mediated ERAD was blocked by enhanced conformational tightness of the substrate. On the basis of these findings, we propose a mechanism underlying polypeptide movement through the endoplasmic reticulum membrane. We anticipate that our biochemical system paves the way for identifying the factors necessary for the retrotranslocation of membrane proteins.

Calmodulin regulates MGRN1-GP78 interaction mediated ubiquitin proteasomal degradation system.

The mechanism by which the endoplasmic reticulum (ER) ubiquitin ligases sense stress to potentiate their activity is poorly understood. GP78, an ER E3 ligase, is best known for its role in ER-associated protein degradation, although its activity is also linked to mitophagy, ER-mitochondria junctions, and MAPK signaling, thus highlighting the importance of understanding its regulation. In healthy cells, Mahogunin really interesting new gene (RING) finger 1 (MGRN1) interacts with GP78 and proteasomally degrades it to alleviate mitophagy. Here, we identify calmodulin (CaM) as the adapter protein that senses fluctuating cytosolic Ca2+ levels and modulates the Ca2+-dependent MGRN1-GP78 interactions. When stress elevates cytosolic Ca2+ levels in cultured and primary neuronal cells, CaM binds to both E3 ligases and inhibits their interaction. Molecular docking, simulation, and biophysical studies show that CaM interacts with both proteins with different affinities and binding modes. The physiological impact of this interaction switch manifests in the regulation of ER-associated protein degradation, ER-mitochondria junctions, and relative distribution of smooth ER and rough ER.-Mukherjee, R., Bhattacharya, A., Sau, A., Basu, S., Chakrabarti, S., Chakrabarti, O. Calmodulin regulates MGRN1-GP78 interaction mediated ubiquitin proteasomal degradation system.

Induction via Functional Protein Stabilization of Hepatic Cytochromes P450 upon gp78/Autocrine Motility Factor Receptor (AMFR) Ubiquitin E3-Ligase Genetic Ablation in Mice: Therapeutic and Toxicological Relevance.

The hepatic endoplasmic reticulum (ER)-anchored monotopic proteins, cytochromes P450 (P450s), are enzymes that metabolize endobiotics (physiologically active steroids and fatty acids), as well as xenobiotics including therapeutic/chemotherapeutic drugs, nutrients, carcinogens, and toxins. Alterations of hepatic P450 content through synthesis, inactivation, or proteolytic turnover influence their metabolic function. P450 proteolytic turnover occurs via ER-associated degradation (ERAD) involving ubiquitin (Ub)-dependent proteasomal degradation (UPD) as a major pathway. UPD critically involves P450 protein ubiquitination by E2/E3 Ub-ligase complexes. We have previously identified the ER-polytopic gp78/AMFR (autocrine motility factor receptor) as a relevant E3 in CYP3A4, CYP3A23, and CYP2E1 UPD. We now document that liver-conditional genetic ablation of gp78/AMFR in male mice disrupts P450 ERAD, resulting in statistically significant stabilization of Cyp2a5 and Cyp2c, in addition to that of Cyp3a and Cyp2e1. More importantly, we establish that such stabilization is of the functionally active P450 proteins, leading to corresponding statistically significant enhancement of their drug-metabolizing capacities. Our findings, with clinically relevant therapeutic drugs (nicotine, coumarin, chlorzoxazone, and acetaminophen) and the prodrug (tamoxifen) as P450 substrates, reveal that P450 ERAD disruption could influence therapeutic drug response and/or toxicity, warranting serious consideration as a potential source of clinically relevant drug-drug interactions (DDIs). Because gp78/AMFR is not only an E3 Ub-ligase, but also a cell-surface prometastatic oncogene that is upregulated in various malignant cancers, our finding that hepatic gp78/AMFR knockout can enhance P450-dependent bioactivation of relevant cancer chemotherapeutic prodrugs is of therapeutic relevance and noteworthy in prospective drug design and development. SIGNIFICANCE STATEMENT: The cell-surface and ER transmembrane protein gp78/AMFR, a receptor for the prometastatic autocrine motility factor (AMF), as well as an E3 ubiquitin-ligase involved in the ER-associated degradation (ERAD) of not only the tumor metastatic suppressor KAI1 but also of hepatic cytochromes P450, is upregulated in various human cancers, enhancing their invasiveness, metastatic potential, and poor prognosis. Liver-specific gp78/AMFR genetic ablation results in functional protein stabilization of several hepatic P450s and consequently enhanced drug and prodrug metabolism, a feature that could be therapeutically exploited in the bioactivation of chemotherapeutic prodrugs through design and development of novel short-term gp78/AMFR chemical inhibitors.

Ring finger protein 145 (RNF145) is a ubiquitin ligase for sterol-induced degradation of HMG-CoA reductase.

Cholesterol biosynthesis is tightly regulated in the cell. For example, high sterol concentrations can stimulate degradation of the rate-limiting cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, HMGCR). HMGCR is broken down by the endoplasmic reticulum membrane-associated protein complexes consisting of insulin-induced genes (Insigs) and the E3 ubiquitin ligase gp78. Here we found that HMGCR degradation is partially blunted in Chinese hamster ovary (CHO) cells lacking gp78 (gp78-KO). To identify other ubiquitin ligase(s) that may function together with gp78 in triggering HMGCR degradation, we performed a small-scale short hairpin RNA-based screening targeting endoplasmic reticulum-localized E3s. We found that knockdown of both ring finger protein 145 (Rnf145) and gp78 genes abrogates sterol-induced degradation of HMGCR in CHO cells. We also observed that RNF145 interacts with Insig-1 and -2 proteins and ubiquitinates HMGCR. Moreover, the tetrapeptide sequence YLYF in the sterol-sensing domain and the Cys-537 residue in the RING finger domain were essential for RNF145 binding to Insigs and RNF145 E3 activity, respectively. Of note, amino acid substitutions in the YLYF or of Cys-537 completely abolished RNF145-mediated HMGCR degradation. In summary, our study reveals that RNF145, along with gp78, promotes HMGCR degradation in response to elevated sterol levels and identifies residues essential for RNF145 function.

The ubiquitin specific protease USP34 protects the ubiquitin ligase gp78 from proteasomal degradation.

The E3 ubiquitin (Ub) ligase gp78 plays an important role in endoplasmic reticulum (ER)-associated degradation (ERAD) and regulation of lipid biogenesis. Although a variety of substrates of gp78 have been described, the regulation of the degradation of gp78 itself remains poorly understood. To address this problem, we used co-immunoprecipitation-coupled liquid chromatography-tandem mass spectrometry (Co-IP/LC-MS/MS) to identify novel proteins interacting with gp78. One of the proteins identified in this study is the deubiquitylating (DUB) enzyme USP34 (Ub-specific protease 34). We demonstrate that knockdown of USP34 facilitates proteasomal degradation of gp78 and consequently impairs the function of gp78 in regulating lipid droplet formation. This study unveils a previously unknown function of USP34 in regulating the metabolic stability of gp78 and adds to our understanding of the relevance of partnering of DUBs and E3s in regulation of protein ubiquitylation.

POLE mutations improve the prognosis of endometrial cancer via regulating cellular metabolism through AMF/AMFR signal transduction.

BACKGROUND: The morbidity and mortality of endometrial tumors, a common type of malignant cancer in women, have increased in recent years. POLE encodes the DNA polymerase ε, which is responsible for the leading strand DNA replication. Somatic mutations of POLE have been acknowledged in numerous cancers, resulting in the accumulation of DNA errors, leading to ultra-mutated tumors. Mutations in the exonuclease domain of POLE have been reported to improve progression-free survival in endometrial cancer. However, the potential relationship and underlying mechanism between POLE mutations and the prognosis of endometrial cancer patients remains unclear. METHODS: The whole exome sequencing data, RNA sequencing data, and clinical information were obtained from the TCGA database and employed for the analyses in this study. The detailed mutational information was analyzed using whole exome sequencing data and the mutated genes were shown with OncoPlot. The survival curves and cox proportional hazards regression analysis were used to accessed patient prognosis, the association of clinical characteristics and prognosis. Differentially expressed genes were analyzed by the edgeR R/Bioconductor package, then the GSEA Pre-ranked tool was used for Gene Set Enrichment Analysis (GSEA) to estimate the function of genes. Expression values were clustered using hierarchical clustering with Euclidean distance and ward linkage by the dendextend R package. RESULTS: POLE mutational status was proven to be an independent prognostic factor for endometrial cancer patients. Patients with somatic POLE mutations presented a favorable prognosis. POLE mutations regulated glycolysis and cytokine secretion, affecting cell metabolism and immune response. Autocrine motility factor (AMF)/PGI and AMFR/gp78 exhibited higher expression levels in POLE mutant patients. The comprehensive high expressions of AMFR/gp78 and low expression of POLE were associated with the favorable prognosis of endometrial cancer patients. CONCLUSIONS: This study showed the POLE mutations a vital factor in endometrial cancer patients, leading to a higher expression of AMF/PGI and AMFR/gp78. These results suggested comprehensive consideration of the POLE mutations, expression of AMF/PGI and AMFR/gp78 may provide a more feasible and effective approach for the treatment of endometrial cancer, which might improve the prognosis.

Autocrine motility factor promotes endometrial cancer progression by targeting GPER-1.

BACKGROUND: Autocrine motility factor (AMF) is a critical factor regulating aggressiveness of endometrial cancer (EC). Multiple pieces of evidence indicate that it is through G protein coupled estrogen receptor (GPER) signaling pathway that some growth factors promoted the migration and proliferation of tumor cells. The aim of this study is to explore the role of GPER-1 in AMF mediated regulatory mechanisms of EC recurrence and progression. METHODS: Real-Time Cell Analysis (RTCA) assays were performed to assess whether AMF depends on Autocrine motility factor recepter (AMFR) signaling in EC cells. A genome-wide expression microarray and Yeast Two-Hybrid assay were used to detect AMF and GPER-1 interaction in the context of AMFR depletion, and co-immunoprecipitation and immunofluorescence experiments were performed to confirm the physical interaction. Isobaric Tags for Relative and Absolute Quantification (iTRAQ) analysis was used for the identification of the target pathway activated by AMF-GPER-1 interaction. Cohorts of mice harboring xenografts derived from modified SPEC2 cell lines were treated with or without exogenous AMF to validate the results of previous experiments. Immunohistochemistry was performed to assess AMF and GPER-1 expression in endometrial cancer specimens and normal endometrium. RESULTS: Our data showed that GPER-1 binds to AMF and the formed complex translocates from the plasma membrane to the cytoplasm. Mechanistic investigations demonstrated that interaction between AMF and GPER-1 triggers phosphoinositide-3-kinase signaling and promotes EC cell growth. More importantly, through animal experiments and human tissue experiments, we found that AMF contributes to GPER-1-mediated EC progression, which is consistent with the above observations. CONCLUSIONS: Our work not only delineated the regulatory mechanisms of endometrial cancer progression by AMF-GPER-1-AKT signaling cascade but also laid the foundation of targeting this pathway for treating endometrial cancer.

Endoplasmic reticulum stress-induced degradation of DNAJB12 stimulates BOK accumulation and primes cancer cells for apoptosis.

DNAJB12 (JB12) is an endoplasmic reticulum (ER)-associated Hsp40 family protein that recruits Hsp70 to the ER surface to coordinate the function of ER-associated and cytosolic chaperone systems in protein quality control. Hsp70 is stress-inducible, but paradoxically, we report here that JB12 was degraded by the proteasome during severe ER stress. Destabilized JB12 was degraded by ER-associated degradation complexes that contained HERP, Sel1L, and gp78. JB12 was the only ER-associated chaperone that was destabilized by reductive stress. JB12 knockdown by siRNA led to the induction of caspase processing but not the unfolded protein response. ER stress-induced apoptosis is regulated by the highly labile and ER-associated BCL-2 family member BOK, which is controlled at the level of protein stability by ER-associated degradation components. We found that JB12 was required in human hepatoma cell line 7 (Huh-7) liver cancer cells to maintain BOK at low levels, and BOK was detected in complexes with JB12 and gp78. Depletion of JB12 during reductive stress or by shRNA from Huh-7 cells was associated with accumulation of BOK and activation of Caspase 3, 7, and 9. The absence of JB12 sensitized Huh-7 to death caused by proteotoxic agents and the proapoptotic chemotherapeutic LCL-161. In summary, JB12 is a stress-sensitive Hsp40 whose degradation during severe ER stress provides a mechanism to promote BOK accumulation and induction of apoptosis.

Ubiquitin-mediated regulation of the E3 ligase GP78 by MGRN1 in trans affects mitochondrial homeostasis.

Cellular quality control provides an efficient surveillance system to regulate mitochondrial turnover. This study elucidates a new interaction between the cytosolic E3 ligase mahogunin RING finger 1 (MGRN1) and the endoplasmic reticulum (ER) ubiquitin E3 ligase GP78 (also known as AMFR). Loss of Mgrn1 function has been implicated in late-onset spongiform neurodegeneration and congenital heart defects, among several developmental defects. Here, we show that MGRN1 ubiquitylates GP78 in trans through non-canonical K11 linkages. This helps maintain constitutively low levels of GP78 in healthy cells, in turn downregulating mitophagy. GP78, however, does not regulate MGRN1. When mitochondria are stressed, cytosolic Ca(2+) increases. This leads to a reduced interaction between MGRN1 and GP78 and its compromised ubiquitylation. Chelating Ca(2+) restores association between the two ligases and the in trans ubiquitylation. Catalytic inactivation of MGRN1 results in elevated levels of GP78 and a consequential increase in the initiation of mitophagy. This is important because functional depletion of MGRN1 by the membrane-associated disease-causing prion protein (Ctm)PrP affects polyubiquitylation and degradation of GP78, also leading to an increase in mitophagy events. This suggests that MGRN1 participates in mitochondrial quality control and could contribute to neurodegeneration in a subset of (Ctm)PrP-mediated prion diseases.

Lunapark Is a Component of a Ubiquitin Ligase Complex Localized to the Endoplasmic Reticulum Three-way Junctions.

The endoplasmic reticulum (ER) network comprises sheets and tubules that are connected by dynamic three-way junctions. Lunapark (Lnp) localizes to and stabilizes ER three-way junctions by antagonizing the small GTPase Atlastin, but how Lnp shapes the ER network is unclear. Here, we used an affinity purification approach and mass spectrometry to identify Lnp as an interacting partner of the ER protein quality control ubiquitin ligase gp78. Accordingly, Lnp purified from mammalian cells has a ubiquitin ligase activity in vitro Intriguingly, biochemical analyses show that this activity can be attributed not only to associated ubiquitin ligase, but also to an intrinsic ubiquitin ligase activity borne by Lnp itself. This activity is contained in the N-terminal 45 amino acids of Lnp although this segment does not share homology to any known ubiquitin ligase motifs. Despite its interaction with gp78, Lnp does not seem to have a broad function in degradation of misfolded ER proteins. On the other hand, the N-terminal ubiquitin ligase-bearing motif is required for the ER three-way junction localization of Lnp. Our study identifies a new type of ubiquitin ligase and reveals a potential link between ubiquitin and ER morphology regulation.

Allosteric regulation of E2:E3 interactions promote a processive ubiquitination machine.

RING finger proteins constitute the large majority of ubiquitin ligases (E3s) and function by interacting with ubiquitin-conjugating enzymes (E2s) charged with ubiquitin. How low-affinity RING-E2 interactions result in highly processive substrate ubiquitination is largely unknown. The RING E3, gp78, represents an excellent model to study this process. gp78 includes a high-affinity secondary binding region for its cognate E2, Ube2g2, the G2BR. The G2BR allosterically enhances RING:Ube2g2 binding and ubiquitination. Structural analysis of the RING:Ube2g2:G2BR complex reveals that a G2BR-induced conformational effect at the RING:Ube2g2 interface is necessary for enhanced binding of RING to Ube2g2 or Ube2g2 conjugated to Ub. This conformational effect and a key ternary interaction with conjugated ubiquitin are required for ubiquitin transfer. Moreover, RING:Ube2g2 binding induces a second allosteric effect, disrupting Ube2g2:G2BR contacts, decreasing affinity and facilitating E2 exchange. Thus, gp78 is a ubiquitination machine where multiple E2-binding sites coordinately facilitate processive ubiquitination.

Human liver cytochrome P450 3A4 ubiquitination: molecular recognition by UBC7-gp78 autocrine motility factor receptor and UbcH5a-CHIP-Hsc70-Hsp40 E2-E3 ubiquitin ligase complexes.

CYP3A4 is an abundant and catalytically dominant human liver endoplasmic reticulum-anchored cytochrome P450 enzyme engaged in the biotransformation of endo- and xenobiotics, including >50% of clinically relevant drugs. Alterations of CYP3A4 protein turnover can influence clinically relevant drug metabolism and bioavailability and drug-drug interactions. This CYP3A4 turnover involves endoplasmic reticulum-associated degradation via the ubiquitin (Ub)-dependent 26 S proteasomal system that relies on two highly complementary E2 Ub-conjugating-E3 Ub-ligase (UBC7-gp78 and UbcH5a-C terminus of Hsc70-interacting protein (CHIP)-Hsc70-Hsp40) complexes, as well as protein kinases (PK) A and C. We have documented that CYP3A4 Ser/Thr phosphorylation (Ser(P)/Thr(P)) by PKA and/or PKC accelerates/enhances its Lys ubiquitination by either of these E2-E3 systems. Intriguingly, CYP3A4 Ser(P)/Thr(P) and ubiquitinated Lys residues reside within the cytosol-accessible surface loop and/or conformationally assembled acidic Asp/Glu clusters, leading us to propose that such post-translational Ser/Thr protein phosphorylation primes CYP3A4 for ubiquitination. Herein, this possibility was examined through various complementary approaches, including site-directed mutagenesis, chemical cross-linking, peptide mapping, and LC-MS/MS analyses. Our findings reveal that such CYP3A4 Asp/Glu/Ser(P)/Thr(P) surface clusters are indeed important for its intermolecular electrostatic interactions with each of these E2-E3 subcomponents. By imparting additional negative charge to these Asp/Glu clusters, such Ser/Thr phosphorylation would generate P450 phosphodegrons for molecular recognition by the E2-E3 complexes, thereby controlling the timing of CYP3A4 ubiquitination and endoplasmic reticulum-associated degradation. Although the importance of phosphodegrons in the CHIP targeting of its substrates is known, to our knowledge this is the first example of phosphodegron involvement in gp78-substrate recruitment, an important step in CYP3A4 proteasomal degradation.

Hepatic cytochromes P450: structural degrons and barcodes, posttranslational modifications and cellular adapters in the ERAD-endgame.

The endoplasmic reticulum (ER)-anchored hepatic cytochromes P450 (P450s) are enzymes that metabolize endo- and xenobiotics i.e. drugs, carcinogens, toxins, natural and chemical products. These agents modulate liver P450 content through increased synthesis or reduction via inactivation and/or proteolytic degradation, resulting in clinically significant drug-drug interactions. P450 proteolytic degradation occurs via ER-associated degradation (ERAD) involving either of two distinct routes: Ubiquitin (Ub)-dependent 26S proteasomal degradation (ERAD/UPD) or autophagic lysosomal degradation (ERAD/ALD). CYP3A4, the major human liver/intestinal P450, and the fast-turnover CYP2E1 species are degraded via ERAD/UPD entailing multisite protein phosphorylation and subsequent ubiquitination by gp78 and CHIP E3 Ub-ligases. We are gaining insight into the nature of the structural determinants involved in CYP3A4 and CYP2E1 molecular recognition in ERAD/UPD [i.e. K48-linked polyUb chains and linear and/or "conformational" phosphodegrons consisting either of consecutive sequences on surface loops and/or disordered regions, or structurally-assembled surface clusters of negatively charged acidic (Asp/Glu) and phosphorylated (Ser/Thr) residues, within or vicinal to which, Lys-residues are targeted for ubiquitination]. Structural inspection of select human liver P450s reveals that such linear or conformational phosphodegrons may indeed be a common P450-ERAD/UPD feature. By contrast, although many P450s such as the slow-turnover CYP2E1 species and rat liver CYP2B1 and CYP2C11 are degraded via ERAD/ALD, little is known about the mechanism of their ALD-targeting. On the basis of our current knowledge of ALD-substrate targeting, we propose a tripartite conjunction of K63-linked Ub-chains, P450 structural "LIR" motifs and selective cellular "cargo receptors" as plausible P450-ALD determinants.