BACKGROUND & AIMS:Increased de novo lipogenesis, largely mediated by sterol regulatory element-binding protein 1 (SREBP1), is a hallmark of metabolic dysfunction-associated steatotic liver disease (MASLD). However, the post-translational mechanisms regulating SREBP1 turnover remain poorly understood. The endoplasmic reticulum-associated degradation (ERAD) pathway ensures protein quality and quantity control, yet its role in hepatic lipid metabolism remains elusive. Here, we investigate the function of the ERAD-specific E3 ubiquitin ligase MARCHF6 in hepatic lipid homeostasis and MASLD pathogenesis. METHODS:Liver-specific Marchf6 knockout (Marchf6Alb) mouse cell lines and organoids were generated to assess the impact of Marchf6 depletion on hepatic lipid metabolism under normal chow, high-fat diet, and Western diet conditions. RNA sequencing, proteomics, biochemical and molecular biological analyses were performed to identify molecular pathways regulated by MARCHF6. Functional studies in primary hepatocytes, human hepatoma cells and organoids were conducted to determine the mechanistic link between MARCHF6 and SREBP1. RESULTS:Hepatic MARCHF6 expression was significantly reduced in both MASLD mouse models and human patients. Liver-specific Marchf6 deletion aggravated hepatic lipid accumulation, fibrosis, and inflammation. Transcriptomic and proteomic analyses revealed upregulation of lipogenic genes in Marchf6Alb livers, with a marked increase in SREBP1 protein levels. Mechanistically, MARCHF6 directly interacted with and ubiquitinated SREBP1, targeting it for proteasomal degradation. Loss of MARCHF6 prolonged SREBP1 half-life, driving excessive de novo lipogenesis. CONCLUSIONS:MARCHF6-ERAD is a critical regulator of hepatic lipid metabolism, functioning as a sterol binding protein to control SREBP1 turnover. Its downregulation promotes hepatic steatosis and MASLD progression, highlighting MARCHF6 as a potential therapeutic target in MASLD. IMPACT AND IMPLICATIONS:This study identifies the endoplasmic reticulum-resident E3 ubiquitin ligase MARCHF6 as a key regulator of SREBP1 stability, hepatic lipid homeostasis and MASLD (metabolic dysfunction-associated steatotic liver disease) progression. We demonstrate that loss of MARCHF6 promotes hepatic steatosis and fibrosis, whereas restoration of MARCHF6 largely reverses these phenotypes, highlighting a reversible and therapeutically targetable pathway. These findings provide new mechanistic insight into lipid dysregulation in MASLD and position the MARCHF6-SREBP1 axis as a promising target for metabolic liver disease intervention.

Hepatic lipid metabolism is regulated by circadian rhythms and dynamically responds to nutrient availability, such that lipid synthesis, oxidation, and storage are temporally coordinated. We demonstrated that the endoplasmic reticulum (ER)-localized E3 ubiquitin ligase HRD1 stimulated lipid accumulation in the liver by decreasing the N6-methyladenosine (m6A) methylation and expression of mRNAs encoding factors involved in lipid metabolism. In mouse livers, m6A RNA modification and the expression of mRNAs encoding the m6A writer METTL14 and the m6A reader YTHDF3 were under circadian control and inversely correlated with the abundance of HRD1. m6A RNA sequencing analyses revealed that HRD1 and the m6A writer METTL14 had opposing roles in the m6A modification and expression of mRNAs encoding factors involved in fatty acid metabolism. In vivo, hepatic lipid accumulation and triglyceride amounts were decreased in mice with hepatic HRD1 deficiency fed a high-fat diet but increased in mice with hepatic METTL14 or YTHDF deficiency fed normal chow. Mechanistically, HRD1 mediated the polyubiquitination and degradation of PPARα, which transcriptionally activated METTL14 and YTHDF3 expression in the liver. Our work identifies a pathway regulated by circadian rhythms or nutrients in which HRD1 promotes the degradation of PPARα to decrease the m6A modification and expression of hepatic mRNAs encoding factors involved in lipid metabolism.

Lung transplantation (lung-tx) offers the last life-saving option for patients with COVID-19-associated lung injury (CALI). We show that the RBD-specific antibodies are dramatically reduced in these CALI lung-tx patients despite a comparable frequency of CD19+ B cells in circulating blood between CALI lung-tx patients and sero-negative controls. In contrast, non-transplant COVID-19 groups maintain a high level of RBD-specific antibodies. Single-cell RNA sequencing of white blood cells (WBCs) reveals three populations expanded in CALI lung-tx patients, including "lung-tx CD14+FCGR3B(CD16b)+ neutrophils," "lung-tx CD8+ T cells," and "KLF2+ CD4+ T cells," alongside reduced conventional T and B lymphocytes. Elevated expression of interferon (IFN)-responsive genes as part of COVID-19 molecular signatures is sustained in some clusters of monocytes, Tregs, and B cells in all COVID-19 groups. Multiplex analysis of cytokines and chemokines detects a statistically significant increase in interleukin (IL)-6, IL-8, MCP-1, and MIP-1α in circulating blood, specifically in CALI lung-tx patients.

Identifying highly efficacious, broad-spectrum antibodies against fast-mutating viral variants remains a major challenge in therapeutic development. Here, we developed AbGen, a machine learning-assisted antibody generation pipeline powered by an antibody language model (AbLM), to accelerate antibody screening and re-design. AbLM, pretrained on protein domain sequences and fine-tuned on paired VH-VL sequences, enables the analysis and prediction of neutralization activity against viruses (specifically SARS-CoV-2 in this study), targeting both wild-type (through antigen interaction prediction [docking]) and emerging variants (through Gaussian process regression [Kriging]). Screening over 1300 RBD-binding IgG sequences from convalescent patients, AbGen efficiently prioritized candidates for experimental validation and/or redesign against wild-type, Delta, and Omicron variants, preventing viral infections in vitro and in vivo. AbLM outperformed other language models in predicting IgGs with low variant susceptibility. Our work advances artificial intelligence-based antibody discovery by synergizing data-driven language models and Kriging with physics-driven docking and design.

While immune checkpoint blockade (ICB) therapy has revolutionized the antitumor therapeutic landscape, it remains successful in only a small subset of patients with cancer. Poor or loss of MHC-I expression has been implicated as a common mechanism of ICB resistance. Yet, the molecular mechanisms underlying impaired MHC-I remain to be fully elucidated. Herein, we identified USP22 as a critical factor responsible for ICB resistance through suppressing MHC-I-mediated neoantigen presentation to CD8+ T cells. Both genetic and pharmacologic USP22 inhibition increased immunogenicity and overcame anti-PD-1 immunotherapeutic resistance. At the molecular level, USP22 functions as a deubiquitinase for the methyltransferase EZH2, leading to transcriptional silencing of MHC-I gene expression. Targeted Usp22 inhibition resulted in increased tumoral MHC-I expression and consequently enhanced CD8+ T cell killing, which was largely abrogated by Ezh2 reconstitution. Multiplexed immunofluorescence staining detected a strong reverse correlation between USP22 expression and both β2M expression and CD8+ T lymphocyte infiltration in solid tumors. Importantly, USP22 upregulation was associated with ICB immunotherapeutic resistance in patients with lung cancer. Collectively, this study highlights the role of USP22 as a diagnostic biomarker for ICB resistance and provides a potential therapeutic avenue to overcome the current ICB resistance through inhibition of USP22.

While immunotherapy has revolutionized cancer treatment, its safety has been hampered by immunotherapy-related adverse events. Unexpectedly, we show that Mediator complex subunit 1 (MED1) is required for T regulatory (Treg) cell function specifically in the tumor microenvironment. Treg cell-specific MED1 deletion does not predispose mice to autoimmunity or excessive inflammation. In contrast, MED1 is required for Treg cell promotion of tumor growth because MED1 is required for the terminal differentiation of effector Treg cells in the tumor. Suppression of these terminally differentiated Treg cells is sufficient for eliciting antitumor immunity. Both human and murine Treg cells experience divergent paths of differentiation in tumors and matched tissues with non-malignant inflammation. Collectively, we identify a pathway promoting the differentiation of a Treg cell effector subset specific to tumors and demonstrate that suppression of a subset of Treg cells is sufficient for promoting antitumor immunity in the absence of autoimmune consequences.

Chronic stress–induced epinephrine (EPI) accelerates breast cancer progression and metastasis, but the molecular mechanisms remain unclear. Herein, we found a strong positive correlation between circulating EPI levels and the tumoral expression of ubiquitin-specific peptidase 22 (USP22) in patients with breast cancer. USP22 facilitated EPI-induced breast cancer progression and metastasis by enhancing adipose triglyceride lipase (ATGL)–mediated lipolysis. Targeted USP22 deletion decreased ATGL expression and lipolysis, subsequently inhibiting EPI-mediated breast cancer lung metastasis. USP22 acts as a bona fide deubiquitinase for the Atgl gene transcription factor FOXO1, and EPI architects a lipolysis signaling pathway to stabilize USP22 through AKT-mediated phosphorylation. Notably, USP22 phosphorylation levels are positively associated with EPI and with downstream pathways involving both FOXO1 and ATGL in breast cancers. Pharmacological USP22 inhibition synergized with β-blockers in treating preclinical xenograft breast cancer models. This study reveals a molecular pathway behind EPI’s tumor-promoting effects and provides a strong rationale for combining USP22 inhibition with β-blockers to treat aggressive breast cancer.

Integrins play critical roles in connecting the extracellular matrix and actin. While the upregulation of integrins is thought to promote cancer stemness and metastasis, the mechanisms underlying their upregulation in cancer stem cells (CSCs) remain poorly understood. Herein, we show that USP22 is essential in maintaining breast cancer cell stemness by promoting the transcription of integrin β1 (ITGB1). Both genetic and pharmacological inhibition of USP22 largely impaired breast CSCs self-renewal and prevented their metastasis. Reconstitution of Integrin β1 partially rescued USP22-null breast cancer metastasis. USP22 functions as a bona fide deubiquitinase to protect the proteasomal degradation of the forkhead box M1 (FoxM1), a transcription factor for tumoral ITGB1 gene transcription. Immunohistochemistry staining detected a positive correlation among USP22, FoxM1 and integrin β1 in human breast cancers. Collectively, our study identifies the USP22-FoxM1-integrin β1 signaling axis as critical for cancer stemness and offers a potential target for antitumor therapy.

Impaired secretion of an essential blood coagulation factor fibrinogen leads to hepatic fibrinogen storage disease (HFSD), characterized by the presence of fibrinogen-positive inclusion bodies and hypofibrinogenemia. However, the molecular mechanisms underlying the biogenesis of fibrinogen in the endoplasmic reticulum (ER) remain unexplored. Here we uncover a key role of SEL1L-HRD1 complex of ER-associated degradation (ERAD) in the formation of aberrant inclusion bodies, and the biogenesis of nascent fibrinogen protein complex in hepatocytes. Acute or chronic deficiency of SEL1L-HRD1 ERAD in the hepatocytes leads to the formation of hepatocellular inclusion bodies. Proteomics studies followed by biochemical assays reveal fibrinogen as a major component of the inclusion bodies. Mechanistically, we show that the degradation of misfolded endogenous fibrinogen Aα, Bβ, and γ chains by SEL1L-HRD1 ERAD is indispensable for the formation of a functional fibrinogen complex in the ER. Providing clinical relevance of these findings, SEL1L-HRD1 ERAD indeed degrades and thereby attenuates the pathogenicity of two disease-causing fibrinogen γ mutants. Together, this study demonstrates an essential role of SEL1L-HRD1 ERAD in fibrinogen biogenesis and provides insight into the pathogenesis of protein-misfolding diseases.

Regulation of tumoral PD-L1 expression is critical to advancing our understanding of tumor immune evasion and the improvement of existing antitumor immunotherapies. Herein, we describe a CRISPR-based screening platform and identified ATXN3 as a positive regulator for PD-L1 transcription. TCGA database analysis revealed a positive correlation between ATXN3 and CD274 in more than 80% of human cancers. ATXN3-induced Pd-l1 transcription was promoted by tumor microenvironmental factors, including the inflammatory cytokine IFN-γ and hypoxia, through protection of their downstream transcription factors IRF1, STAT3, and HIF-2α. Moreover, ATXN3 functioned as a deubiquitinase of the AP-1 transcription factor JunB, indicating that ATNX3 promotes PD-L1 expression through multiple pathways. Targeted deletion of ATXN3 in cancer cells largely abolished IFN-γ– and hypoxia-induced PD-L1 expression and consequently enhanced antitumor immunity in mice, and these effects were partially reversed by PD-L1 reconstitution. Furthermore, tumoral ATXN3 suppression improved the preclinical efficacy of checkpoint blockade antitumor immunotherapy. Importantly, ATXN3 expression was increased in human lung adenocarcinoma and melanoma, and its levels were positively correlated with PD-L1 as well as its transcription factors IRF1 and HIF-2α. Collectively, our study identifies what we believe to be a previously unknown deubiquitinase, ATXN3, as a positive regulator for PD-L1 transcription and provides a rationale for targeting ATXN3 to sensitize checkpoint blockade antitumor immunotherapy.

Iron homeostasis is critical for cellular and organismal function and is tightly regulated to prevent toxicity or anemia due to iron excess or deficiency, respectively. However, subcellular regulatory mechanisms of iron remain largely unexplored. Here, we report that SEL1L-HRD1 protein complex of endoplasmic reticulum (ER)-associated degradation (ERAD) in hepatocytes controls systemic iron homeostasis in a ceruloplasmin (CP)-dependent, and ER stress-independent, manner. Mice with hepatocyte-specific Sel1L deficiency exhibit altered basal iron homeostasis and are sensitized to iron deficiency while resistant to iron overload. Proteomics screening for a factor linking ERAD deficiency to altered iron homeostasis identifies CP, a key ferroxidase involved in systemic iron distribution by catalyzing iron oxidation and efflux from tissues. Indeed, CP is highly unstable and a bona fide substrate of SEL1L-HRD1 ERAD. In the absence of ERAD, CP protein accumulates in the ER and is shunted to refolding, leading to elevated secretion. Providing clinical relevance of these findings, SEL1L-HRD1 ERAD is responsible for the degradation of a subset of disease-causing CP mutants, thereby attenuating their pathogenicity. Together, this study uncovers the role of SEL1L-HRD1 ERAD in systemic iron homeostasis and provides insights into protein misfolding-associated proteotoxicity.

Over two years into the COVID-19 pandemic, the human immune response to SARS-CoV-2 during the active disease phase has been extensively studied. However, the long-term impact after recovery, which is critical to advance our understanding SARS-CoV-2 and COVID-19-associated long-term complications, remains largely unknown. Herein, we characterized multi-omic single-cell profiles of circulating immune cells in the peripheral blood of 100 patients, including covenlesent COVID-19 and sero-negative controls. The reduced frequencies of both short-lived monocytes and long-lived regulatory T (Treg) cells are significantly associated with the patients recovered from severe COVID-19. Consistently, sc-RNA seq analysis reveals seven heterogeneous clusters of monocytes (M0-M6) and ten Treg clusters (T0-T9) featuring distinct molecular signatures and associated with COVID-19 severity. Asymptomatic patients contain the most abundant clusters of monocyte and Treg expressing high CD74 or IFN-responsive genes. In contrast, the patients recovered from a severe disease have shown two dominant inflammatory monocyte clusters with S100 family genes: S100A8 & A9 with high HLA-I whereas S100A4 & A6 with high HLA-II genes, a specific non-classical monocyte cluster with distinct IFITM family genes, and a unique TGF-β high Treg Cluster. The outpatients and seronegative controls share most of the monocyte and Treg clusters patterns with high expression of HLA genes. Surprisingly, while presumably short-ived monocytes appear to have sustained alterations over 4 months, the decreased frequencies of long-lived Tregs (high HLA-DRA and S100A6) in the outpatients restore over the tested convalescent time (>= 4 months). Collectively, our study identifies sustained and dynamically altered monocytes and Treg clusters with distinct molecular signatures after recovery, associated with COVID-19 severity.

The tumor microenvironment (TME) enhances regulatory T (T reg ) cell stability and immunosuppressive functions through up-regulation of lineage transcription factor Foxp3, a phenomenon known as T reg fitness or adaptation. Here, we characterize previously unknown TME-specific cellular and molecular mechanisms underlying T reg fitness. We demonstrate that TME-specific stressors including transforming growth factor–β (TGF-β), hypoxia, and nutrient deprivation selectively induce two Foxp3-specific deubiquitinases, ubiquitin-specific peptidase 22 ( Usp22 ) and Usp21 , by regulating TGF-β, HIF, and mTOR signaling, respectively, to maintain T reg fitness. Simultaneous deletion of both USPs in T reg cells largely diminishes TME-induced Foxp3 up-regulation, alters T reg metabolic signatures, impairs T reg -suppressive function, and alleviates T reg suppression on cytotoxic CD8 + T cells. Furthermore, we developed the first Usp22 -specific small-molecule inhibitor, which dramatically reduced intratumoral T reg Foxp3 expression and consequently enhanced antitumor immunity. Our findings unveil previously unappreciated mechanisms underlying T reg fitness and identify Usp22 as an antitumor therapeutic target that inhibits T reg adaptability in the TME.

Summary: SEL1L-HRD1 ERAD controls hepatocyte proliferation and tumorigenesis, in part via mediating quality control of the maturation process of known tumor suppressor WNT5A in the hepatic ER. Proteomics analysis of SEL1L-deficient livers reveals WNT5A to accumulate, forming disulfide-bonded, high molecular weight aggregates, and potentially leading to cancer pathogenesis in the liver. ABSTRACT Endoplasmic reticulum (ER) homeostasis has been implicated in the pathogenesis of various forms of cancer; however, our understanding of the role of ER quality-control mechanisms in tumorigenesis remains incomplete. Here, we show that the SEL1L-HRD1 complex of ER-associated degradation (ERAD) suppresses hepatocyte proliferation and tumorigenesis in mice. Hepatocyte-specific deletion of Sel1L or Hrd1 predisposed mice to diet/chemical-induced tumors. Proteomics screen from SEL1L-deficient livers revealed WNT5A, a tumor suppressor, as an ERAD substrate. Indeed, nascent WNT5A was misfolding prone and degraded by SEL1L-HRD1 ERAD in a quality control capacity. In the absence of ERAD, WNT5A misfolds, is largely retained in the ER and forms high molecular-weight aggregates, thereby depicting a loss-of-function effect and attenuating WNT5A-mediated suppression of hepatocyte proliferation. In humans, SEL1L-HRD1 ERAD expression correlated positively with survival time for liver cancer patients. Overall, our data reveal a key role of SEL1L-HRD1 ERAD in suppressing hepatocyte proliferation and liver cancer. in tumor tissues compared to adjacent normal tissues. We speculate that this increased SEL1L-HRD1 levels could be an adaptive response to elevated protein synthesis in the ER during tumorigenesis (Kim et al. , 2015). This change may enhance ERAD-mediated clearance of misfolded WNT5A in the ER, thereby increasing WNT5A suppressive activity and leading to better prognosis. The answers to these questions will be explored in future studies. Nonetheless, this study provides an important framework for future studies to directly assess the relevance, significance, and molecular mechanism of ERAD in various cancers in vivo peroxidase quenching was carried out before antigen retrieval, and avidin-biotin kit was used to amplify the signal obtained using the DAB substrate kit. The next day, sections were washed thrice in PBS-0.03% TritonX-100 and treated with secondary antibody at room temperature for 2 hr. Counterstaining and/or mounting were done using Permount. Trichrome staining was carried out using the TRM-1 kit from ScyTek. H&E and DAB stained sections were scanned with the help of Aperio Scanscope (Leica Biosystems). All images were analyzed using ImageJ plugin (FIJI). the Genotype-Tissue Expression (GTEx) Project (Consortium, 2013). Non-log scale was used for correlation coefficient calculations and log scale for visualization. Analysis of single nucleotide polymorphisms (SNPs) for SEL1L and HRD1 was done from the Liver Hepatocellular Carcinoma TCGA Pan Cancer Atlas dataset (353 samples) using cBio Portal for Cancer Genomics (www.cbioportal.org). endoplasmic reticulum

Cancer cells evade the immune system by expressing inhibitory immune checkpoint receptors such as ecto-5'-nucleotidase (NT5E), also known as CD73, which consequently suppress tumor neoantigen-specific immune response. Blockade of CD73 in mouse models of breast cancer showed a reduction in tumor growth and metastasis. CD73 expression is elevated in a variety of human tumors including breast cancer. While the regulation of CD73 expression at the transcriptional level has been well understood, the factors involved in regulating CD73 expression at the post-transcriptional level have not been identified. Herein, we discovered that the ubiquitin-specific peptidase 22 (USP22), a deubiquitinase associated with poor prognosis and overexpressed in breast cancers, is a positive regulator for CD73. Targeted USP22 deletion resulted in a statistically significant reduction in CD73 protein expression. In contrast, CD73 mRNA expression levels were not reduced, but even slightly increased by USP22 deletion. Further analysis demonstrated that USP22 is a deubiquitinase that specifically interacts with and inhibits CD73 ubiquitination. Consequently, USP22 protects CD73 from ubiquitin-mediated proteasomal degradation in breast cancer cells. Targeted USP22 deletion, inhibits syngeneic breast cancer growth. Collectively, our study reveals USP22 as a positive regulator to promote CD73 expression in breast cancer and provides a rationale to target USP22 in antitumor immune therapy.

Endoplasmic reticulum (ER) homeostasis has been implicated in the pathogenesis of various forms of cancer; however, our understanding of the role of ER quality control mechanisms in tumorigenesis remains incomplete. Here, we show that the SEL1L-HRD1 complex of ER-associated degradation (ERAD) suppresses hepatocyte proliferation and tumorigenesis in mice. Hepatocyte-specific deletion of Sel1L or Hrd1 predisposed mice to diet/chemical-induced tumors. Proteomics screen from SEL1L-deficient livers revealed WNT5A, a tumor suppressor, as an ERAD substrate. Indeed, nascent WNT5A was misfolding prone and degraded by SEL1L-HRD1 ERAD in a quality control capacity. In the absence of ERAD, WNT5A misfolds is largely retained in the ER and forms high-molecular weight aggregates, thereby depicting a loss-of-function effect and attenuating WNT5A-mediated suppression of hepatocyte proliferation. In humans, SEL1L-HRD1 ERAD expression correlated positively with survival time for patients with liver cancer. Overall, our data reveal a key role of SEL1L-HRD1 ERAD in suppressing hepatocyte proliferation and liver cancer.

Hepatocellular carcinoma (HCC), also known as hepatoma, is a primary malignancy of the liver and the third leading cause of cancer mortality globally. Although much attention has focused on HCC, its pathogenesis remains largely obscure. The endoplasmic reticulum (ER) is a cellular organelle important for regulating protein synthesis, folding, modification and trafficking, and lipid metabolism. ER stress occurs when ER homeostasis is disturbed by numerous environmental, physiological, and pathological challenges. In response to ER stress due to misfolded/unfolded protein accumulation, unfolded protein response (UPR) is activated to maintain ER function for cell survival or, in cases of excessively severe ER stress, initiation of apoptosis. The liver is especially susceptible to ER stress given its protein synthesis and detoxification functions. Experimental data suggest that ER stress and unfolded protein response are involved in HCC development, aggressiveness and response to treatment. Herein, we highlight recent findings and provide an overview of the evidence linking ER stress to the pathogenesis of HCC.

Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen triggers an unfolded protein response (UPR) for stress adaptation, the failure of which induces cell apoptosis and tissue/organ damage. The molecular switches underlying how the UPR selects for stress adaptation over apoptosis remain unknown. Here, we discovered that accumulation of unfolded/misfolded proteins selectively induces N-6-adenosine-methyltransferase-14 (METTL14) expression. METTL14 promotes C/EBP-homologous protein (CHOP) mRNA decay through its 3' UTR N-6-methyladenosine (m(6)A) to inhibit its downstream pro-apoptotic target gene expression. UPR induces METTL14 expression by competing against the HRD1-ER-associated degradation (ERAD) machinery to block METTL14 ubiquitination and degradation. Therefore, mice with livers-pecific METTL14 deletion are highly susceptible to both acute pharmacological and alpha-1 antitrypsin (AAT) deficiency-induced ER proteotoxic stress and liver injury. Further hepatic CHOP deletion protects METTL14 knockout mice from ER-stress-induced liver damage. Our study reveals a crosstalk between ER stress and mRNA m(6)A modification pathways, termed the ERm(6)A pathway, for ER stress adaptation to proteotoxicity.

Objective: The endoplasmic reticulum (ER)-resident E3 ligase HRD1 and its co-activator Sel1L are major components of ER-associated degradation (ERAD) machinery. Here, we investigated the molecular mechanism and functional significance underlying the circadian regulation of HRD1/Sel1L-mediated protein degradation program in hepatic energy metabolism. Methods: Genetically engineered animal models as well as gain- and loss-of-function studies were employed to address the circadian regulatory mechanism and functional significance. Gene expression, transcriptional activation, protein-protein interaction, and animal metabolic phenotyping analyses were performed to dissect the molecular network and metabolic pathways. Results: Hepatic HRD1 and Sel1L expression exhibits circadian rhythmicity that is controlled by the ER-tethered transcriptional activator CREBH, the nuclear receptor peroxisome proliferator-activated receptor a (PPARa), and the core clock oscillator BMAL1 in mouse livers. HRD1/Sel1L mediates polyubiquitination and degradation of the CREBH protein across the circadian cycle to modulate rhythmic expression of the genes encoding the rate-limiting enzymes or regulators in fatty acid (FA) oxidation, triglyceride (TG) lipolysis, lipophagy, and gluconeogenesis. HRD1 liver-specific knockout (LKO) mice displayed increased expression of the genes involved in lipid and glucose metabolism and impaired circadian profiles of circulating TG, FA, and glucose due to overproduction of CREBH. The circadian metabolic activities of HRD1 LKO mice were inversely correlated with those of CREBH KO mice. Suppressing CREBH overproduction in the livers of HRD1 LKO mice restored the diurnal levels of circulating TG and FA of HRD1 LKO mice. Conclusion: Our work revealed a key circadian-regulated molecular network through which the E3 ubiquitin ligase HRD1 and its co-activator Sel1L regulate hepatic circadian metabolism. (c) 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).