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Hepatic ischemia followed by reperfusion (I/R), a major clinical problem during liver surgical procedures, can induce liver injury with severe cell death including ferroptosis which is characterized by iron-dependent accumulation of lipid peroxidation. The HECT domain-containing ubiquitin E3 ligase HUWE1 (also known as MULE) was initially shown to promote apoptosis. However, our preliminary study demonstrates that high expression of HUWE1 in the liver donors corelates with less injury and better hepatic function after liver transplantation in patients. Thus, we investigate the role of HUWE1 in acute liver injury, and identify HUWE1 as a negative ferroptosis modulator through transferrin receptor 1(TfR1). Deficiency of Huwe1 in mice hepatocytes (HKO) exacerbated I/R and CCl4-induced liver injury with more ferroptosis occurrence. Moreover, Suppression of Huwe1 remarkably enhances cellular sensitivity to ferroptosis in primary hepatocytes and mouse embryonic fibroblasts. Mechanistically, HUWE1 specifically targets TfR1 for ubiquitination and proteasomal degradation, thereby regulates iron metabolism. Importantly, chemical and genetic inhibition of TfR1 dramatically diminishes the ferroptotic cell death in Huwe1 KO cells and Huwe1 HKO mice. Therefore, HUWE1 is a potential protective factor to antagonize both aberrant iron accumulation and ferroptosis thereby mitigating acute liver injury. These findings may provide clinical implications for patients with the high-expression Huwe1 alleles.

The selective removal of undesired or damaged mitochondria by autophagy, known as mitophagy, is crucial for cellular homoeostasis, and prevents tumour diffusion, neurodegeneration and ageing. The pro-autophagic molecule AMBRA1 (autophagy/beclin-1 regulator-1) has been defined as a novel regulator of mitophagy in both PINK1/PARKIN-dependent and -independent systems. Here, we identified the E3 ubiquitin ligase HUWE1 as a key inducing factor in AMBRA1-mediated mitophagy, a process that takes place independently of the main mitophagy receptors. Furthermore, we show that mitophagy function of AMBRA1 is post-translationally controlled, upon HUWE1 activity, by a positive phosphorylation on its serine 1014. This modification is mediated by the IKKα kinase and induces structural changes in AMBRA1, thus promoting its interaction with LC3/GABARAP (mATG8) proteins and its mitophagic activity. Altogether, these results demonstrate that AMBRA1 regulates mitophagy through a novel pathway, in which HUWE1 and IKKα are key factors, shedding new lights on the regulation of mitochondrial quality control and homoeostasis in mammalian cells. Mitophagy is crucial for mitochondrial quality control and maintenance of cellular homeostasis. Here the authors identify an E3 ubiquitin ligase, HUWE1, that collaborates with LC3-interacting protein AMBRA1 to induce mitochondrial clearance.

Background The proliferation antigen Ki-67 has been widely used in clinical settings for cancer staging for many years, but investigations on its biological functions have lagged. Recently, Ki-67 has been shown to regulate both the composition of the chromosome periphery and chromosome behaviour in mitosis as well as to play a role in heterochromatin organisation and gene transcription. However, how the different roles for Ki-67 across the cell cycle are regulated and coordinated remain poorly understood. The progress towards understanding Ki-67 function have been limited by the tools available to deplete the protein, coupled to its abundance and fluctuation during the cell cycle. Results Here, we use a doxycycline-inducible E3 ligase together with an auxin-inducible degron tag to achieve a rapid, acute and homogeneous degradation of Ki-67 in HCT116 cells. This system, coupled with APEX2 proteomics and phospho-proteomics approaches, allows us to show that Ki-67 plays a role during DNA replication. In its absence, DNA replication is severely delayed, the replication machinery is unloaded, causing DNA damage that is not sensed by the canonical pathways and dependent on HUWE1 ligase. This leads to defects in replication and sister chromatids cohesion, but it also triggers an interferon response mediated by the cGAS/STING pathway in all the cell lines tested. Conclusions We unveil a new function of Ki-67 in DNA replication and genome maintenance that is independent of its previously known role in mitosis and gene regulation.

Immune checkpoint blockade therapies targeting the PD-L1/PD-1 axis have demonstrated clear clinical benefits. Improved understanding of the underlying regulatory mechanisms might contribute new insights into immunotherapy. Here, we identify transmembrane and ubiquitin-like domain-containing protein 1 (TMUB1) as a modulator of PD-L1 post-translational modifications in tumor cells. Mechanistically, TMUB1 competes with HECT, UBA and WWE domain-containing protein 1 (HUWE1), a E3 ubiquitin ligase, to interact with PD-L1 and inhibit its polyubiquitination at K281 in the endoplasmic reticulum. Moreover, TMUB1 enhances PD-L1 N-glycosylation and stability by recruiting STT3A, thereby promoting PD-L1 maturation and tumor immune evasion. TMUB1 protein levels correlate with PD-L1 expression in human tumor tissue, with high expression being associated with poor patient survival rates. A synthetic peptide engineered to compete with TMUB1 significantly promotes antitumor immunity and suppresses tumor growth in mice. These findings identify TMUB1 as a promising immunotherapeutic target. Cancer cells exploit immune checkpoint pathways, such as PD-1/PD-L1, to evade elimination by the immune system. Here, the authors demonstrate that TMUB1 regulates post-translational modifications of PD-L1 and that targeting the TMUB1/PD-L1 interaction promotes anti-tumour T cells responses

Ubiquitination modulates a large repertoire of cellular functions and thus, dysregulation of the ubiquitin system results in multiple human diseases, including cancer. Ubiquitination requires an E3 ligase, which is responsible for substrate recognition and conferring specificity to ubiquitination. HUWE1 is a multifaceted HECT domain-containing ubiquitin E3 ligase, which catalyzes both mono-ubiquitination and K6-, K48- and K63-linked poly-ubiquitination of its substrates. Many of the substrates of HUWE1 play a crucial role in maintaining the homeostasis of cellular development. Not surprisingly, dysregulation of HUWE1 is associated with tumorigenesis and metastasis. HUWE1 is frequently overexpressed in solid tumors, but can be downregulated in brain tumors, suggesting that HUWE1 may possess differing cell-specific functions depending on the downstream targets of HUWE1. This review introduces some important discoveries of the HUWE1 substrates, including those controlling proliferation and differentiation, apoptosis, DNA repair, and responses to stress. In addition, we review the signaling pathways HUWE1 participates in and obstacles to the identification of HUWE1 substrates. We also discuss up-to-date potential therapeutic designs using small molecules or ubiquitin variants (UbV) against the HUWE1 activity. These molecular advances provide a translational platform for future bench-to-bed studies. HUWE1 is a critical ubiquitination modulator during the tumor progression and may serve as a possible therapeutic target for cancer treatment.

HUWE1, a member of HECT E3 ubiquitin ligase family, is implicated in a variety of cellular processes. Recent studies find that HUWE1 also plays critical roles in germ cell development and inactivation of HUWE1 causes germ cell loss in both male and female mice. In this study, we found that Huwe1 was also highly expressed in testicular Sertoli cells. Inactivation of Huwe1 in Sertoli cells resulted in loss of cell polarity, which in turn caused germ cells loss and male infertility. Further study revealed that dysregulation in the expression of cytoskeletal and adhesion-related molecules, as well as a significant increase in EMT-related trans-factors SNAI1&2 in Huwe1 -deficient Sertoli cells. Intriguingly, the protein level of WT1 was significantly increased in Huwe1 -deficient Sertoli cells, and overexpression of Wt1 in Sertoli cells also caused the defects in spermatogenesis which was consistent with Huwe1 CKO mouse model. Furthermore, the defect of spermatogenesis in Huwe1 CKO mice was partially rescued by deleting one allele of Wt1 gene. Mechanistic studies revealed that WT1 interacts with HUWE1 protein and it could be ubiquitinated by HUWE1. Our study demonstrates that HUWE1 is involved in the establishment of Sertoli cell polarity mainly by regulating the protein level of WT1 gene.

The mechanism by which inflammasome activation is modulated remains unclear. In this study, we identified an AIM2-interacting protein, the E3 ubiquitin ligase HUWE1, which was also found to interact with NLRP3 and NLRC4 through the HIN domain of AIM2 and the NACHT domains of NLRP3 and NLRC4. The BH3 domain of HUWE1 was important for its interaction with NLRP3, AIM2, and NLRC4. Caspase-1 maturation, IL-1β release, and pyroptosis were reduced in Huwe1-deficient bone marrow-derived macrophages (BMDMs) compared with WT BMDMs in response to stimuli to induce NLRP3, NLRC4, and AIM2 inflammasome activation. Furthermore, the activation of NLRP3, NLRC4, and AIM2 inflammasomes in both mouse and human cells was remarkably reduced by treatment with the HUWE1 inhibitor BI8622. HUWE1 mediated the K27-linked polyubiquitination of AIM2, NLRP3, and NLRC4, which led to inflammasome assembly, ASC speck formation, and sustained caspase-1 activation. Huwe1-deficient mice had an increased bacterial burden and decreased caspase-1 activation and IL-1β production upon Salmonella, Francisella, or Acinetobacter baumannii infection. Our study provides insights into the mechanisms of inflammasome activation as well as a potential therapeutic target against bacterial infection.

Precisely controlling the activation of transcription factors is crucial for physiology. After a transcription factor is activated and carries out its transcriptional activity, it also needs to be properly deactivated. Here, we report a deactivation mechanism of HIF-1 and several other oncogenic transcription factors. HIF-1 promotes the transcription of an ADP ribosyltransferase, TiPARP, which serves to deactivate HIF-1. Mechanistically, TiPARP forms distinct nuclear condensates or nuclear bodies in an ADP ribosylation-dependent manner. The TiPARP nuclear bodies recruit both HIF-1α and an E3 ubiquitin ligase HUWE1, which promotes the ubiquitination and degradation of HIF-1α. Similarly, TiPARP promotes the degradation of c-Myc and estrogen receptor. By suppressing HIF-1α and other oncogenic transcription factors, TiPARP exerts strong antitumor effects both in cell culture and in mouse xenograft models. Our work reveals TiPARP as a negative-feedback regulator for multiple oncogenic transcription factors, provides insights into the functions of protein ADP-ribosylation, and suggests activating TiPARP as an anticancer strategy.

Receptor-mediated mitophagy is a crucial process involved in mitochondria quality control. AMBRA1 is a mitophagy receptor for the selective removal of damaged mitochondria in mammalian cells. A critical unresolved issue is how AMBRA1-mediated mitophagy is controlled in response to cellular stress. Here, we investigated the role of BCL2-family proteins on AMBRA1-dependent mitophagy and showed that MCL1 delays AMBRA1-dependent mitophagy. Indeed, MCL1 overexpression is sufficient to inhibit recruitment to mitochondria of the E3 Ubiquitin ligase HUWE1, a crucial dynamic partner of AMBRA1, upon AMBRA1-mediated mitophagy induction. In addition, we found that during mitophagy induced by AMBRA1, MCL1 levels decreased but were sustained by inhibition of the GSK-3β kinase, which delayed AMBRA1-mediated mitophagy. Also, we showed that MCL1 was phosphorylated by GSK-3β at a conserved GSK-3 phosphorylation site (S159) during AMBRA1-mediated mitophagy and that this event was accompanied by HUWE1-dependent MCL1 degradation. Altogether, our results demonstrate that MCL1 stability is regulated by the kinase GSK-3β and the E3 ubiquitin ligase HUWE1 in regulating AMBRA1-mediated mitophagy. Our work thus defines MCL1 as an upstream stress-sensitive protein, functional in AMBRA1-mediated mitophagy.

SummaryE3 ligases are a class of critical enzymes that can catalyse the transfer of ubiquitin (Ub) from an E2 enzyme to the substrate and are essential to cellular processes. The E3 ligase HUWE1 (also known as ARF-BP1, HECTH9, HSPC272, Ib772, LASU1, MULE, URE-B1, UREB1, and HECT, UBA and WWE domain-containing E3 ubiquitin protein ligase 1) is encoded by the huwe1 gene. HUWE1 is a key regulator of the DNA damage response, transcription, autophagy, apoptosis and metabolism in a variety of cancers. Due to its pivotal role in conferring substrate specificity, HUWE1 has attracted enormous attention as a promising anticancer drug target. In this review, we indicate the specific molecular structure of HUWE1 and its role in various cellular signalling pathways and highlight new insights into HUWE1 in cancer. Finally, we discuss outstanding questions regarding HUWE1 in oncology and highlight its limitations in drug development and clinical guidance to better define the role of HUWE1 in multiple cancers.