E3 Ubiquitin Ligase HUWE1 Research Articles

Regulation of primary cilia disassembly through HUWE1-mediated TTBK2 degradation plays a crucial role in cerebellar development and medulloblastoma growth

Development of the cerebellum requires precise regulation of granule neuron progenitor (GNP) proliferation. Although it is known that primary cilia are necessary to support GNP proliferation, the exact molecular mechanism governing primary cilia dynamics within GNPs remains elusive. Here, we establish the pivotal roles for the centrosomal kinase TTBK2 (Tau tubulin kinase-2) and the E3 ubiquitin ligase HUWE1 in GNP proliferation. We show that TTBK2 is highly expressed in proliferating GNPs under Sonic Hedgehog (SHH) signaling, coinciding with active GNP proliferation and the presence of primary cilia. TTBK2 stabilizes primary cilia by inhibiting their disassembly, thereby promoting GNP proliferation in response to SHH. Mechanistically, we identify HUWE1 as a novel centrosomal E3 ligase that facilitates primary cilia disassembly by targeting TTBK2 degradation. Disassembly of primary cilia serves as a trigger for GNP differentiation, allowing their migration from the external granule layer (EGL) of the cerebellum to the internal granule layer (IGL) for subsequent maturation. Moreover, we have established a link between TTBK2 and SHH-type medulloblastoma (SHH-MB), a tumor characterized by uncontrolled GNP proliferation. TTBK2 depletion inhibits SHH-MB proliferation, indicating that TTBK2 may be a potential therapeutic target for this cancer type. In summary, our findings reveal the mechanism governing cerebellar development and highlight a potential anti-cancer strategy for SHH-MB.

Distinct Regulation of ASCL1 by the Cell Cycle and Chemotherapy in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is an aggressive and lethal malignancy. Achaete-scute homolog 1 (ASCL1) is essential for the initiation of SCLC in mice and the development of pulmonary neuroendocrine cells (PNEC), which are the major cells of origin for SCLC. However, the regulatory mechanism of ASCL1 in SCLC remains elusive. Here, we found that ASCL1 expression gradually increases as the tumors grow in a mouse SCLC model, and is regulated by the cell cycle. Mechanistically, CDK2-CyclinA2 complex phosphorylates ASCL1, which results in increased proteasome-mediated ASCL1 protein degradation by E3 ubiquitin ligase HUWE1 during mitosis. TCF3 promotes the multisite phosphorylation of ASCL1 through the CDK2-CyclinA2 complex and the interaction between ASCL1 and TCF3 protects ASCL1 from degradation. The dissociation of TCF3 from ASCL1 during mitosis accelerates the degradation of ASCL1. In addition, chemotherapy drugs greatly reduce the transcription of ASCL1 in SCLC cells. Depletion of ASCL1 sensitizes SCLC cells to chemotherapy drugs. Together, our study demonstrates that ASCL1 is a cell-cycle-regulated protein and provides a theoretical basis for applying cell-cycle-related antitumor drugs in SCLC treatment. Implications:Our study revealed a novel regulatory mechanism of ASCL1 by cell cycle and chemotherapy drugs in SCLC. Treating patients with SCLC with a combination of ASCL1-targeting therapy and chemotherapy drugs could potentially be beneficial.

Suppression of interferon α and γ response by Huwe1-mediated Miz1 degradation promotes SARS-CoV-2 replication.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been demonstrated to limit the host interferon response; however, the underlying mechanism remains unclear. Here, we found that SARS-CoV-2 infection upregulated the E3 ubiquitin ligase Huwe1, which in turn facilitated the degradation of the transcription factor Miz1. The degradation of Miz1 hampered interferon alpha and gamma responses, consequently fostering viral replication and impeding viral clearance. Conversely, silencing or inhibiting Huwe1 enhanced the interferon responses, effectively curbing viral replication. Consistently, overexpressing Miz1 augmented the interferon responses and limited viral replication, whereas silencing Miz1 had the opposite effect. Targeting Huwe1 or overexpressing Miz1 elicited transcriptomic alterations characterized by enriched functions associated with bolstered antiviral response and diminished virus replication. Further study revealed Miz1 exerted epigenetic control over the transcription of specific interferon signaling molecules, which acted as common upstream regulators responsible for the observed transcriptomic changes following Huwe1 or Miz1 targeting. These findings underscore the critical role of the Huwe1-Miz1 axis in governing the host antiviral response, with its dysregulation contributing to the impaired interferon response observed during COVID-19.

Monocarboxylate transporter 1 in the liver modulates high-fat diet-induced obesity and hepatic steatosis in mice

BackgroundThe monocarboxylate transporter 1 (MCT1) is a member of the MCT family and is implicated in the transport of lactate and a few other monocarboxylates across the cell membrane. How hepatic MCT1 regulates the metabolic functions of the body is currently unknown. MethodsThe functions of hepatic MCT1 on metabolism were analyzed using a mouse model with liver-specific deletion of Slc16a1 that encodes MCT1. Obesity and hepatosteatosis of the mice were induced by high-fat diet (HFD). The function of MCT1 on lactate transport was analyzed by measuring lactate level in hepatocytes and mouse liver. Degradation and polyubiquitination of PPARα protein were investigated by biochemical methods. ResultsHepatic deletion of Slc16a1 aggravated high-fat diet (HFD)-induced obesity in female mice, but not in male mice. However, the increased adiposity in Slc16a1-deleted mice was not associated with obvious reductions in metabolic rate and activity. The lactate level of the liver was significantly increased by Slc16a1 deletion in the female mice under HFD condition, suggesting that MCT1 mainly mediated the efflux of lactate in hepatocytes. Deficiency of MCT1 in the liver aggravated HFD-induced hepatic steatosis in both female and male mice. Mechanistically, deletion of Slc16a1 was associated with reduced expressions of genes involved in fatty acid oxidation (FAO) in the liver. The degradation rate and polyubiquitination of PPARα protein were enhanced by Slc16a1 deletion. Blocking the MCT1 function elevated the interaction of PPARα with an E3 ubiquitin ligase HUWE1. ConclusionsOur findings suggested that the enhanced polyubiquitination and degradation of PPARα upon Slc16a1 deletion likely contributes to the reduced expression of FAO-related genes and aggravation of HFD-induced hepatic steatosis.

The E3 ubiquitin ligase HUWE1 acts through the N-Myc-DLL1-NOTCH1 signaling axis to suppress glioblastoma progression.

BackgroundElucidation of the post‐transcriptional modification has led to novel strategies to treat intractable tumors, especially glioblastoma (GBM). The ubiquitin‐proteasome system (UPS) mediates a reversible, stringent and stepwise post‐translational modification which is closely associated with malignant processes of GBM. To this end, developing novel therapeutic approaches to target the UPS may contribute to the treatment of this disease. This study aimed to screen the vital and aberrantly regulated component of the UPS in GBM. Based on the molecular identification, functional characterization, and mechanism investigation, we sought to elaborate a novel therapeutic strategy to target this vital factor to combat GBM.MethodsWe combined glioma datasets and human patient samples to screen and identify aberrantly regulated E3 ubiquitin ligase. Multidimensional database analysis and molecular and functional experiments in vivo and in vitro were used to evaluate the roles of HECT, UBA and WWE domain‐containing E3 ubiquitin ligase 1 (HUWE1) in GBM. dCas9 synergistic activation mediator system and recombinant adeno‐associated virus (rAAV) were used to endogenously overexpress full‐length HUWE1 in vitro and in glioma orthotopic xenografts.ResultsLow expression of HUWE1 was closely associated with worse prognosis of GBM patients. The ubiquitination and subsequent degradation of N‐Myc mediated by HUWE1, leading to the inactivation of downstream Delta‐like 1 (DLL1)‐NOTCH1 signaling pathways, inhibited the proliferation, invasion, and migration of GBM cells in vitro and in vivo. A rAAV dual‐vector system for packaging and delivery of dCas9‐VP64 was used to augment endogenous HUWE1 expression in vivo and showed an antitumor activity in glioma orthotopic xenografts.ConclusionsThe E3 ubiquitin ligase HUWE1 acts through the N‐Myc‐DLL1‐NOTCH1 signaling axis to suppress GBM progression. Antitumor activity of rAAV dual‐vector delivering dCas9‐HUWE1 system uncovers a promising therapeutic strategy for GBM.

HELZ2 promotes K63-linked polyubiquitination of c-Myc to induce retinoblastoma tumorigenesis.

Retinoblastoma is a rare ocular tumor in children that originates in the retina. Several core transcriptional regulators maintain the expansion of retinoblastoma tumors, including c-Myc. Here, we demonstrated that Helicase with zinc finger domain 2 (HELZ2) promoted retinoblastoma tumorigenesis by targeting c-Myc. HELZ2-deficient inhibited retinoblastoma cell proliferation, whereas overexpression of HELZ2 promoted retinoblastoma cell proliferation. In addition, high levels of HELZ2 promoted xenograft retinoblastoma tumorigenesis and inhibited animal survival. Mechanistically, HELZ2 interacted with c-Myc and promoted its K63-linked polyubiquitination. We indicated that HELZ2 promoted the interaction between E3 ubiquitin ligase HUWE1 and c-Myc, and HELZ2-mediated K63-linked polyubiquitination and activation of c-Myc were dependent on HUWE1. Taken together, HELZ2 plays a critical role in the regulation of retinoblastoma tumorigenesis by enhancing the activity of c-Myc.

SGK2, 14-3-3, and HUWE1 Cooperate to Control the Localization, Stability, and Function of the Oncoprotein PTOV1.

PTOV1 is an oncogenic protein, initially identified in prostate cancer, that promotes proliferation, cell motility, and invasiveness. However, the mechanisms that regulate PTOV1 remain unclear. Here, we identify 14-3-3 as a PTOV1 interactor and show that high levels of 14-3-3 expression, like PTOV1, correlate with prostate cancer progression. We discover an SGK2-mediated phosphorylation of PTOV1 at S36, which is required for 14-3-3 binding. Disruption of the PTOV1-14-3-3 interaction results in an accumulation of PTOV1 in the nucleus and a proteasome-dependent reduction in PTOV1 protein levels. We find that loss of 14-3-3 binding leads to an increase in PTOV1 binding to the E3 ubiquitin ligase HUWE1, which promotes proteasomal degradation of PTOV1. Conversely, our data suggest that 14-3-3 stabilizes PTOV1 protein by sequestering PTOV1 in the cytosol and inhibiting its interaction with HUWE1. Finally, our data suggest that stabilization of the 14-3-3-bound form of PTOV1 promotes PTOV1-mediated expression of cJun, which drives cell-cycle progression in cancer. Together, these data provide a mechanism to understand the regulation of the oncoprotein PTOV1. IMPLICATIONS: These findings identify a potentially targetable mechanism that regulates the oncoprotein PTOV1.

Abstract 2295: SGK2, 14-3-3, and HUWE1 coordinately regulate the localization and stability of PTOV1

Abstract PTOV1 is an oncogenic protein, initially identified in prostate cancer, that promotes proliferation, cell motility, and invasiveness. However, the mechanism of PTOV1 regulation is poorly understood. To understand these mechanisms, we identify 14-3-3 as a PTOV1 interactor and show that high levels of 14-3-3 expression, like PTOV1, correlate with prostate cancer progression. Further, we found that SGK2-mediated phosphorylation of PTOV1 is required for 14-3-3 binding. This phosphorylation occurs in a cell cycle-dependent manner and peaks in the G1 phase. Disruption of the PTOV1-14-3-3 interaction results in an accumulation of PTOV1 in the nucleus and a proteasome-dependent reduction in PTOV1 protein levels. To understand the effect of 14-3-3 on PTOV1 stability, we found that loss of 14-3-3 binding leads to an increase in PTOV1 binding to the E3 ubiquitin ligase HUWE1, which promotes proteasomal degradation of PTOV1. Conversely, our data suggest that 14-3-3 stabilizes PTOV1 protein by sequestering PTOV1 in the cytosol and inhibiting its interaction with HUWE1. Finally, our data suggest that stabilization of the 14-3-3-bound form of PTOV1 promotes PTOV1-mediated expression of cJun. Together, these data provide a first mechanism to understand the regulation of PTOV1 stability, localization, and function within the cell. Citation Format: Katie L. Pennington, James Woods, Colin Muir, Colten M. McEwan, Pramoda S. Aththota Gamage, Riley J. Eastmond, Crissy M. Egbert, Tyler Heaton, Stephen R. Piccolo, Joshua L. Andersen. SGK2, 14-3-3, and HUWE1 coordinately regulate the localization and stability of PTOV1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2295.

The future belongs to those who prepare for it today

Hippocampal neural stem cells (NSCs) and neurogenesis decline sharply with age, though a small population remains. Two articles in this issue of Cell Stem Cell by Ibrayeva etal. (2021) and Harris etal. (2021) indicate the presence of subpopulations of NSCs whose dynamics of activation and self-renewal change over time and may be key to NSC preservation.

Increased p53 signaling impairs neural differentiation in HUWE1-promoted intellectual disabilities

SummaryEssential E3 ubiquitin ligase HUWE1 (HECT, UBA, and WWE domain containing 1) regulates key factors, such as p53. Although mutations in HUWE1 cause heterogenous neurodevelopmental X-linked intellectual disabilities (XLIDs), the disease mechanisms common to these syndromes remain unknown. In this work, we identify p53 signaling as the central process altered in HUWE1-promoted XLID syndromes. By focusing on Juberg-Marsidi syndrome (JMS), one of the severest XLIDs, we show that increased p53 signaling results from p53 accumulation caused by HUWE1 p.G4310R destabilization. This further alters cell-cycle progression and proliferation in JMS cells. Modeling of JMS neurodevelopment reveals majorly impaired neural differentiation accompanied by increased p53 signaling. The neural differentiation defects can be successfully rescued by reducing p53 levels and restoring the expression of p53 target genes, in particular CDKN1A/p21. In summary, our findings suggest that increased p53 signaling underlies HUWE1-promoted syndromes and impairs XLID JMS neural differentiation.

HUWE1 mediates inflammasome activation and promotes host defense against bacterial infection.

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.

Protease Activity Profiling via Programmable Phage Display of Comprehensive Proteome-Scale Peptide Libraries

Endopeptidases catalyze the internal cleavage of proteins, playing pivotal roles in protein turnover, substrate maturation, and the activation of signaling cascades. A broad range of biological functions in health and disease are controlled by proteases, yet assays to characterize their activities at a proteomic scale do not exist. To address this unmet need, we developed Sensing EndoPeptidase Activity via Release and recapture using flAnking Tag Epitopes (SEPARATE), which uses a monovalent phage display of the human proteome at a 90-aa peptide resolution. We demonstrate that SEPARATE is compatible with several human proteases from distinct catalytic classes, including caspase-1, ADAM17, and thrombin. Both well-characterized and newly identified substrates of these enzymes were detected in the assay. SEPARATE was used to discover a non-canonical caspase-1 substrate, the E3 ubiquitin ligase HUWE1, a key mediator of apoptotic cell death. SEPARATE enables efficient, unbiased assessment of endopeptidase activity by using a phage-displayed proteome. A record of this paper's Transparent Peer Review process is included in the Supplemental Information.

TiPARP forms nuclear condensates to degrade HIF-1α and suppress tumorigenesis

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.

Proteogenomics analysis unveils a TFG-RET gene fusion and druggable targets in papillary thyroid carcinomas

Papillary thyroid cancer (PTC) is the most common type of endocrine malignancy. By RNA-seq analysis, we identify a RET rearrangement in the tumour material of a patient who does not harbour any known RAS or BRAF mutations. This new gene fusion involves exons 1–4 from the 5′ end of the Trk fused Gene (TFG) fused to the 3′ end of RET tyrosine kinase leading to a TFG-RET fusion which transforms immortalized human thyroid cells in a kinase-dependent manner. TFG-RET oligomerises in a PB1 domain-dependent manner and oligomerisation of TFG-RET is required for oncogenic transformation. Quantitative proteomic analysis reveals the upregulation of E3 Ubiquitin ligase HUWE1 and DUBs like USP9X and UBP7 in both tumor and metastatic lesions, which is further confirmed in additional patients. Expression of TFG-RET leads to the upregulation of HUWE1 and inhibition of HUWE1 significantly reduces RET-mediated oncogenesis.

The structure and regulation of the E3 ubiquitin ligase HUWE1 and its biological functions in cancer.

E3 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.

Prolyl hydroxylase 3 controls the intestine goblet cell generation through stabilizing ATOH1.

Intestinal epithelia self-renew constantly and generate differentiated cells such as secretary goblet cells. The intestine goblet cells secrete gel-forming mucins that form mucus to create a barrier of defense. We reported previously that loss of prolyl hydroxylase (PHD) 3 led to disruption of the intestinal epithelial barrier function. However, the underlying mechanism remains elusive. Here, we demonstrate that PHD3 controls the generation of intestine goblet cell. We found that genetic ablation of Phd3 in mice intestine epithelial cells reduced the amount of goblet cells. Mechanistically, PHD3 bounds the E3 ubiquitin ligase HUWE1 and prevented HUWE1 from mediating ubiquitination and degradation of ATOH1, an essential driver for goblet cell differentiation. The prolyl hydroxylase activity-deficient variant PHD3(H196A) also prevented ATOH1 destruction. A genetic intestine epithelial PHD3(H196A)-knockin had no effect on ATOH1 expression or goblet cell amount in mice, suggesting that the PHD3 prolyl hydroxylase activity is dispensable for its ability to control ATOH1 expression and goblet cell generation. In dextran sulfate sodium (DSS)-induced experimental colitis, PHD3-knockout rather than PHD3(H196A)-knockin sensitized the mice to DSS treatment. Our results reveal an additional critical mechanism underlying the regulation of ATOH1 expression and goblet cell generation and highlight that PHD3 plays a role in controlling intestine goblet cell generation in a hydroxylase-independent manner.

Direct regulation of Chk1 protein stability by E3 ubiquitin ligase HUWE1.

The HECT E3 ubiquitin ligase HUWE1 is required for a wide array of important functions in cell biology. Although HUWE1 is known to play a role in DNA damage signaling, the mechanism(s) that underlie this function remain elusive. HUWE1 regulates effectors of DNA replication and genotoxic stress tolerance. However, the loss of HUWE1 can also result in the accrual of significant endogenous DNA damage due to insufficient remediation of replication stress induced by an overabundance of key substrates. We discovered that HUWE1 depletion leads to a significant increase in levels of the single-strand break effector kinase Chk1, independent of the DNA damage response, activation of apical DNA damage repair (DDR) signaling kinases (ATM and ATR), and the tumor suppressor p53. We also identified multiple lysine residues on Chk1 that are polyubiquitinated by HUWE1 invitro, many of which are within the kinase domain. HUWE1 knockdown also markedly prolonged the protein half-life of Chk1 in steady-state conditions and resulted in greater stabilization of Chk1 protein than depletion of Cul4A, an E3 ubiquitin ligase previously described to control Chk1 abundance. Moreover, prolonged replication stress induced by hydroxyurea or camptothecin resulted in a reduction of Chk1 protein levels, which was rescued by HUWE1 knockdown. Our study indicates that HUWE1 plays a significant role in the regulation of the DDR signaling pathway by directly modulating the abundance of Chk1 protein.

HUWE1 controls MCL1 stability to unleash AMBRA1-induced mitophagy.

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.

MTORC1-Regulated and HUWE1-Mediated WIPI2 Degradation Controls Autophagy Flux

mTORC1, the major homeostatic sensor and responder, regulates cell catabolism mainly by targeting autophagy. Here, we show that mTORC1 directly controls autophagosome formation via phosphorylation of WIPI2, a critical protein in isolation membrane growth and elongation. mTORC1 phosphorylates Ser395 of WIPI2, directing WIPI2 to interact specifically with the E3 ubiquitin ligase HUWE1 for ubiquitination and proteasomal degradation. Physiological or pharmacological inhibition of mTORC1 in cells promotes WIPI2 stabilization, autophagosome formation, and autophagic degradation. In mouse liver, fasting significantly increases the WIPI2 protein level, while silencing HUWE1 enhances autophagy, and introducing WIPI2 improves lipid clearance. Thus, regulation of the intracellular WIPI2 protein level by mTORC1 and HUWE1 is a key determinant of autophagy flux and may coordinate the initiation, progression, and completion of autophagy.

HUWE1 E3 ligase promotes PINK1/PARKIN-independent mitophagy by regulating AMBRA1 activation via IKK\u03b1

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.