Ubiquitin ligase E3 HUWE1/MULE targets transferrin receptor for degradation and suppresses ferroptosis in acute liver injury.

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 role of mitochondrial apoptotic pathway in islet amyloid-induced ß-cell death.

Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to ß-cell death in type 2 diabetes. We previously showed that extracellular hIAPP aggregates promote Fas-mediated ß-cell apoptosis. Here, we tested if hIAPP aggregates can trigger the mitochondrial apoptotic pathway (MAP). hIAPP aggregation in Ad-hIAPP transduced INS-1 and human islet ß-cells promoted cytochrome c release, caspase-9 activation and apoptosis, which were reduced by Bax inhibitor. Amyloid formation in hIAPP-expressing mouse islets during culture increased caspase-9 activation in ß-cells. Ad-hIAPP transduced islets from CytcKA/KA and BaxBak ßDKO mice (models of blocked MAP), had lower caspase-9-positive and apoptotic ß-cells than transduced wild-type islets, despite comparable amyloid formation. Blocking Fas (markedly) and Bax or caspase-9 (modestly) reduced ß-cell death induced by extracellular hIAPP aggregates. These findings suggest a role for MAP in amyloid-induced ß-cell death and a potential strategy to reduce intracellular amyloid ß-cell toxicity by blocking cytochrome c apoptotic function.

The Mitochondrial Transacylase, Tafazzin, Regulates for AML Stemness by Modulating Intracellular Levels of Phospholipids.

Tafazzin (TAZ) is a mitochondrial transacylase that remodels the mitochondrial cardiolipin into its mature form. Through a CRISPR screen, we identified TAZ as necessary for the growth and viability of acute myeloid leukemia (AML) cells. Genetic inhibition of TAZ reduced stemness and increased differentiation of AML cells both in vitro and in vivo. In contrast, knockdown of TAZ did not impair normal hematopoiesis under basal conditions. Mechanistically, inhibition of TAZ decreased levels of cardiolipin but also altered global levels of intracellular phospholipids, including phosphatidylserine, which controlled AML stemness and differentiation by modulating toll-like receptor (TLR) signaling.

HDAC2 Regulates Site-Specific Acetylation of MDM2 and Its Ubiquitination Signaling in Tumor Suppression.

Histone deacetylases (HDACs) are promising targets for cancer therapy, although their individual actions remain incompletely understood. Here, we identify a role for HDAC2 in the regulation of MDM2 acetylation at previously uncharacterized lysines. Upon inactivation of HDAC2, this acetylation creates a structural signal in the lysine-rich domain of MDM2 to prevent the recognition and degradation of its downstream substrate, MCL-1 ubiquitin ligase E3 (MULE). This mechanism further reveals a therapeutic connection between the MULE ubiquitin ligase function and tumor suppression. Specifically, we show that HDAC inhibitor treatment promotes the accumulation of MULE, which diminishes the t(X; 18) translocation-associated synovial sarcomagenesis by directly targeting the fusion product SS18-SSX for degradation. These results uncover a new HDAC2-dependent pathway that integrates reversible acetylation signaling to the anticancer ubiquitin response.

Glutathione Primes T Cell Metabolism for Inflammation.

Activated T cells produce reactive oxygen species (ROS), which trigger the antioxidative glutathione (GSH) response necessary to buffer rising ROS and prevent cellular damage. We report that GSH is essential for T cell effector functions through its regulation of metabolic activity. Conditional gene targeting of the catalytic subunit of glutamate cysteine ligase (Gclc) blocked GSH production specifically in murine T cells. Gclc-deficient T cells initially underwent normal activation but could not meet their increased energy and biosynthetic requirements. GSH deficiency compromised the activation of mammalian target of rapamycin-1 (mTOR) and expression of NFAT and Myc transcription factors, abrogating the energy utilization and Myc-dependent metabolic reprogramming that allows activated T cells to switch to glycolysis and glutaminolysis. In vivo, T-cell-specific ablation of murine Gclc prevented autoimmune disease but blocked antiviral defense. The antioxidative GSH pathway thus plays an unexpected role in metabolic integration and reprogramming during inflammatory T cell responses.

The E3 ligase Mule protects the heart against oxidative stress and mitochondrial dysfunction through Myc-dependent inactivation of Pgc-1α and Pink1.

Cardiac homeostasis requires proper control of protein turnover. Protein degradation is principally controlled by the Ubiquitin-Proteasome System. Mule is an E3 ubiquitin ligase that regulates cellular growth, DNA repair and apoptosis to maintain normal tissue architecture. However, Mule's function in the heart has yet to be described. In a screen, we found reduced Mule expression in left ventricular samples from end-stage heart failure patients. Consequently, we generated conditional cardiac-specific Mule knockout (Mule fl/fl(y);mcm) mice. Mule ablation in adult Mule fl/fl(y);mcm mice prevented myocardial c-Myc polyubiquitination, leading to c-Myc accumulation and subsequent reduced expression of Pgc-1α, Pink1, and mitochondrial complex proteins. Furthermore, these mice developed spontaneous cardiac hypertrophy, left ventricular dysfunction, and early mortality. Co-deletion of Mule and c-Myc rescued this phenotype. Our data supports an indispensable role for Mule in cardiac homeostasis through the regulation of mitochondrial function via maintenance of Pgc-1α and Pink1 expression and persistent negative regulation of c-Myc.

E3 ubiquitin ligase Mule targets ß-catenin under conditions of hyperactive Wnt signaling.

Wnt signaling, named after the secreted proteins that bind to cell surface receptors to activate the pathway, plays critical roles both in embryonic development and the maintenance of homeostasis in many adult tissues. Two particularly important cellular programs orchestrated by Wnt signaling are proliferation and stem cell self-renewal. Constitutive activation of the Wnt pathway resulting from mutation or improper modulation of pathway components contributes to cancer development in various tissues. Colon cancers frequently bear inactivating mutations of the adenomatous polyposis coli (APC) gene, whose product is an important component of the destruction complex that regulates ß-catenin levels. Stabilization and nuclear localization of ß-catenin result in the expression of a panel of Wnt target genes. We previously showed that Mule/Huwe1/Arf-BP1 (Mule) controls murine intestinal stem and progenitor cell proliferation by modulating the Wnt pathway via c-Myc. Here we extend our investigation of Mule's influence on oncogenesis by showing that Mule interacts directly with ß-catenin and targets it for degradation under conditions of hyperactive Wnt signaling. Our findings suggest that Mule uses various mechanisms to fine-tune the Wnt pathway and provides multiple safeguards against tumorigenesis.

K48-linked KLF4 ubiquitination by E3 ligase Mule controls T-cell proliferation and cell cycle progression.

T-cell proliferation is regulated by ubiquitination but the underlying molecular mechanism remains obscure. Here we report that Lys-48-linked ubiquitination of the transcription factor KLF4 mediated by the E3 ligase Mule promotes T-cell entry into S phase. Mule is elevated in T cells upon TCR engagement, and Mule deficiency in T cells blocks proliferation because KLF4 accumulates and drives upregulation of its transcriptional targets E2F2 and the cyclin-dependent kinase inhibitors p21 and p27. T-cell-specific Mule knockout (TMKO) mice develop exacerbated experimental autoimmune encephalomyelitis (EAE), show impaired generation of antigen-specific CD8+ T cells with reduced cytokine production, and fail to clear LCMV infections. Thus, Mule-mediated ubiquitination of the novel substrate KLF4 regulates T-cell proliferation, autoimmunity and antiviral immune responses in vivo.

IDH1 deficiency attenuates gluconeogenesis in mouse liver by impairing amino acid utilization.

Although the enzymatic activity of isocitrate dehydrogenase 1 (IDH1) was defined decades ago, its functions in vivo are not yet fully understood. Cytosolic IDH1 converts isocitrate to α-ketoglutarate (α-KG), a key metabolite regulating nitrogen homeostasis in catabolic pathways. It was thought that IDH1 might enhance lipid biosynthesis in liver or adipose tissue by generating NADPH, but we show here that lipid contents are relatively unchanged in both IDH1-null mouse liver and IDH1-deficient HepG2 cells generated using the CRISPR-Cas9 system. Instead, we found that IDH1 is critical for liver amino acid (AA) utilization. Body weights of IDH1-null mice fed a high-protein diet (HPD) were abnormally low. After prolonged fasting, IDH1-null mice exhibited decreased blood glucose but elevated blood alanine and glycine compared with wild-type (WT) controls. Similarly, in IDH1-deficient HepG2 cells, glucose consumption was increased, but alanine utilization and levels of intracellular α-KG and glutamate were reduced. In IDH1-deficient primary hepatocytes, gluconeogenesis as well as production of ammonia and urea were decreased. In IDH1-deficient whole livers, expression levels of genes involved in AA metabolism were reduced, whereas those involved in gluconeogenesis were up-regulated. Thus, IDH1 is critical for AA utilization in vivo and its deficiency attenuates gluconeogenesis primarily by impairing α-KG-dependent transamination of glucogenic AAs such as alanine.

Mutant IDH1 Downregulates ATM and Alters DNA Repair and Sensitivity to DNA Damage Independent of TET2.

Mutations in the isocitrate dehydrogenase-1 gene (IDH1) are common drivers of acute myeloid leukemia (AML) but their mechanism is not fully understood. It is thought that IDH1 mutants act by inhibiting TET2 to alter DNA methylation, but there are significant unexplained clinical differences between IDH1- and TET2-mutant diseases. We have discovered that mice expressing endogenous mutant IDH1 have reduced numbers of hematopoietic stem cells (HSCs), in contrast to Tet2 knockout (TET2-KO) mice. Mutant IDH1 downregulates the DNA damage (DD) sensor ATM by altering histone methylation, leading to impaired DNA repair, increased sensitivity to DD, and reduced HSC self-renewal, independent of TET2. ATM expression is also decreased in human IDH1-mutated AML. These findings may have implications for treatment of IDH-mutant leukemia.

Mule Regulates the Intestinal Stem Cell Niche via the Wnt Pathway and Targets EphB3 for Proteasomal and Lysosomal Degradation.

The E3 ubiquitin ligase Mule is often overexpressed in human colorectal cancers, but its role in gut tumorigenesis is unknown. Here, we show in vivo that Mule controls murine intestinal stem and progenitor cell proliferation by modulating Wnt signaling via c-Myc. Mule also regulates protein levels of the receptor tyrosine kinase EphB3 by targeting it for proteasomal and lysosomal degradation. In the intestine, EphB/ephrinB interactions position cells along the crypt-villus axis and compartmentalize incipient colorectal tumors. Our study thus unveils an important new avenue by which Mule acts as an intestinal tumor suppressor by regulation of the intestinal stem cell niche.

System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes.

HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins.

Idh1 mutations contribute to the development of T-cell malignancies in genetically engineered mice.

Gain-of-function mutations in isocitrate dehydrogenase 1 (IDH1) are key drivers of hematopoietic malignancies. Although these mutations are most commonly associated with myeloid diseases, they also occur in malignancies of the T-cell lineage. To investigate their role in these diseases and provide tractable disease models for further investigation, we analyzed the T-cell compartment in a conditional knock-in (KI) mouse model of mutant Idh1. We observed the development of a spontaneous T-cell acute lymphoblastic leukemia (T-ALL) in these animals. The disease was transplantable and maintained expression of mutant IDH1. Whole-exome sequencing revealed the presence of a spontaneous activating mutation in Notch1, one of the most common mutations in human T-ALL, suggesting Idh1 mutations may have the capacity to cooperate with Notch1 to drive T-ALL. To further investigate the Idh1 mutation as an oncogenic driver in the T-cell lineage, we crossed Idh1-KI mice with conditional Trp53 null mice, a well-characterized model of T-cell malignancy, and found that T-cell lymphomagenesis was accelerated in mice bearing both mutations. Because both IDH1 and p53 are known to affect cellular metabolism, we compared the requirements for glucose and glutamine in cells derived from these tumors and found that cells bearing the Idh1 mutation have an increased dependence on both glucose and glutamine. These data suggest that mutant IDH1 contributes to malignancy in the T-cell lineage and may alter the metabolic profile of malignant T cells.

The Rho-guanine nucleotide exchange factor PDZ-RhoGEF governs susceptibility to diet-induced obesity and type 2 diabetes.

Adipose tissue is crucial for the maintenance of energy and metabolic homeostasis and its deregulation can lead to obesity and type II diabetes (T2D). Using gene disruption in the mouse, we discovered a function for a RhoA-specific guanine nucleotide exchange factor PDZ-RhoGEF (Arhgef11) in white adipose tissue biology. While PDZ-RhoGEF was dispensable for a number of RhoA signaling-mediated processes in mouse embryonic fibroblasts, including stress fiber formation and cell migration, it's deletion led to a reduction in their proliferative potential. On a whole organism level, PDZ-RhoGEF deletion resulted in an acute increase in energy expenditure, selectively impaired early adipose tissue development and decreased adiposity in adults. PDZ-RhoGEF-deficient mice were protected from diet-induced obesity and T2D. Mechanistically, PDZ-RhoGEF enhanced insulin/IGF-1 signaling in adipose tissue by controlling ROCK-dependent phosphorylation of the insulin receptor substrate-1 (IRS-1). Our results demonstrate that PDZ-RhoGEF acts as a key determinant of mammalian metabolism and obesity-associated pathologies.

Inhibition of the Mitochondrial Protease ClpP as a Therapeutic Strategy for Human Acute Myeloid Leukemia.

From an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has structural similarity to the cytoplasmic proteosome is increased in leukemic cells from approximately half of all patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found that ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism.

Autophagy-independent functions of UVRAG are essential for peripheral naive T-cell homeostasis.

UV radiation resistance-associated gene (UVRAG) encodes a tumor suppressor with putative roles in autophagy, endocytic trafficking, and DNA damage repair but its in vivo role in T cells is unknown. Because conditional homozygous deletion of Uvrag in mice results in early embryonic lethality, we generated T-cell-specific UVRAG-deficient mice that lacked UVRAG expression specifically in T cells. This loss of UVRAG led to defects in peripheral homeostasis that could not be explained by the increased sensitivity to cell death and impaired proliferation observed for other autophagy-related gene knockout mice. Instead, UVRAG-deficient T-cells exhibited normal mitochondrial clearance and activation-induced autophagy, suggesting that UVRAG has an autophagy-independent role that is critical for peripheral naive T-cell homeostatic proliferation. In vivo, T-cell-specific loss of UVRAG dampened CD8(+) T-cell responses to LCMV infection in mice, delayed viral clearance, and impaired memory T-cell generation. Our data provide novel insights into the control of autophagy in T cells and identify UVRAG as a new regulator of naïve peripheral T-cell homeostasis.

T-cell STAT3 is required for the maintenance of humoral immunity to LCMV.

STAT3 is a critical transcription factor activated downstream of cytokine signaling and is integral for the function of multiple immune cell types. Human mutations in STAT3 cause primary immunodeficiency resulting in impaired control of a variety of infections, including reactivation of latent viruses. In this study, we investigate how T-cell functions of STAT3 contribute to responses to viral infection by inducing chronic lymphocytic choriomeningitis virus (LCMV) infection in mice lacking STAT3 specifically in T cells. Although mice with conditional disruption of STAT3 in T cells were able to mount early responses to viral infection similar to control animals, including expansion of effector T cells, we found generation of T-follicular helper (Tfh) cells to be impaired. As a result, STAT3 T cell deficient mice produced attenuated germinal center reactions, and did not accumulate bone marrow virus specific IgG-secreting cells, resulting in failure to maintain levels of virus-specific IgG or mount neutralizing responses to LCMV in the serum. These effects were associated with reduced control of viral replication and prolonged infection. Our results demonstrate the importance of STAT3 in T cells for the generation of functional long-term humoral immunity to viral infections.

XB130 deficiency affects tracheal epithelial differentiation during airway repair.

The repair and regeneration of airway epithelium is important for maintaining homeostasis of the respiratory system. XB130 is an adaptor protein involved in the regulation of cell proliferation, survival and migration. In the human trachea, XB130 is expressed on the apical site of ciliated epithelial cells. We hypothesize that XB130 may play a role in epithelial repair and regeneration after injury. Xb130 knockout (KO) mice were generated, and a mouse isogenic tracheal transplantation model was used. Adult Xb130 KO mice did not show any significant anatomical and physiological phenotypes in comparison with their wild type (WT) littermates. The tracheal epithelium in Xb130 KO mice, however, was significantly thicker than that in WT mice. Severe ischemic epithelial injury was observed immediately after the tracheal transplantation, which was followed by epithelial cell flattening, proliferation and differentiation. No significant differences were observed in terms of initial airway injury and apoptosis. However, at Day 10 after transplantation, the epithelial layer was significantly thicker in Xb130 KO mice, and associated with greater proliferative (Ki67+) and basal (CK5+) cells, as well as thickening of the connective tissue and fibroblast layer between the epithelium and tracheal cartilages. These results suggest that XB130 is involved in the regulation of airway epithelial differentiation, especially during airway repair after injury.