Polyubiquitination of the demethylase Jhd2 controls histone methylation and gene expression

The identification of histone methyltransferases and demethylases has uncovered a dynamic methylation system needed to modulate appropriate levels of gene expression. Gene expression levels of various histone demethylases, such as the JARID1 family, show distinct patterns of embryonic and adult expression and respond to different environmental cues, suggesting that histone demethylase protein levels must be tightly regulated for proper development. In our study, we show that the protein level of the yeast histone H3 Lys 4 (H3 K4) demethylase Jhd2/Kdm5 is modulated through polyubiquitination by the E3 ubiquitin ligase Not4 and turnover by the proteasome. We determine that polyubiquitin-mediated degradation of Jhd2 controls in vivo H3 K4 trimethylation and gene expression levels. Finally, we show that human NOT4 can polyubiquitinate human JARID1C/SMCX, a homolog of Jhd2, suggesting that this is likely a conserved mechanism. We propose that Not4 is an E3 ubiquitin ligase that monitors and controls a precise amount of Jhd2 protein so that the proper balance between histone demethylase and histone methyltransferase activities occur in the cell, ensuring appropriate levels of H3 K4 trimethylation and gene expression.     

WD40 protein FBW5 promotes ubiquitination of tumor suppressor TSC2 by DDB1-CUL4-ROC1 ligase

Tuberous sclerosis (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs and is caused by mutations targeting either the TSC1 or TSC2 genes. TSC1 and TSC2 proteins form a functionally interdependent dimeric complex. Phosphorylation of either TSC subunit by different kinases regulates the function of TSC and represents a major mechanism to integrate various signals into a centralized cell growth pathway. The majority of disease-associated mutations targeting either TSC1 or TSC2 results in a substantial decrease in protein level, suggesting that protein turnover also plays a critical role in TSC regulation. Here we report that TSC2 protein binds to FBW5, a DDB1-binding WD40 (DWD) protein, and is recruited by FBW5 to the DDB1-CUL4-ROC1 E3 ubiquitin ligase. Overexpression of FBW5 or CUL4A promotes TSC2 protein degradation, and this is abrogated by the coexpression of TSC1. Conversely, depletion of FBW5, DDB1, or CUL4A/B stabilizes TSC2. Ddb1 or Cul4 mutations in Drosophila result in Gigas/TSC2 protein accumulation and cause growth defects that can be partially rescued by Gigas/Tsc2 reduction. These results indicate that FBW5-DDB1-CUL4-ROC1 is an E3 ubiquitin ligase regulating TSC2 protein stability and TSC complex turnover.     

Nicotine increases apoptosis in HUVECs cultured in high glucose/high fat via Akt ubiquitination and degradation

It is well‐documented that nicotine, the main active ingredient in cigarettes, results in endothelial cell injury in numerous diseases. However, whether nicotine plays a crucial role in endothelial cell injury in diabetes and the exact molecular mechanism that mediates this process have not been fully elucidated. The current study aimed to investigate the effects of nicotine on endothelial cell injury in diabetes and the specific molecular mechanism by which it plays a role. Human umbilical vein endothelial cells (HUVECs) were incubated in HG/HF media and treated with nicotine, PYR‐41 (a selective ubiquitin E1 inhibitor), Akt‐overexpressing adenovirus, or TTC3 and MUL1 shRNA adenovirus. Cell viability was subsequently detected by the CCK8 assay, and apoptosis was examined by caspase‐3 cleavage and activity analysis. Compared to the HG/HF incubated group, nicotine incubation significantly decreased cell survival and increased apoptosis. Moreover, nicotine induced Akt degradation via UPS, and Akt overexpression blocked nicotine‐induced apoptosis in HUVECs cultured in HG/HF media. Furthermore, the TTC3 and MUL1 shRNA adenovirus dramatically decreased the Akt ubiquitination and apoptosis induced by nicotine. These results indicate that nicotine‐induced Akt ubiquitination and degradation occurs through TTC3 and MUL1 and results in a dramatic increase in apoptosis in HUVECs cultured in HG/HF media.     

A patent review of the ubiquitin ligase system: 2015–2018

ABSTRACT

Introduction: Ubiquitin-proteasome system (UPS) has been validated as a novel anticancer drug target in the past 20 years. The UPS contains two distinct steps: ubiquitination of a substrate protein by ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3), and substrate degradation by the 26S proteasome complex. The E3 enzyme is the central player in the ubiquitination step and has a wide range of specific substrates in cancer cells, offering great opportunities for discovery and development of selective drugs.

Areas covered: This review summarizes the recent advances in small molecule inhibitors of E1s, E2s, and E3s, with a focus on the latest patents (from 2015 to 2018) of E3 inhibitors and modulators.

Expert opinion: One strategy to overcome limitations of current 20S proteasome inhibitors is to discover inhibitors of the upstream key components of the UPS, such as E3 enzymes. E3s play important roles in cancer development and determine the specificity of substrate ubiquitination, offering novel target opportunities. E3 modulators could be developed by rational design, natural compound or library screening, old drug repurposes, and application of other novel technologies. Further understanding of mechanisms of E3–substrate interaction will be essential for discovering and developing next-generation E3 inhibitors as effective anticancer drugs.     

HRD1 inhibits fatty acid oxidation and tumorigenesis by ubiquitinating CPT2 in triple‐negative breast cancer

Dependence on glutamine and acceleration of fatty acid oxidation (FAO) are both metabolic characteristics of triple‐negative breast cancer (TNBC). With the rapid growth of tumors, accelerated glutamine catabolism depletes local glutamine, resulting in glutamine deficiency. Studies have shown that the use of alternative energy sources, such as fatty acids, enables tumor cells to continue to proliferate rapidly in a glutamine‐deficient microenvironment. However, the detailed mechanisms behind this metabolic change are still unclear. Herein, we identified HRD1 as a regulatory protein for FAO that specifically inhibits TNBC cell proliferation under glutamine‐deficient conditions. Furthermore, we observed that HRD1 expression is significantly downregulated under glutamine deprivation and HRD1 directly ubiquitinates and stabilizes CPT2 through K48‐linked ubiquitination. In addition, the inhibition of CPT2 expression dramatically suppresses TNBC cell proliferation mediated by HRD1 knockdown in vitro and in vivo. Finally, we found that the glutaminase inhibitor CB839 significantly inhibited TNBC cell tumor growth, but not in the HRD1 knock‐downed TNBC cells. These findings provide an invaluable insight into HRD1 as a regulator of lipid metabolism and have important implications for TNBC therapeutic targeting.

Abbreviations

CPT1A

carnitine palmitoyltransferase 1A

CPT2

carnitine palmitoyltransferase 2

FAO

fatty acid oxidation

GLS

glutaminase

HRD1

HMG‐CoA reductase degradation protein 1

LC‐MS

liquid chromatography mass spectrometry

TNBC

triple‐negative breast cancer

     

The role of ubiquitin pathway-mediated regulation of immune checkpoints in cancer immunotherapy

With the continuous cognition of the relationship between tumor cells and tumor immune microenvironment, immunotherapy based on the immune checkpoint blockade has achieved great breakthroughs, led to improved clinical outcomes, and prolonged survival for cancer patients in recent years. Nevertheless, the de novo or acquired resistance to immunotherapy has greatly counteracted the efficacy, leading to a 20%–40% overall response rate. Thus, further in-depth understanding of the regulation of the tumor microenvironment and antitumor immunity is urgently warranted. Ubiquitination-mediated protein degradation plays vital roles in protein stabilization, activation, and dynamics as well as in cellular homeostasis modulation. The dysregulated ubiquitination and deubiquitination are closely related to the changes in physiological and pathological processes, which subsequently result in a variety of diseases including cancer. In this review, the authors first summarize the current knowledge about the involvement of the ubiquitin-proteasome system in tumor development with the ubiquitin conjugation-regulated stability of p53, phosphatase and tensin homolog, and Myc protein as examples, then dissect the potential implications of ubiquitination-mediated immune checkpoints degradation in tumor microenvironment and immune responses, and finally discuss the effects of therapeutically targeting the ubiquitin-proteasome pathway on immunotherapy, with the goal of providing deep insights into the exploitation of more precise and effective combinational therapy against cancer.     

FRK inhibits glioblastoma progression via phosphorylating YAP and inducing its ubiquitylation and degradation by Siah1

BackgroundWe previously report that yes-associated protein (YAP), the core downstream effector of Hippo pathway, promotes the malignant progression of glioblastoma (GBM). However, although classical regulatory mechanisms of YAP are well explored, how YAP is modulated by the Hippo-independent manner remains poorly understood. Meanwhile, the nonreceptor tyrosine kinase Fyn-related kinase (FRK), which exhibits low expression and possesses tumor suppressor effects in GBM, is reported to be involved in regulation of protein phosphorylation. Here, we examined whether FRK could impede tumor progression by modulating YAP activities.MethodsHuman GBM cells and intracranial GBM model were used to assess the effects of FRK and YAP on the malignant biological behaviors of GBM. Immunoblotting and immunohistochemistry were used to detect the expression of core proteins in GBM tissues. Co-immunoprecipitation, proximity ligation assay, luciferase assay and ubiquitination assay were utilized to determine the protein–protein interactions and related molecular mechanisms.ResultsThe expression levels of FRK and YAP were inversely correlated with each other in glioma tissues. In addition, FRK promoted the ubiquitination and degradation of YAP, leading to tumor suppression in vitro and in vivo. Mechanistically, FRK interacted with and phosphorylated YAP on Tyr391/407/444, which recruited the classical E3 ubiquitin ligase Siah1 to catalyze ubiquitination and eventually degradation of YAP. Siah1 is required for YAP destabilization initiated by FRK.ConclusionsWe identify a novel mechanism by which FRK orchestrates tumor-suppression effect through phosphorylating YAP and inducing its ubiquitination by Siah1. FRK-Siah1-YAP signaling axis may serve as a potential therapeutic target for GBM treatment.