Inhibition of cIAP1 as a strategy for targeting c-MYC–driven oncogenic activity

Protooncogenec-MYC, a master transcription factor, is a major driver of human tumorigenesis. Development of pharmacological agents for inhibiting c-MYC as an anticancer therapy has been a longstanding but elusive goal in the cancer field. E3 ubiquitin ligase cIAP1 has been shown to mediate the activation of c-MYC by destabilizing MAD1, a key antagonist of c-MYC. Here we developed a high-throughput assay for cIAP1 ubiquitination and identified D19, a small-molecule inhibitor of E3 ligase activity of cIAP1. We show that D19 binds to the RING domain of cIAP1 and inhibits the E3 ligase activity of cIAP1 by interfering with the dynamics of its interaction with E2. Blocking cIAP1 with D19 antagonizes c-MYC by stabilizing MAD1 protein in cells. Furthermore, we show that D19 and an improved analog (D19-14) promote c-MYC degradation and inhibit the oncogenic function of c-MYC in cells and xenograft animal models. In contrast, we show that activating E3 ubiquitin ligase activity of cIAP1 by Smac mimetics destabilizes MAD1, the antagonist of MYC, and increases the protein levels of c-MYC. Our study provides an interesting example using chemical biological approaches for determining distinct biological consequences from inhibiting vs. activating an E3 ubiquitin ligase and suggests a potential broad therapeutic strategy for targeting c-MYC in cancer treatment by pharmacologically modulating cIAP1 E3 ligase activity.

Download Full-text

PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity

The Journal of Cell Biology ◽

10.1083/jcb.201402104 ◽

2014 ◽

Vol 205 (2) ◽

pp. 143-153 ◽

Cited By ~ 610

Author(s):

Lesley A. Kane ◽

Michael Lazarou ◽

Adam I. Fogel ◽

Yan Li ◽

Koji Yamano ◽

...

Keyword(s):

Mass Spectrometry ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

E3 Ligase ◽

Feed Forward ◽

Selective Autophagy ◽

Ubiquitin Ligase Activity ◽

In Cells

PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin’s ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity.

Download Full-text

Targeting ADT-Induced Activation of the E3 Ubiquitin Ligase Siah2 to Delay the Occurrence of Castration-Resistant Prostate Cancer

Frontiers in Oncology ◽

10.3389/fonc.2021.637040 ◽

2021 ◽

Vol 11 ◽

Author(s):

Tingmang Yan ◽

Dapeng Zhou ◽

Youwei Shi ◽

Di Cui ◽

Juntao Jiang ◽

...

Keyword(s):

Prostate Cancer ◽

Protein Expression ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

E3 Ligase ◽

Castration Resistant Prostate Cancer ◽

Vitamin K3 ◽

Protein Levels ◽

Castration Resistant

Siah2 is an E3 ubiquitin ligase that targets androgen receptor (AR) and plays an important role in the development of castration-resistant prostate cancer (CRPC). However, the regulation of Siah2 in prostate cancer (PCa) is largely unknown. In this study, we used AR-dependent and -independent cells lines to investigate the cellular roles of AR and androgen deprivation therapy (ADT) on Siah2 protein levels and E3 ligase activity using Western blotting and co-immunoprecipitation. We also validated our findings using patient samples taken before and after ADT. Finally, we used xenograft tumor models to test the effects of ADT combined with vitamin K3 (Vit K3) on tumor growth in vivo. Our results showed that AR stabilizes Siah2 protein by attenuating its self-ubiquitination and auto-degradation, likely by blocking its E3 ubiquitin ligase activity. Conversely, ADT decreased Siah2 protein expression but enhanced its E3 ligase activity in PCa cells. Notably, the findings that ADT decreasing Siah2 protein expression were verified in a series of paired PCa samples from the same patient. Additionally, we found that ADT-induced Siah2 activation could be abolished by Vit K3. Strikingly, ADT combined with Vit K3 treatment delayed the occurrence of CRPC and dramatically inhibited the growth of tumor xenografts compared with ADT treatment alone. AR is an inhibitor of Siah2 in PCa, and ADT leads to the continuous activation of Siah2, which may contribute to CRPC. Finally, ADT+Vit K3 may be a potential approach to delay the occurrence of CRPC.

Download Full-text

Latency-Associated Nuclear Antigen E3 Ubiquitin Ligase Activity Impacts Gammaherpesvirus-Driven Germinal Center B Cell Proliferation

Journal of Virology ◽

10.1128/jvi.00813-16 ◽

2016 ◽

Vol 90 (17) ◽

pp. 7667-7683 ◽

Cited By ~ 3

Author(s):

Sofia A. Cerqueira ◽

Min Tan ◽

Shijun Li ◽

Franceline Juillard ◽

Colin E. McVey ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Ubiquitin Ligase ◽

Germinal Center ◽

E3 Ubiquitin Ligase ◽

E3 Ligase ◽

Nuclear Antigen ◽

Ubiquitin Ligase Activity ◽

Socs Box

ABSTRACTViruses have evolved mechanisms to hijack components of cellular E3 ubiquitin ligases, thus modulating the ubiquitination pathway. However, the biological relevance of such mechanisms for viral pathogenesisin vivoremains largely unknown. Here, we utilized murid herpesvirus 4 (MuHV-4) infection of mice as a model system to address the role of MuHV-4 latency-associated nuclear antigen (mLANA) E3 ligase activity in gammaherpesvirus latent infection. We show that specific mutations in the mLANA SOCS box (V199A, V199A/L202A, or P203A/P206A) disrupted mLANA's ability to recruit Elongin C and Cullin 5, thereby impairing the formation of the Elongin BC/Cullin 5/SOCS (EC5SmLANA) complex and mLANA's E3 ligase activity on host NF-κB and Myc. Although these mutations resulted in considerably reduced mLANA binding to viral terminal repeat DNA as assessed by electrophoretic mobility shift assay (EMSA), the mutations did not disrupt mLANA's ability to mediate episome persistence.In vivo, MuHV-4 recombinant viruses bearing these mLANA SOCS box mutations exhibited a deficit in latency amplification in germinal center (GC) B cells. These findings demonstrate that the E3 ligase activity of mLANA contributes to gammaherpesvirus-driven GC B cell proliferation. Hence, pharmacological inhibition of viral E3 ligase activity through targeting SOCS box motifs is a putative strategy to control gammaherpesvirus-driven lymphoproliferation and associated disease.IMPORTANCEThe gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause lifelong persistent infection and play causative roles in several human malignancies. Colonization of B cells is crucial for virus persistence, and access to the B cell compartment is gained by virus-driven proliferation in germinal center (GC) B cells. Infection of B cells is predominantly latent, with the viral genome persisting as a multicopy episome and expressing only a small subset of viral genes. Here, we focused on latency-associated nuclear antigen (mLANA) encoded by murid herpesvirus-4 (MuHV-4), which exhibits homology in sequence, structure, and function to KSHV LANA (kLANA), thereby allowing the study of LANA-mediated pathogenesis in mice. Our experiments show that mLANA's E3 ubiquitin ligase activity is necessary for efficient expansion of latency in GC B cells, suggesting that the development of pharmacological inhibitors of LANA E3 ubiquitin ligase activity may allow strategies to interfere with gammaherpesvirus-driven lymphoproliferation and associated disease.

Download Full-text

PICK1 inhibits the E3 ubiquitin ligase activity of Parkin and reduces its neuronal protective effect

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1716506115 ◽

2018 ◽

Vol 115 (30) ◽

pp. E7193-E7201 ◽

Cited By ~ 7

Author(s):

Jing He ◽

Mengying Xia ◽

Patrick K. K. Yeung ◽

Jiahua Li ◽

Zhifang Li ◽

...

Keyword(s):

Protective Effect ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Loss Of Function ◽

Protective Function ◽

Bar Domain ◽

Protein Levels ◽

Ubiquitin Ligase Activity ◽

New Gene ◽

Familial Parkinson’S Disease

Parkin functions as a multipurpose E3 ubiquitin ligase, and Parkin loss of function is associated with both sporadic and familial Parkinson’s disease (PD). We report that the Bin/Amphiphysin/Rvs (BAR) domain of protein interacting with PRKCA1 (PICK1) bound to the really interesting new gene 1 (RING1) domain of Parkin and potently inhibited the E3 ligase activity of Parkin by disrupting its interaction with UbcH7. Parkin translocated to damaged mitochondria and led to their degradation in neurons, whereas PICK1 robustly inhibited this process. PICK1 also impaired the protective function of Parkin against stresses in SH-SY5Y cells and neurons. The protein levels of several Parkin substrates were reduced in young PICK1-knockout mice, and these mice were resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated toxicity. Taken together, the results indicate that PICK1 is a potent inhibitor of Parkin, and the reduction of PICK1 enhances the protective effect of Parkin.

Download Full-text

Discovery of Small-Molecule Inhibitors Targeting the E3 Ubiquitin Ligase Activity of the Herpes Simplex Virus 1 ICP0 Protein Using anIn VitroHigh-Throughput Screening Assay

Journal of Virology ◽

10.1128/jvi.00619-19 ◽

2019 ◽

Vol 93 (13) ◽

Cited By ~ 2

Author(s):

Thibaut Deschamps ◽

Hope Waisner ◽

Christos Dogrammatzis ◽

Anuradha Roy ◽

Shibin Chacko ◽

...

Keyword(s):

Gene Expression ◽

Herpes Simplex ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

E3 Ligase ◽

Screening Assay ◽

Herpes Simplex Virus 1 ◽

Ubiquitin Ligase Activity ◽

Simplex Virus ◽

Hsv 1

ABSTRACTHerpes simplex virus 1 (HSV-1) has infected more than 80% of the population. Reactivation of the virus causes diseases ranging in severity from benign cold sores to fatal encephalitis. Current treatments involve viral DNA replication inhibitors, but the emergence of drug-resistant mutants is observed frequently, highlighting the need for novel antiviral therapies. Infected cell protein 0 (ICP0) of HSV-1 is encoded by an immediate early gene and plays a fundamental role during infection, because it enables viral gene expression and blocks antiviral responses. One mechanism by which ICP0 functions is through an E3 ubiquitin ligase activity that induces the degradation of targeted proteins. A ΔICP0 virus or mutants with deficiencies in E3 ligase activity cannot counteract beta interferon (IFN-β)-induced restriction of viral infection, are highly immunogenic, are avirulent, and fail to spread. Thus, small molecules interfering with essential and conserved ICP0 functions are expected to compromise HSV-1 infection. We have developed a high-throughput screening assay, based on the autoubiquitination properties of ICP0, to identify small-molecule inhibitors of ICP0 E3 ubiquitin ligase activity. Through a pilot screening procedure, we identified nine compounds that displayed dose-dependent inhibitory effects on ICP0 but not on Mdm2, a control E3 ubiquitin ligase. Following validation, one compound displayed ICP0-dependent inhibition of HSV-1 infection. This compound appeared to bind ICP0 in a cellular thermal shift assay, it blocked ICP0 self-elimination, and it blocked wild-type but not ICP0-null virus gene expression. This scaffold displays specificity and could be used to develop optimized ICP0 E3 ligase inhibitors.IMPORTANCESince acyclovir and its derivatives were launched for herpesviruses control almost four decades ago, the search for novel antivirals has waned. However, as human life expectancy has increased, so has the number of immunocompromised individuals who receive prolonged treatment for HSV recurrences. This has led to an increase in unresponsive patients due to acquired viral drug resistance. Thus, novel treatments need to be explored. Here we explored the HSV-1 ICP0 E3 ligase as a potential antiviral target because (i) ICP0 is expressed before virus replication, (ii) it is essential for infectionin vivo, (iii) it is required for efficient reactivation of the virus from latency, (iv) inhibition of its E3 ligase activity would sustain host immune responses, and (v) it is shared by other herpesviruses. We report a compound that inhibits HSV-1 infection in an ICP0-dependent manner by inhibiting ICP0 E3 ligase activity.

Download Full-text

Itch WW Domains Inhibit Its E3 Ubiquitin Ligase Activity by Blocking E2-E3 Ligase Trans-thiolation

Journal of Biological Chemistry ◽

10.1074/jbc.m115.649269 ◽

2015 ◽

Vol 290 (39) ◽

pp. 23875-23887 ◽

Cited By ~ 40

Author(s):

Christopher Riling ◽

Hari Kamadurai ◽

Suresh Kumar ◽

Claire E. O'Leary ◽

Kuen-Phon Wu ◽

...

Keyword(s):

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

E3 Ligase ◽

Ww Domains ◽

Ubiquitin Ligase Activity

Download Full-text

Faculty Opinions recommendation of The E3 ubiquitin ligase activity of RING1B is not essential for early mouse development.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725799223.793526294 ◽

2016 ◽

Author(s):

Anton Wutz

Keyword(s):

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Mouse Development ◽

Ubiquitin Ligase Activity ◽

Early Mouse

Download Full-text

Therapeutically actionable signaling node to rescue AURKA driven loss of primary cilia in VHL-deficient cells

Scientific Reports ◽

10.1038/s41598-021-89933-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Pratim Chowdhury ◽

Dimuthu Perera ◽

Reid T. Powell ◽

Tia Talley ◽

Durga Nand Tripathi ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Primary Cilia ◽

Mtor Inhibitor ◽

E3 Ubiquitin Ligase ◽

Xenograft Model ◽

Aurora Kinase ◽

Tumor Burden ◽

Aurora Kinase A ◽

Von Hippel Lindau ◽

In Cells

AbstractLoss of primary cilia in cells deficient for the tumor suppressor von Hippel Lindau (VHL) arise from elevated Aurora Kinase A (AURKA) levels. VHL in its role as an E3 ubiquitin ligase targets AURKA for degradation and in the absence of VHL, high levels of AURKA result in destabilization of the primary cilium. We identified NVP-BEZ235, a dual PI3K/AKT and mTOR inhibitor, in an image-based high throughput screen, as a small molecule that restored primary cilia in VHL-deficient cells. We identified the ability of AKT to modulate AURKA expression at the transcript and protein level. Independent modulation of AKT and mTOR signaling decreased AURKA expression in cells confirming AURKA as a new signaling node downstream of the PI3K cascade. Corroborating these data, a genetic knockdown of AKT in cells deficient for VHL rescued the ability of these cells to ciliate. Finally, inhibition of AKT/mTOR using NVP-BEZ235 was efficacious in reducing tumor burden in a 786-0 xenograft model of renal cell carcinoma. These data highlight a previously unappreciated signaling node downstream of the AKT/mTOR pathway via AURKA that can be targeted in VHL-null cells to restore ciliogenesis.

Download Full-text