Study On De - Ubiquitination Of PTEN And Regulation Of Protein Stability

PTEN(phosphatase and tension homolog deleted on chromosome 10) has been identified as one of the most frequently disrupted or mutated tumor suppressors in multiple tumor types, including endometrial carcinomas, glioblastomas, prostate cancer, and breast, colon, and lung tumors. PTEN germline mutations are also found in patients with cancer predisposition syndromes such as Cowden disease and Bannayan-Zonana syndrome. Heterozygous deletion of Pten leads to multiple tumors in mice, whereas homozygous mice die as embryos. PTEN is a phosphatase that dephosphorylates Ptd Ins(3,4,5)P3 and inhibits PI3K-Akt signaling in the cytoplasm. PTEN also has phosphatase-independent activities and PI3K-Akt-independent functions in the nucleus.PTEN function has been suggested to be a continuum that is related to the level of expression or activity of the PTEN protein rather than as a discrete series of step-wise changes in the PTEN gene copy number. It is very interest that regulation of PTEN stability, because tremendous consequences in tumor development are derived from slight reduction of PTEN protein levels. In contrast to p53 which is degraded rapidly, PTEN is a rather stable protein. A critical question in this field is why PTEN protein is so stable. PTEN expression, activity and localization are essencially regulated by posttranslational modifications, such as ubiquitination and phosphorylation. Several E3 ubiquitin ligases, for example, Nedd4-1, WWP2, XIAP, CHIP and TRIM27, have been shown promoting PTEN either polyubiquitination for proteasomal degradation or monoubiquitination for nuclear import.It is well documented that the ubiquitination of many proteins can be reversed by deubiquitinases(DUBs), which play an important role in preventing protein degradation. There are about 90 DUBs in the human proteome. However, the mechanisms regulating PTEN deubiquitination remain enigmatic. It has been shown that the ubiquitin-specific protease HAUSP removes the PTEN monoubiquitination and induces its nuclear export, however, it does not regulate PTEN protein stability thus is a DUB specific for PTEN de-monoubiquitination. To date, the DUB for de-polyubiquitination of PTEN and consequent maintenance of PTEN stability still remains largely unclear.Here we report OTUD3 as a deubiquitinase that regulates PTEN stability and functions as a tumor suppressor. Our main findings are as follows:(1) OTUD3 binds to PTEN, catalyzes the removal of polyubiquitin chains of PTEN and stabilizes PTEN protein. Depletion of OTUD3 destabilizes PTEN, leading to the activation of Akt signaling.(2) OTUD3 as a tumor suppressor inhibits cancer progression and metastasis.First, ectopic expression of OTUD3 WT, but not the C76 A enzyme-inactive mutant, in MCF7 breast cancer cells effectively inhibited tumor formation in nude mice. On the contrary, depletion of OTUD3 from MCF10 A mammary epithelial cells resulted in the cell transformation(soft agar assay) and tumor formation in nude mice.Second, depletion of OTUD3 in three types of tumor cells(including human colon cancer HCT116 cells, human hepatoma Hep G2 cells and human cervical cancer He La cells) all resulted in enhanced tumorigenicity and lung or liver metastasis, indicated by nude mice data. These results suggest that the tumor suppressor role of OTUD3 was not restricted in breast cancers.Third, we generated successfully the OTUD3 transgenic mice model. To evaluate the tumor suppressive function of OTUD3 in vivo, we crossed these mice with MMTV-Py MT(polyomavirus middle T antigen) mice, which have been widely used to study mammary tumorigenesis and metastasis. OTUD3 transgenic mice displayed less susceptibility against MMTV-Py MT-induced breast tumor development. In addition, OTUD3 transgenic mice-derived MEF cells and various tissues displayed upregulated PTEN protein levels, enhanced PTEN stability and decreased PTEN ubiquitination. OTUD3 TG-MEF displayed less duration of Akt phosphorylation upon EGF stimulation. These genetic evidences strongly support the conclusion that OTUD3 is a critical stabilizing regulator of PTEN and OTUD3 possesses tumor suppressor functions in vivo.(3) OTUD3 expression is downregulated in a high percentage of breast cancer cases, which are known to have few PTEN mutations. Moreover, OTUD3 expression correlated with PTEN protein levels and inversely with p-Akt. Mutations of OTUD3 or PTEN in breast cancer, which abolish OTUD3 enzymatic activity or the OTUD3-PTEN interaction, result in PTEN destabilization. Patients bearing tumors with relatively low levels of OTUD3 showed poorer overall survival than those bearing tumors with high OTUD3 expression. In patients with metastasis, OTUD3 levels were relatively lower than those without metastasis.OTUD3 and PTEN gene have mutantions in breast cancers. Ectopic expression of OTUD3-E86 K mutant(but not WT) in MCF10 A cells promoted the tumor formation in nude mice, suggesting that E86 K is a transforming mutation. The E86 K mutation seems to switch OTUD3 from a tumor suppressor to a potential oncogene. This is interesting and similar to the case of tumor suppressor p53 and PTEN.These findings demonstrate that OTUD3 is an essential positive regulator of PTEN and the OTUD3-PTEN axis plays a critical role in tumor suppression. Overall, our studies have addressed an important previously unanswered question in the field of PTEN and revealed a missing piece in the mechanism of regulation of PTEN stability. OTUD3 is located at the 1p36.13 and this region is frequently deleted in breast tumors and associated with cancer metastasis, poor prognosis and high recurrence rates. Since OTUD3 is mutated in breast cancer and mutant OTUD3 loses its functions, we suggest that OTUD3 is a novel tumor suppressor.Coronaviruses are mainly associated with respiratory, enteric, hepatic, and central nervous system diseases. However, until the late 1960 s, coronaviruses were not recognized as pathogens that are responsible for human diseases, and it was only in 2003 when human coronaviruses(HCo Vs) received worldwide attention after the emergence of severe and acute respiratory syndrome(SARS), which is caused by the coronavirus SARS-Co V. SARS-Co V has infected more than 8,000 people in 32 countries, with a mortality rate of up to 10%. The increasing amounts of research on coronaviruses soon led to the discovery of another human coronavirus, HCo V-NL63. Infection by the NL63 virus is prevalent in 7% of hospitalized patients and is associated with both upper and lower respiratory tract diseases, bronchiolitis and possibly conjunctivitis in children and adults. Currently, no antiviral drugs are available to treat coronavirus infections; thus, potential drug targets need to be identified and characterized.Coronaviruses are enveloped viruses with large RNA genomes(28 to 32 kb). Upon entry, coronavirus genomic RNA is translated to produce two large polyproteins, pp1 a and pp1 ab. These polyproteins are processed by viral cysteine proteases, both papain-like(PLPs/PLpro) and picornavirus 3C-like(3CLpro), to generate mature nonstructural proteins that assemble with host cell membranes to form double membrane vesicles. We previously identified HCo V-NL63 replicase gene products and characterized two viral PLPs, PLP1 and PLP2, which process the viral replicase polyprotein. HCo V-NL63 replicase can be detected at 24 hours post-infection. These proteins accumulate in the perinuclear region, consistent with the function of membrane-associated replication complexes. Furthermore, NL63-PLP2 was found to exhibit deubiquitinase(DUB) activity and inhibit the expression of type I IFN.Previous studies have shown that type I interferons can inhibit the replication of coronaviruses and that IFNβ is more effective than IFNα. However, clinical studies have revealed that coronavirus infections only induce very low levels of type I IFNs, which most likely contributes to rampant viral replication and a weakened immune response. The low-level IFN response to this vigorously replicating RNA virus suggests that coronaviruses might either evade or inactivate the innate immune response. However, the molecular mechanism of the low dosage IFN production remains unclear.The tumor suppressor protein p53 is widely known as “the guardian of the genome” due to its ability to prevent the emergence of transformed cells by inducing cell cycle arrest and apoptosis. Recent studies indicate that P53 is also a direct target gene of the type I interferon(IFNα/β) pathway, and thus, it is activated by certain cytokines upon viral infection. This provides new insight into the function of p53 in antiviral innate immunity. Because virus infection activates p53, this tumor suppressor has been recently introduced as a new component of the cellular antiviral defense mechanism. In fact, p53 is a key player in antiviral innate immunity by both inducing apoptosis in infected cells and enforcing type I IFN production. Both actions coordinated by this tumor suppressor help thwart the replication of a wide variety of viruses both in vitro and in vivo. The finding that p53 is involved in antiviral immunity may help explain why this protein is conserved in invertebrate organisms, which do not suffer from cancer-related diseases, and why it is so frequently targeted by viral proteins.Here, we provide evidence that PLP2, a catalytic domain of the nonstructural protein 3 of human coronavirus NL63(HCo V-NL63), deubiquitinates and stabilizes the cellular oncoprotein MDM2 and induces the proteasomal degradation of p53. Meanwhile, we identify IRF7(interferon regulatory factor 7) as a bona fide target gene of p53 to mediate the p53-directed production of type I interferon and the innate immune response. By promoting p53 degradation, PLP2 inhibits the p53-mediated antiviral response and apoptosis to ensure viral growth in infected cells. Thus, our study reveals that coronavirus engages PLPs to escape from the innate antiviral response of the host by inhibiting p53-IRF7-IFNβ signaling.