SUMOylation attenuates human ß-arrestin 2 inhibition of IL-1R/TRAF6 signaling.

ß-Arrestin 2 as an adaptor plays a role in the regulation of receptor desensitization, trafficking, and signaling. Bovine ß-arrestin 2 has been shown to be SUMOylated on the lysine 400 residue, which links it to the endocytosis of the ß2-adrenergic receptor. Here we identify a major SUMOylation site, lysine 295, on human ß-arrestin 2. SUMOylation on this site attenuates ß-arrestin 2 binding to TRAF6, then enhances TRAF6 oligomerization and autoubiquitination, and consequently leads to the increase of TRAF6-mediated NF-κB/AP-1 activation. We further determine SENP1 as a specific de-SUMOylation protease that can reverse the SUMOylation of ß-arrestin 2-mediated processes. Our study reveals SUMOylation as a novel mechanism in the regulation of ß-arrestin 2-mediated IL-1R/TRAF6 signaling.

Induction of SENP1 in myocardium contributes to abnormities of mitochondria and cardiomyopathy.

Defect in mitochondrial biogenesis and cardiac energy metabolism is a critical contributing factor to cardiac hypertrophy and heart failure. Sentrin/SUMO specific protease 1 (SENP1) mediated regulation of PGC-1α transcriptional activity plays an essential role in mitochondrial biogenesis and mitochondrial function. However, whether SENP1 plays a role in cardiac hypertrophy and failure is unknown. We investigated whether alteration in SENP1 expression affects cardiomyopathy and the underlying mechanism. In our present study, we found that the expression of SENP1 was induced in mouse and human failing hearts associated with induced expression of mitochondrial genes. SENP1 expression in cardiomyocytes was induced by hypertrophic stimuli through calcium/calcineurin-NFAT3. SENP1 regulated mitochondrial gene expression by de-SUMOylation of MEF-2C, which enhanced MEF-2C-mediated PGC-1α transcription. Genetic induction of SENP1 led to mitochondrial dysregulation and cardiac dysfunction in vivo. Our data showed that pathogenesis of cardiomyopathy is attributed by SENP1 mediated regulation of mitochondrial abnormities. SENP1 up-regulation in diseased heart is mediated via calcineurin-NFAT/MEF2C-PGC-1α pathway.

Small ubiquitin-like modifier (SUMO) protein-specific protease 1 de-SUMOylates Sharp-1 protein and controls adipocyte differentiation.

Adipocyte differentiation is regulated by a transcriptional cascade that mainly includes CCAAT/enhancer-binding protein family members and the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ). Here we show the defects in adipocyte differentiation as well as PPARγ expression in Senp1(-/-) mouse embryonic fibroblast cells induced by adipogenic stimuli. We further determine that SENP1 is a specific de-SUMOylation protease for Sharp-1, a repressor for PPARγ transcription and adipogenesis. SENP1 enhances adipogenesis through de-SUMOylation of Sharp-1, which then releases Sharp-1 repression of PPARγ expression and adipocyte differentiation. These results reveal SENP1 as a novel regulator in adipogenesis.

Co-expression of P1A35-43/beta2m fusion protein and co-stimulatory molecule CD80 elicits effective anti-tumor immunity in the P815 mouse mastocytoma tumor model.

A strong CTL response is dependent upon a high level of expression of specific class I major histocompatibility complex (MHC)/peptide complexes at the cell surface. An epitope-linked beta2-microglobulin (beta2m) molecule could provide a simple and more efficient means to enhance the formation of defined MHC/peptide complexes. However, the ability of an epitope-linked beta2m molecule to elicit primary CTL responses in vivo is still unknown. In this study, we modified the P1A tumor cell vaccine by addition of the tumor-associated epitope (TAE)-linked beta2m molecule and co-stimulatory molecule CD80 to improve the efficiency in the application of the vaccine. A eukaryotic co-expression vector consisting of the P1A35-43-linked beta2m molecule and the murine CD80 gene was constructed. P815 cell lines stably expressing P1A35-43-linked beta2m molecule and/or CD80 were established after transfection, by selection under G418. Administration of these inactivated tumor cell vaccines allowed the TAE-specific CD8+ T cell responses to be examined in vivo. Our results indicate that immunization with P815 cells expressing both the P1A35-43-linked beta2m molecule and the murine CD80 gene elicited a significantly stronger antitumor immune response than the single-modified tumor cell vaccines (expressing either P1A35-43-linked beta2m or CD80 alone). These findings support the feasibility and effectiveness of developing a dual-modified tumor cell vaccine consisting of the epitope-linked beta2m molecule and a co-stimulatory molecule.

NRF2 SUMOylation promotes de novo serine synthesis and maintains HCC tumorigenesis.

Nuclear factor erythroid-2 related factor 2 (NRF2) is a pivotal transcription factor that maintains cellular redox homeostasis and facilitates the development of malignant tumor phenotypes. At the molecular level, NRF2 promotes de novo serine synthesis and SUMOylation affects its function. Our results indicated that the SUMO1 acceptor site of NRF2 is the conserved lysine residue 110 (K110), and that NRF2 SUMOylation deficiency inhibited tumorigenesis in hepatocellular carcinoma (HCC). Mechanistically, NRF2 SUMOylation promoted de novo serine synthesis in HCC by enhancing the clearance of intracellular reactive oxygen species (ROS) and up-regulating phosphoglycerate dehydrogenase (PHGDH). More importantly, serine starvation increased the level of NRF2 SUMOylation, leading to sustained HCC growth. Collectively, our results indicate the presence of a novel NRF2 SUMOylation-mediated signaling process that maintains HCC tumorigenesis in normal conditions and in response to metabolic stress.

Cell Transformation by PTP1B Truncated Mutants Found in Human Colon and Thyroid Tumors.

Expression of wild-type protein tyrosine phosphatase (PTP) 1B may act either as a tumor suppressor by dysregulation of protein tyrosine kinases or a tumor promoter through Src dephosphorylation at Y527 in human breast cancer cells. To explore whether mutated PTP1B is involved in human carcinogenesis, we have sequenced PTP1B cDNAs from human tumors and found splice mutations in ~20% of colon and thyroid tumors. The PTP1BΔE6 mutant expressed in these two tumor types and another PTP1BΔE5 mutant expressed in colon tumor were studied in more detail. Although PTP1BΔE6 revealed no phosphatase activity compared with wild-type PTP1B and the PTP1BΔE5 mutant, its expression induced oncogenic transformation of rat fibroblasts without Src activation, indicating that it involved signaling pathways independent of Src. The transformed cells were tumourigenic in nude mice, suggesting that the PTP1BΔE6 affected other molecule(s) in the human tumors. These observations may provide a novel therapeutic target for colon and thyroid cancer.

Tumor-specific, hypoxia-regulated, WW domain-containing oxidoreductase-expressing adenovirus inhibits human non-small cell lung cancer growth in vivo.

An elevated level of hypoxia-inducible factor 1 (HIF-1) is common in solid tumors and correlates with poor prognosis. Therefore, targeting of HIF-1 presents an appealing approach for cancer therapy. In this study, we developed an adenoviral vector carrying a fusion of human WW domain-containing oxidoreductase (hWWOX) and the HIF-1alpha oxygen-dependent degradation domain (ODD) under the control of a synthetic human recombinant telomerase reverse transcriptase promoter (hrTRTP). Luciferase reporter assay showed elevated promoter activity of the synthetic hrTRTP in tested tumor cell lines, but not in WI-38, a nontransformed cell line. Furthermore, adenoviral hrTRTP-hWWOX-Linker-ODD (Ad-TWLH) expression induced apoptosis in a variety of human cancer cell lines under hypoxic conditions dose dependently. Importantly, Ad-TWLH injection into xenografts of A549 tumor cells dramatically reduced tumor size in vivo. Western blot and immunohistochemistry assays also confirmed that hWWOX-Linker-ODD fusion protein was expressed in the A549 xenografts. And the protein level in center part was much higher than that of the circumjacent area. In conclusion, our dual-regulated adenovirus specifically induced apoptosis in human cancer cell lines under hypoxic conditions in vitro and repressed ectopic xenograft tumor growth in vivo, thus providing a novel strategy for hypoxia-targeted cancer gene therapy.

[Activity analysis of human beta ß2-microglobulin gene promoter in P815 cells].

AIM: To clone the human beta2-microglobulin(beta2m) gene promoter and study its activity in P815 cells. METHODS: PCR amplification was performed using primers based on human beta2m gene sequence from GenBank and human genomic DNA as a template. The PCR product was directedly ligated into pBluescript II vector for sequencing. The promoter fragment was subcloned into a pcDNA3-EGFP plasmid after it had been identified correctly. A mouse mastocytoma cell line P815 was transiently and stably transfected with the plasmid containing human beta2m gene promoter and enhanced green fluorescence protein(EGFP) gene. The mRNA expression of EGFP in transiently transfected cells was quantified by RT-PCR and that in stably transfected cells was detected by fluorescence microscope and flow cytometry(FCM). RESULTS: A 302 bp DNA fragment was amplified and cloned into the pcDNA3-EGFP vector. RT-PCR analysis showed that EGFP mRNA expression was induced by IFN-gamma in a dose-dependent manner. There was no difference in the fluorescence positive cell rate between the IFN-gamma-treated group and the control group. But the fluorescence intensity of the 5 x 10(5) U/L IFN-gamma-treated group increased about 2 folds compared with that of the control group. CONCLUSION: Human beta2m gene promoter is active in mouse mastocytoma P815 cells. It can regulate the expression of reporter gene under the control of IFN-gamma.