Dong-Soo Im

The Arginine-1493 Residue in QRRGRTGR1493G Motif IV of the Hepatitis C Virus NS3 Helicase Domain Is Essential for NS3 Protein Methylation by the Protein Arginine Methyltransferase 1

ABSTRACT The NS3 protein of hepatitis C virus (HCV) contains protease and RNA helicase activities, both of which are likely to be essential for HCV propagation. An arginine residue present in the arginine-glycine (RG)-rich region of many RNA-binding proteins is posttranslationally methylated by protein arginine methyltransferases (PRMTs). Amino acid sequence analysis revealed that the NS3 protein contains seven RG motifs, including two potential RG motifs in the 1486-QRRGRTGRG-1494 motif IV of the RNA helicase domain, in which arginines are potentially methylated by PRMTs. Indeed, we found that the full-length NS3 protein is arginine methylated in vivo. The full-length NS3 protein and the NS3 RNA helicase domain were methylated by a crude human cell extract. The purified PRMT1 methylated the full-length NS3 and the RNA helicase domain, but not the NS3 protease domain. The NS3 helicase bound specifically and comigrated with PRMT1 in vitro. Mutational analyses indicate that the Arg1493 in the QRR1488GRTGR1493G region of the NS3 RNA helicase is essential for NS3 protein methylation and that Arg1488 is likely methylated. NS3 protein methylation by the PRMT1 was decreased in the presence of homoribopolymers, suggesting that the arginine-rich motif IV is involved in RNA binding. The results suggest that an arginine residue(s) in QRXGRXGR motif IV conserved in the virus-encoded RNA helicases can be posttranslationally methylated by the PRMT1.

Enigma negatively regulates p53 through MDM2 and promotes tumor cell survival in mice

The human E3 ubiquitin ligase murine double minute 2 (MDM2) targets the tumor suppressor p53 for ubiquitination and degradation but also promotes its own ubiquitination and subsequent degradation. As the balance between MDM2 and p53 levels plays a crucial role in regulating cell proliferation and apoptosis, we sought to identify factors selectively inhibiting MDM2 self-ubiquitination. Here we have shown that the LIM domain protein Enigma directly interacts with MDM2 to form a ternary complex with p53 in vitro and in human hepatoma and colon carcinoma cell lines and mouse embryonic fibroblasts. We found that Enigma elicited p53 degradation by inhibiting MDM2 self-ubiquitination and increasing its ubiquitin ligase activity toward p53 in cells. Moreover, mitogenic stimuli such as serum, FGF, and HGF increased Enigma transcription via induction of serum response factor (SRF), leading to MDM2 stabilization and subsequent p53 degradation. We observed similar results in the livers of mice treated with HGF. In humans, we found SRF and Enigma coexpressed with MDM2 but not p53 in several liver and stomach tumors. Finally, we showed that Enigma promoted cell survival and chemoresistance by suppressing p53-mediated apoptosis in both cell lines and a mouse xenograft model. Our findings suggest a role for Enigma in tumorigenesis and uncover a mechanism whereby mitogens attenuate p53 antiproliferative activity through an SRF/Enigma/MDM2 pathway.

PRMT3 inhibits ubiquitination of ribosomal protein S2 and together forms an active enzyme complex

Protein arginine methyltransferase 3 (PRMT3) comprises a region not required for catalytic activity in its amino-terminus and the core domain catalyzing protein arginine methylation. PRMT3 has been shown to interact with the 40S ribosomal protein S2 (rpS2) and methylate arginine residues in the arginine-glycine (RG) repeat region in the amino-terminus of rpS2. We investigated the biological implications of this interaction by delineating the domains that mediate binding between PRMT3 and rpS2. The rpS2 (100-293 amino acids) domain, but not the amino-terminus of rpS2 that includes the RG repeat region was essential for binding to PRMT3 and was susceptible to degradation. The amino-terminus of PRMT3, but not its catalytic core was required for binding to and the stability of rpS2. Overexpressed rpS2 was ubiquitinated in cells, but expression of PRMT3 reduced this ubiquitination and stabilized the rpS2 protein. Recombinant PRMT3 formed an active enzyme complex with endogenous rpS2 in vitro. Recombinant rpS2 in molar excess modestly increased the enzymatic activity of PRMT3 in vitro. Our results suggest that in addition to its catalytic function, PRMT3 may control the level of rpS2 protein in cells by inhibiting ubiquitin-mediated proteolysis of rpS2, while rpS2 may regulate the enzymatic activity of PRMT3 as a likely non-catalytic subunit.

Adenovirus-mediated interleukin-18 mutant in vivo gene transfer inhibits tumor growth through the induction of T cell immunity and activation of natural killer cell cytotoxicity

We report here that gene transfer using recombinant adenoviruses encoding interleukin (IL)-18 mutants induces potent antitumor activity in vivo. The precursor form of IL-18 (ProIL-18) is processed by caspase-1 to produce bioactive IL-18, but its cleavage by caspase-3 (CPP32) produces an inactive form. To prepare IL-18 molecules with an effective antitumor activity, a murine IL-18 mutant with the signal sequence of murine granulocyte-macrophage (GM)- colony stimulating factor (CSF) at the 5′-end of mature IL-18 cDNA (GMmIL-18) and human IL-18 mutant with the prepro leader sequence of trypsin (PPT), which is not cleaved by caspase-3 (PPThIL-18CPP32−), respectively, were constructed. Adenovirus vectors carrying GMmIL-18 or PPThIL-18CPP32− produced bioactive IL-18. Ad.GMmIL-18 had a more potent antitumor effect than Ad.mProIL-18 encoding immature IL-18 in renal cell adenocarcinoma (Renca) tumor-bearing mice. Tumor-specific cytotoxic T lymphocytes, the induction of Th1 cytokines, and an augmented natural killer (NK) cell activity were detected in Renca tumor-bearing mice treated with Ad.GMmIL-18. An immunohistological analysis revealed that CD4+ and CD8+ T cells abundantly infiltrated into tumors of mice treated with Ad.GMmIL-18. Huh-7 human hepatoma tumor growth in nude mice with a defect of T cell function was significantly inhibited by Ad.PPThIL-18CPP32− compared with Ad.hProIL-18 encoding immature IL-18. Nude mice treated with Ad.PPThIL-18CPP32− contained NK cells with increased cytotoxicity. The results suggest that the release of mature IL-18 in tumors is required for achieving an antitumor effect including tumor-specific cellular immunity and augmented NK cell-mediated cytotoxicity. These optimally designed IL-18 mutants could be useful for improving the antitumor effectiveness of wild-type IL-18.

E2-EPF UCP regulates stability and functions of missense mutant pVHL via ubiquitin mediated proteolysis

BackgroundMissense mutation of VHL gene is frequently detected in type 2 VHL diseases and linked to a wide range of pVHL functions and stability. Certain mutant pVHLs retain ability to regulate HIFs but lose their function by instability. In this case, regulating of degradation of mutant pVHLs, can be postulated as therapeutic method.MethodThe stability and cellular function of missense mutant pVHLs were determine in HEK293T transient expressing cell and 786-O stable cell line. Ubiquitination assay of mutant VHL proteins was performed in vitro system. Anticacner effect of adenovirus mediated shUCP expressing was evaluated using ex vivo mouse xenograft assay.ResultsThree VHL missense mutants (V155A, L158Q, and Q164R) are directly ubiquitinated by E2-EPF UCP (UCP) in vitro. Mutant pVHLs are more unstable than wild type in cell. Missense mutant pVHLs interact with UCP directly in both in vitro and cellular systems. Lacking all of lysine residues of pVHL result in resistance to ubiquitination thereby increase its stability. Missense mutant pVHLs maintained the function of E3 ligase to ubiquitinate HIF-1α in vitro. In cells expressing mutant pVHLs, Glut-1 and VEGF were relatively upregulated compared to their levels in cells expressing wild-type. Depletion of UCP restored missense mutant pVHLs levels and inhibited cell growth. Adenovirus-mediated shUCP RNA delivery inhibited tumor growth in ex vivo mouse xenograft model.ConclusionThese data suggest that targeting of UCP can be one of therapeutic method in type 2 VHL disease caused by unstable but functional missense mutant pVHL.

Adenovirus-mediated E2-EPF UCP Gene Transfer Prevents Autoamputation in a Mouse Model of Hindlimb Ischemia

E2-EPF ubiquitin carrier protein (UCP) stabilizes hypoxia-inducible factor-1α (HIF-1α) inducing ischemic vascular responses. Here, we investigated the effect of UCP gene transfer on therapeutic angiogenesis. Adenovirus-encoded UCP (Ad-F-UCP) increased the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in cells and mice. Conditioned media from UCP-overexpressing cells promoted proliferation, tubule formation, and invasion of human umbilical-vascular-endothelial cells (HUVECs), and vascularization in chorioallantoic membrane (CAM) assay. Ad-F-UCP increased the vessel density in the Martigel plug assay, and generated copious vessel-like structures in the explanted muscle. The UCP effect on angiogenesis was dependent on VEGF and FGF-2. In mouse hindlimb ischemia model (N = 30/group), autoamputation (limb loss) occurred in 87% and 68% of the mice with saline and Ad encoding β-galactosidase (Ad-LacZ), respectively, whereas only 23% of the mice injected with Ad-F-UCP showed autoamputation after 21 days of treatment. Ad-F-UCP increased protein levels of HIF-1α, platelet-endothelial cell adhesion molecule-1 (PECAM-1), smooth muscle cell actin (SMA) in the ischemic muscle, and augmented blood vessels doubly positive for PECAM-1 and SMA. Consequently, UCP gene transfer prevented muscle degeneration and autoamputation of ischemic limb. The results suggest that E2-EPF UCP may be a target for therapeutic angiogenesis.

E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue.

Here, we show that E2-EPF ubiquitin carrier protein (UCP) elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL) and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196). A UCP mutant in which Cys118 was changed to alanine (UCPC118A) did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation.