Laboratory for Infectious Immunity

Project

Recent Publications

Yonashiro, R., S. Ishido, S. Kyo, T. Fukuda, E. Goto, Y. Matsuki, M. Ohmura-Hoshino, K. Sada, H. Hotta, H. Yamamura, R. Inatome, and S. Yanagi. 2006. A novel mitochondrial ubiquitin ligase plays a critical role in mitochondrial dynamics. EMBO J. Aug 9;25(15):3618-26.
Ohmura-Hoshino, M., Y. Matsuki, M. Aoki, E. Goto, M. Mito, M. Uematsu, T. Kakiuchi, H. Hotta, and S. Ishido. 2006. Inhibition of MHC class II expression and immune responses by c-MIR. J Immunol. Jul 1;177(1):341-54.
Goto, E., S. Ishido, Y. Sato, S. Ohgimoto, K. Ohgimoto, M. Nagano-Fujii, and H. Hotta. 2003. c-MIR, a human E3 ubiquitin ligase, is a functional homolog of herpesvirus proteins MIR1 and MIR2 and has similar activity. J Biol. Chem. 278:14657.
Ishido, S., C. Wang, B. S. Lee, G. B. Cohen, and J. U. Jung. 2000. Downregulation of major histocompatibility complex class I molecules by Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins. J Virol 74:5300.
Ishido, S., J. K. Choi, B. S. Lee, C. Wang, M. DeMaria, R. P. Johnson, G. B. Cohen, and J. U. Jung. 2000. Inhibition of natural killer cell-mediated cytotoxicity by Kaposi's sarcoma-associated herpesvirus K5 protein. Immunity 13:365.

　　Recently, we have discovered a novel E3 ubiquitin ligase family that consists of viral E3 ubiquitin ligases (E3s) and their mammalian homologues. These novel E3s are membrane-bound molecules that share the secondary structure and catalytic domain for E3 activity. All family members have two transmembrane regions at the center and a RING-CH domain at the amino terminus. Overexpression of these novel E3s has been shown to reduce the surface expression of various membrane proteins through ubiquitination of target molecules. Initial examples of viral E3s were identified in Kaposi’s sarcoma-associated herpesvirus (KSHV) and murine γ-herpesvirus 68 (MHV-68) and have been designated as Modulator of Immune Recognition (MIR) 1, 2 and mK3, respectively. MIR 1, 2 and mK3 are able to downregulate MHC class I molecule expression, and mK3 is required to establish an effective latent viral infection in vivo. The first characterized mammalian homologue to MIR 1, 2 and mK3 is c-MIR1. Overexpression of c-MIR1 downregulates B7-2, a costimulatory molecule important for antigen presentation. Subsequently, several mammalian molecules related to c-MIR1 have been characterized and named as MIR family. However, the precise physiological function of MIR family members remains unknown. Therefore, we intended to elucidate the physiological functions of MIR family members by generating genetically modified mice.

Major observations

　　Kaposi’s sarcoma-associated herpes virus (KSHV) encodes two proteins, modulator of immune recognition 1 and 2 (MIR1 and MIR2), which are involved in the evasion of host immunity. MIR1 and 2 have been shown to function as an E3 ubiquitin ligase for immune recognition-related molecules (e.g., MHC class I, B7-2 and ICAM-1) through the RINGv domain. We showed that the human genome also encodes a novel RINGv domain-containing protein that functions as an E3 ubiquitin ligase and whose putative substrate is the B7-2 costimulatory molecule. This novel E3 ubiquitin ligase was designated as c-MIR (cellular-MIR) based on its functional and structural similarity to MIR1 and 2. Forced expression of c-MIR induced specific downregulation of B7-2 surface expression through ubiquitination, rapid endocytosis and lysosomal degradation of the target molecule. This specific targeting was dependent on the binding of c-MIR to B7-2. Replacing the RINGv domain of MIR1 with the corresponding domain of c-MIR did not alter MIR1 function.
　Thus, we identified a novel E3 ubiquitin ligase (E3), designated as c-MIR, which targets B7-2 to lysosomal degradation and downregulates the B7-2 surface expression through ubiquitination of its cytoplasmic tail. B7-2 is well known as a costimulatory molecule for antigen presentation, suggesting that the manipulation of c-MIR expression modulates immune responses in vivo. To examine this hypothesis, we generated genetically modified mice in which c-MIR1 was expressed under an invariant chain promoter. Dendritic cells derived from genetically engineered mice showed low ability to present antigens. In addition, these mice showed resistance to the onset of experimental autoimmune encephalomyelitis and an impaired development of CD4 T cells in the thymus and the periphery. These findings led us to conclude that MHC class II (MHC II) is an additional target for c-MIR1. Indeed, forced expression of c-MIR1 in several B cell lines downregulated the surface expression of MHC II, and downregulation was found to depend on the presence of a single lysine residue in the cytoplasmic tail of the I-A β chain. In a reconstitution system using 293T cells, we found that the lysine residue at position 225 in the I-A β chain was ubiquitinated by c-MIR1.

Conclusion with perspectives

　　Based on our recent observations, we concluded that forcibly expressed c-MIR functions as a potent immune modulator in vivo and in vitro, and that MHC II and B7-2 are targets of c-MIR. To our knowledge, c-MIR is the first example of an E3 that is capable of targeting MHC II. Hence, further analysis of c-MIR might provide new insight into the molecular mechanism of antigen presentation. In addition, the discovery of c-MIR, a novel E3 ubiquitin ligase, highlights the possibility that viral immune regulatory proteins originated in the host genome and presents unique functions of RINGv domain-containing proteins in mammals.
　At present, the physiological role of c-MIR is as yet unknown. Since another research group reported that immature DCs degrade MHC II-peptide complexes much faster than mature DCs, and we found that c-MIR is moderately expressed in splenic macrophages and splenic DCs, which are physiological APCs in vivo, we hypothesize that c-MIR might function as a modulator of MHC II expression in immature DCs. To examine this hypothesis, we are presently generating c-MIR-deficient mice.
　Given the striking effect of c-MIR overexpression on the immune system, manipulation of the function and/or expression of c-MIR might be used as a strategy for artificial immune modulation in vivo. At present, there are several projects that are attempting to identify and produce the novel subsets of DCs that induce immunological tolerance. These DCs, which have been designated as tolerogenic DCs, or regulatory DCs (DCreg), show a very limited capability of antigen presentation. In this connection, bone marrow-derived DCs (BMDCs) in c-MIR transgenic mice (c-MIR Tg) did not present several antigens to T cells efficiently, suggesting that APCs forcibly expressing c-MIR might function as tolerogenic DCs. Preliminary experiments by our group showed that allogenic T cells stimulated with c-MIR Tg-derived mature BMDCs were not able to proliferate efficiently upon stimulation when compared to control littermate mice-derived BMDCs, while T cell responsiveness was improved by adding an excess amount of IL-2. Therefore, we are now investigating possible applications of c-MIR for immune tolerance induction.