MITOL regulates endoplasmic reticulum-mitochondria contacts via Mitofusin2.

The mitochondrial ubiquitin ligase MITOL regulates mitochondrial dynamics. We report here that MITOL regulates mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) domain formation through mitofusin2 (Mfn2). MITOL interacts with and ubiquitinates mitochondrial Mfn2, but not ER-associated Mfn2. Mutation analysis identified a specific interaction between MITOL C-terminal domain and Mfn2 HR1 domain. MITOL mediated lysine-63-linked polyubiquitin chain addition to Mfn2, but not its proteasomal degradation. MITOL knockdown inhibited Mfn2 complex formation and caused Mfn2 mislocalization and MAM dysfunction. Sucrose-density gradient centrifugation and blue native PAGE retardation assay demonstrated that MITOL is required for GTP-dependent Mfn2 oligomerization. MITOL knockdown reduced Mfn2 GTP binding, resulting in reduced GTP hydrolysis. We identified K192 in the GTPase domain of Mfn2 as a major ubiquitination site for MITOL. A K192R mutation blocked oligomerization even in the presence of GTP. Taken together, these results suggested that MITOL regulates ER tethering to mitochondria by activating Mfn2 via K192 ubiquitination.

Ubiquitination of CD86 is a key mechanism in regulating antigen presentation by dendritic cells.

Dendritic cells (DCs) require costimulatory molecules such as CD86 to efficiently activate T cells for the induction of adaptive immunity. DCs maintain minimal levels of CD86 expression at rest, but upregulate levels upon LPS stimulation. LPS-stimulated DCs produce the immune suppressive cytokine IL-10 that acts in an autocrine manner to regulate CD86 levels. Interestingly, the underlying molecular mechanism behind the tight control of CD86 is not completely understood. In this study, we report that CD86 is ubiquitinated in DCs via MARCH1 E3 ubiquitin ligase and that this ubiquitination plays a key role in CD86 regulation. Ubiquitination at lysine 267 played the most critical role for this regulation. CD86 is ubiquitinated in MARCH1-deficient DCs to a much lesser degree than in wild-type DCs, which also correlated with a significant increase in CD86 expression. Importantly, CD86 is continuously ubiquitinated in DCs following activation by LPS, and this was due to the autocrine IL-10 inhibition of MARCH1 downregulation. Accordingly, DCs lacking MARCH1 and DCs expressing ubiquitination-resistant mutant CD86 both failed to regulate CD86 in response to autocrine IL-10. DCs expressing ubiquitination-resistant mutant CD86 failed to control their T cell-activating abilities at rest as well as in response to autocrine IL-10. These studies suggest that ubiquitination serves as an important mechanism by which DCs control CD86 expression and regulate their Ag-presenting functions.

Ubiquitination regulates MHC class II-peptide complex retention and degradation in dendritic cells.

The expression and turnover of MHC class II-peptide complexes (pMHC-II) on the surface of dendritic cells (DCs) is essential for their ability to activate CD4 T cells efficiently. The half-life of surface pMHC-II is significantly greater in activated (mature) DCs than in resting (immature) DCs, but the molecular mechanism leading to this difference remains unknown. We now show that ubiquitination of pMHC-II by the E3 ubiquitin ligase membrane-associated RING-CH 1 (March-I) regulates surface expression, intracellular distribution, and survival of pMHC-II in DCs. DCs isolated from March-I-KO mice express very high levels of pMHC-II on the plasma membrane even before DC activation. Although ubiquitination does not affect the kinetics of pMHC-II endocytosis from the surface of DCs, the survival of pMHC-II is enhanced in DCs obtained from March-I-deficient and MHC-II ubiquitination-mutant mice. Using pMHC-II-specific mAb, we show that immature DCs generate large amounts of pMHC-II that are remarkably stable under conditions in which pMHC-II ubiquitination is blocked. Thus, the cellular distribution and stability of surface pMHC-II in DCs is regulated by ubiquitin-dependent degradation of internalized pMHC-II.

Contribution of lysine 11-linked ubiquitination to MIR2-mediated major histocompatibility complex class I internalization.

The polyubiquitin chain is generated by the sequential addition of ubiquitin moieties to target molecules, a reaction between specific lysine residues that is catalyzed by E3 ubiquitin ligase. The Lys(48)-linked and Lys(63)-linked polyubiquitin chains are well established inducers of proteasome-dependent degradation and signal transduction, respectively. The concept has recently emerged that polyubiquitin chain-mediated regulation is even more complex because various types of atypical polyubiquitin chains have been discovered in vivo. Here, we demonstrate that a novel complex ubiquitin chain functions as an internalization signal for major histocompatibility complex class I (MHC I) membrane proteins in vivo. Using a tetracycline-inducible expression system and quantitative mass spectrometry, we show that the polyubiquitin chain generated by the viral E3 ubiquitin ligase of Kaposi sarcoma-associated herpesvirus, MIR2, is a Lys(11) and Lys(63) mixed-linkage chain. This novel ubiquitin chain can function as an internalization signal for MHC I through its association with epsin1, an adaptor molecule containing ubiquitin-interacting motifs.

MARCH-I: a new regulator of dendritic cell function.

We and other groups have demonstrated that the expression level of MHC class II (MHC II) is regulated through ubiquitination of the MHC II beta chain. We also reported that MARCH-I, an E3 ubiquitin ligase, is critical for this process. At present, however, the importance of MARCH-I-mediated MHC II regulation in vivo is still unknown. In this review, we will summarize recent advances in our understanding of MARCH-I-mediated MHC II ubiquitination, and discuss how we can overcome the difficulties inherent in these studies.

Cutting edge: requirement of MARCH-I-mediated MHC II ubiquitination for the maintenance of conventional dendritic cells.

MARCH-I (membrane-associated RING-CH I) has been suggested as a physiological E3 ubiquitin ligase for both MHC class II (MHC II) and B7-2. In this study, we show that MARCH-I-mediated MHC II ubiquitination is necessary for the maintenance of conventional dendritic cell (cDC) functions in the steady state. MARCH-I-deficient cDCs accumulated MHC II and B7-2 and exhibited low Ag-presenting ability for exogenous Ags and low cytokine-producing ability upon stimulation in vivo. Importantly, MHC II, but not B7-2, was required for impaired cDC function induced by loss of MARCH-I in vivo. Moreover, MHC II knockin mice whose MHC II was not ubiquitinated showed dysfunction of cDC similar to that of MARCH-I knockout mice. These results suggest that the accumulation of MHC II resulting from loss of ubiquitination caused cDC abnormality; therefore, MARCH-I may function as a housekeeper of cDC in the steady state.

E3 ubiquitin ligases for MHC molecules.

Recently, novel E3 ubiquitin ligases that target MHC molecules for lysosomal degradation have been discovered by several groups. All these E3s are membrane-bound and possess a variant type RING domain, termed the RING-CH or RING variant (RINGv) domain. They belong to a new E3 family designated Modulator of Immune Recognition (MIR), based on the name of the first identified family members. The discovery of the MIR family has provided fresh insight into viral pathogenesis and immune regulation.

Differential MHC class II synthesis and ubiquitination confers distinct antigen-presenting properties on conventional and plasmacytoid dendritic cells.

The importance of conventional dendritic cells (cDCs) in the processing and presentation of antigen is well established, but the contribution of plasmacytoid dendritic cells (pDCs) to these processes, and hence to T cell immunity, remains unclear. Here we showed that unlike cDCs, pDCs continued to synthesize major histocompatibility complex (MHC) class II molecules and the MHC class II ubiquitin ligase MARCH1 long after activation. Sustained MHC class II-peptide complex formation, ubiquitination and turnover rendered pDCs inefficient in the presentation of exogenous antigens but enabled pDCs to continuously present endogenous viral antigens in their activated state. As the antigen-presenting abilities of cDCs and pDCs are fundamentally distinct, these two cell types may activate largely nonoverlapping repertoires of CD4(+) T cells.

An excellent monitoring system for surface ubiquitination-induced internalization in mammals.

BACKGROUND: At present, it is difficult to visualize the internalization of surface receptors induced by ubiquitination that is taken place at the plasma membrane in mammals. This problem makes it difficult to reveal molecular basis for ubiquitination-mediated internalization in mammals. METHODOLOGY/PRINCIPLE FINDINGS: In order to overcome it, we have generated T-REx-c-MIR, a novel mammalian Tet-on B cell line using a constitutively active E3 ubiquitin ligase, c-MIR, and its artificial target molecule. By applying the surface biotinylation method to T-REx-c-MIR, we succeeded to monitor the fate of surface target molecules after initiation of ubiquitination process by doxycycline (Dox)-induced c-MIR expression. Target molecules that pre-existed at the plasma membrane before induction of c-MIR expression were oligo-ubiquitinated and degraded by Dox-induced c-MIR expression. Dox-induced c-MIR expression initiated rapid internalization of surface target molecules, and blockage of the internalization induced the accumulation of the surface target molecules that were newly ubiquitinated by c-MIR. Inhibition of the surface ubiquitination by down-regulating ubiquitin conjugating enzyme E2 impaired the internalization of target molecules. Finally, a complex of c-MIR and target molecule was detected at the plasma membrane. CONCLUSIONS/SIGNIFICANCES: These results demonstrate that in T-REx-c-MIR, surface target molecule is ubiquitinated at the plasma membrane and followed by being internalized from the plasma membrane. Thus, T-REx-c-MIR is a useful experimental tool to analyze how surface ubiquitination regulates internalization in mammals.

Novel regulation of MHC class II function in B cells.

The presence of post-translational regulation of MHC class II (MHC II) under physiological conditions has been demonstrated recently in dendritic cells (DCs) that potently function as antigen-presenting cells (APCs). Here, we report that MARCH-I, an E3 ubiquitin ligase, plays a pivotal role in the post-translational regulation of MHC II in B cells. MARCH-I expression was particularly high in B cells, and the forced expression of MARCH-I induced the ubiquitination of MHC II. In B cells from MARCH-I-deficient mice (MARCH-I KO), the half-life of surface MHC II was prolonged and the ubiquitinated form of MHC II completely disappeared. In addition, MARCH-I-deficient B cells highly expressed exogenous antigen-loaded MHC II on their surface and showed high ability to present exogenous antigens. These results suggest that the function of MHC II in B cells is regulated through ubiquitination by MARCH-I.

A novel family of membrane-bound E3 ubiquitin ligases.

A novel E3 ubiquitin ligase family that consists of viral E3 ubiquitin ligases (E3s) and their mammalian homologues was recently discovered. 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. Forced expression 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 gamma-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 down-regulate 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-MIR/MARCH VIII. Forced expression of c-MIR/MARCH VIII down-regulates B7-2, a co-stimulatory molecule important for antigen presentation. Subsequently, several mammalian molecules related to c-MIR/MARCH VIII have been characterized and named as membrane associated RING-CH (MARCH) family. However, the precise physiological function of MARCH family members remains as yet unknown.

A novel mitochondrial ubiquitin ligase plays a critical role in mitochondrial dynamics.

In this study, we have identified a novel mitochondrial ubiquitin ligase, designated MITOL, which is localized in the mitochondrial outer membrane. MITOL possesses a Plant Homeo-Domain (PHD) motif responsible for E3 ubiquitin ligase activity and predicted four-transmembrane domains. MITOL displayed a rapid degradation by autoubiquitination activity in a PHD-dependent manner. HeLa cells stably expressing a MITOL mutant lacking ubiquitin ligase activity or MITOL-deficient cells by small interfering RNA showed an aberrant mitochondrial morphology such as fragmentation, suggesting the enhancement of mitochondrial fission by MITOL dysfunction. Indeed, a dominant-negative expression of Drp1 mutant blocked mitochondrial fragmentation induced by MITOL depletion. We found that MITOL associated with and ubiquitinated mitochondrial fission protein hFis1 and Drp1. Pulse-chase experiment showed that MITOL overexpression increased turnover of these fission proteins. In addition, overexpression phenotype of hFis1 could be reverted by MITOL co-overexpression. Our finding indicates that MITOL plays a critical role in mitochondrial dynamics through the control of mitochondrial fission proteins.

Inhibition of MHC class II expression and immune responses by c-MIR.

We previously reported a novel E3 ubiquitin ligase (E3), designated as c-MIR, which targets B7-2 to lysosomal degradation and down-regulates the B7-2 surface expression through ubiquitination of its cytoplasmic tail. B7-2 is well known as a costimulatory molecule for Ag 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-MIR was expressed under an invariant chain (Ii) promoter. Dendritic cells derived from genetically engineered mice showed low ability to present Ags. 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-MIR. Indeed, forced expression of c-MIR in several B cell lines down-regulated the surface expression of MHC II, and down-regulation was found to depend on the presence of a single lysine residue in the cytoplasmic tail of the I-A beta-chain. In a reconstitution system using 293T cells, we found that the lysine residue at position 225 in the I-A beta-chain was ubiquitinated by c-MIR. To our knowledge, c-MIR is the first example of an E3 that is capable of inhibiting MHC II expression. Our findings suggest that c-MIR might potently regulate immune responses in vivo.