Ryoko Inatome

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.

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.

Involvement of Fes/Fps tyrosine kinase in semaphorin3A signaling

Collapsin response mediator proteins (CRMPs)/TOAD64/Ulips/DRPs and CRAM have emerged as strong candidates for a role in semaphorin signaling. In this study we identified Fes/Fps (Fes) tyrosine kinase in the CRMP–CRAM complex and investigated whether Fes was involved in semaphorin3A (Sema3A) signaling. In COS‐7 cells, the interaction between Fes and plexinA1 (PlexA1) and the tyrosine phosphorylation of PlexA1 by Fes were observed; however, these events were significantly attenuated by co‐expression of neuropilin‐1 (NP‐1). Even with NP‐1 co‐expression, Sema3A was able to enhance the association of Fes with PlexA1 and Fes‐mediated tyrosine phosphorylation of PlexA1, CRAM and CRMP2. Co‐expression of Fes with PlexA1 exhibited COS‐7 cell contraction activity, indicating that Fes can convert inactive PlexA1 to its active form, whereas combination of Fes/NP‐1/PlexA1 or Fes kinase‐negative mutants/PlexA1 did not alter cell morphology. Finally, Sema3A‐induced growth cone collapse of dorsal root ganglion neurons was suppressed by expression of Fes kinase‐negative mutants. Taken together, our findings suggest that Fes links Sema3A signals to CRMP–CRAM, and that NP‐1 negatively regulates PlexA1 activation by Fes in resting condition.

Mitochondrial Ubiquitin Ligase MITOL Ubiquitinates Mutant SOD1 and Attenuates Mutant SOD1-induced Reactive Oxygen Species Generation

We have previously identified a novel mitochondrial ubiquitin ligase, MITOL, which is localized in the mitochondrial outer membrane and is involved in the control of mitochondrial dynamics. In this study, we examined whether MITOL eliminates misfolded proteins localized to mitochondria. Mutant superoxide dismutase1 (mSOD1), one of misfolded proteins, has been shown to localize in mitochondria and induce mitochondrial dysfunction, possibly involving in the onset and progression of amyotrophic lateral sclerosis. We found that in the mitochondria, MITOL interacted with and ubiquitinated mSOD1 but not wild-type SOD1. In vitro ubiquitination assay revealed that MITOL directly ubiquitinates mSOD1. Cycloheximide-chase assay in the Neuro2a cells indicated that MITOL overexpression promoted mSOD1 degradation and suppressed both the mitochondrial accumulation of mSOD1 and mSOD1-induced reactive oxygen species (ROS) generation. Conversely, the overexpression of MITOL CS mutant and MITOL knockdown by specific siRNAs resulted in increased accumulation of mSOD1 in mitochondria, which enhanced mSOD1-induced ROS generation and cell death. Thus, our findings indicate that MITOL plays a protective role against mitochondrial dysfunction caused by the mitochondrial accumulation of mSOD1 via the ubiquitin-proteasome pathway.

Protein-tyrosine kinase Syk expressed in human nasal fibroblasts and its effect on RANTES production.

Fibroblasts, a rich source of chemokines, interact with eosinophils and play a key role in the pathogenesis of airway disease. RANTES is produced by fibroblasts to attract and activate eosinophils. LPS is known to induce RANTES and cause protein tyrosine phosphorylation. Nonreceptor protein tyrosine kinase Syk is widely expressed and an important role in intracellular signal transduction in hemopoietic cells. In the present study, we examined whether Syk was expressed in a number of primary human nasal polyp tissue-derived fibroblast lines and whether it played some role in cellular function. Syk proteins were expressed in human nasal fibroblasts, but the expression level varied. There were positive correlations between the level of Syk expression and RANTES production induced by LPS. Overexpression of wild-type Syk by gene transfer enhanced RANTES production from nasal fibroblasts stimulated with LPS. The decrease of Syk expression by the administration of Syk antisense inhibited RANTES production. These results suggest that Syk expression affects RANTES production in fibroblasts of nasal polyps.

Distinct regulation of mitochondrial localization and stability of two human Sirt5 isoforms

Seven human Sir2 homologues (sirtuin) have been identified to date. In this study, we clarified the mechanism of subcellular localization of two SIRT5 isoforms (i.e., SIRT5iso1 and SIRT5iso2) encoded by the human SIRT5 gene and whose C‐termini slightly differ from each other. Although both isoforms contain cleavable mitochondrial targeting signals at their N‐termini, we found that the cleaved SIRT5iso2 was localized mainly in mitochondria, whereas the cleaved SIRT5iso1 was localized in both mitochondria and cytoplasm. SIRT5ΔC, which is composed of only the common domain, showed the same mitochondrial localization as that of SIRT5iso2. These results suggest that the cytoplasmic localization of cleaved SIRT5iso1 is dependent on the SIRT5iso1‐specific C‐terminus. Further analysis showed that the C‐terminus of SIRT5iso2, which is rich in hydrophobic amino acid residues, functions as a mitochondrial membrane insertion signal. In addition, a de novo protein synthesis inhibition experiment using cycloheximide showed that the SIRT5iso1‐specific C‐terminus is necessary for maintaining the stability of SIRT5iso1. Moreover, genome sequence analysis from each organism examined indicated that SIRT5iso2 is a primate‐specific isoform. Taken together, these results indicate that human SIRT5 potentially controls various primate‐specific functions via two isoforms with different intracellular localizations or stabilities.

IL-1 Induced Chemokine Production Through the Association of Syk with TNF Receptor-Associated Factor-6 in Nasal Fibroblast Lines

The fibroblasts stimulated by cytokines released the chemokine and recruited the infiltrating cells, including eosinophils, that play a key role in the pathogenesis of airway disease. We established the human fibroblast lines showing high Syk expression and the lines showing low Syk expression from pieces of nasal polyp. IL-1 induces the interaction of TNFR-associated factor (TRAF) 6 with IL-1R-associated kinase, which is rapidly recruited to the IL-1R after IL-1 induction, whereas TRAF2 participates in TNF-α-signaling. In the present study, we found that Syk played a different role in IL-1- and TNF-α-induced chemokine production through a signaling complex involving Syk and TRAF6. Overexpression of wild-type Syk by gene transfer enhanced RANTES production from nasal fibroblasts stimulated with IL-1. The decrease of Syk expression by the administration of Syk antisense inhibited RANTES production in response to IL-1. However, the change of Syk expression did not affect RANTES production by TNF-α stimulation. We concluded that Syk is required for the IL-1-induced chemokine production through the association with TRAF-6 in fibroblasts of nasal polyps.

A mitochondrial ubiquitin ligase MITOL controls cell toxicity of polyglutamine-expanded protein

Expansion of a polyglutamine tract in ataxin-3 (polyQ) causes Machado-Joseph disease, a late-onset neurodegenerative disorder characterized by ubiquitin-positive aggregate formation. Several lines of evidence demonstrate that polyQ also accumulates in mitochondria and causes mitochondrial dysfunction. To uncover the mechanism of mitochondrial quality-control via the ubiquitin-proteasome pathway, we investigated whether MITOL, a novel mitochondrial ubiquitin ligase localized in the mitochondrial outer membrane, is involved in the degradation of pathogenic ataxin-3 in mitochondria. In this study, we used N-terminal-truncated pathogenic ataxin-3 with a 71-glutamine repeat (ΔNAT-3Q71) and found that MITOL promoted ΔNAT-3Q71 degradation via the ubiquitin-proteasome pathway and attenuated mitochondrial accumulation of ΔNAT-3Q71. Conversely, MITOL knockdown induced an accumulation of detergent-insoluble ΔNAT-3Q71 with large aggregate formation, resulting in cytochrome c release and subsequent cell death. Thus, MITOL plays a protective role against polyQ toxicity, and thereby may be a potential target for therapy in polyQ diseases. Our findings indicate a protein quality-control mechanism at the mitochondrial outer membrane via a MITOL-mediated ubiquitin-proteasome pathway.

A novel GTPase, CRAG, mediates promyelocytic leukemia protein–associated nuclear body formation and degradation of expanded polyglutamine protein

Polyglutamine diseases are inherited neurodegenerative diseases caused by the expanded polyglutamine proteins (polyQs). We have identified a novel guanosine triphosphatase (GTPase) named CRAG that contains a nuclear localization signal (NLS) sequence and forms nuclear inclusions in response to stress. After ultraviolet irradiation, CRAG interacted with and induced an enlarged ring-like structure of promyelocytic leukemia protein (PML) body in a GTPase-dependent manner. Reactive oxygen species (ROS) generated by polyQ accumulation triggered the association of CRAG with polyQ and the nuclear translocation of the CRAG–polyQ complex. Furthermore, CRAG promoted the degradation of polyQ at PML/CRAG bodies through the ubiquitin–proteasome pathway. CRAG knockdown by small interfering RNA in neuronal cells consistently blocked the nuclear translocation of polyQ and enhanced polyQ-mediated cell death. We propose that CRAG is a modulator of PML function and dynamics in ROS signaling and is protectively involved in the pathogenesis of polyglutamine diseases.

Syk protein-tyrosine kinase is involved in neuron-like differentiation of embryonal carcinoma P19 cells

Syk has been implicated in activated immunoreceptors to downstream signaling events in hematopoietic cells. Here we report that Syk is expressed in neuron‐like cells and involved in neuron‐like differentiation of embryonal carcinoma P19 cells. Immunoblot, RT‐PCR, and Northern analysis indicated that Syk is expressed in mouse brain, PC12 and P19 cells. In addition, Syk was found to be tyrosine phosphorylated during neuron‐like differentiation of P19 cells. Furthermore, adenovirus‐mediated overexpression of Syk induced supernumerary neurite formation and extracellular signal‐regulated kinase (ERK) activation in P19 cells. These results suggest that Syk plays an important role in signaling steps leading to ERK activation in P19 cells.