MITOL prevents ER stress-induced apoptosis by IRE1α ubiquitylation at ER-mitochondria contact sites.

Unresolved endoplasmic reticulum (ER) stress shifts the unfolded protein response signaling from cell survival to cell death, although the switching mechanism remains unclear. Here, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) inhibits ER stress-induced apoptosis through ubiquitylation of IRE1α at the mitochondria-associated ER membrane (MAM). MITOL promotes K63-linked chain ubiquitination of IRE1α at lysine 481 (K481), thereby preventing hyper-oligomerization of IRE1α and regulated IRE1α-dependent decay (RIDD). Therefore, under ER stress, MITOL depletion or the IRE1α mutant (K481R) allows for IRE1α hyper-oligomerization and enhances RIDD activity, resulting in apoptosis. Similarly, in the spinal cord of MITOL-deficient mice, ER stress enhances RIDD activity and subsequent apoptosis. Notably, unresolved ER stress attenuates IRE1α ubiquitylation, suggesting that this directs the apoptotic switch of IRE1α signaling. Our findings suggest that mitochondria regulate cell fate under ER stress through IRE1α ubiquitylation by MITOL at the MAM.

DNA Damage Response Regulation by Histone Ubiquitination.

Cells are constantly exposed to numerous genotoxic stresses that induce DNA damage. DNA double-strand breaks (DSBs) are among the most serious damages and should be systematically repaired to preserve genomic integrity. The efficiency of repair is closely associated with chromatin structure, which is regulated by posttranslational modifications of histones, including ubiquitination. Recent evidence shows crosstalk between histone ubiquitination and DNA damage responses, suggesting an integrated model for the systematic regulation of DNA repair. There are two major pathways for DSB repair, viz., nonhomologous end joining and homologous recombination, and the choice of the pathway is partially controlled by posttranslational modifications of histones, including ubiquitination. Histone ubiquitination changes chromatin structure in the vicinity of DSBs and serves as a platform to select and recruit repair proteins; the removal of these modifications by deubiquitinating enzymes suppresses the recruitment of repair proteins and promotes the convergence of repair reactions. This article provides a comprehensive overview of the DNA damage response regulated by histone ubiquitination in response to DSBs.

Retusone A, a Guaiane-Type Sesquiterpene Dimer from Wikstroemia retusa and Its Inhibitory Effects on Histone Acetyltransferase HBO1 Expression.

Retusone A (1), a new sesquiterpene dimer consisting of two guaiane-type sesquiterpenoids, and oleodaphnal (2) were isolated from heartwood of Wikstroemia retusa (Thymelaeaceae). The planar structure of 1 was elucidated on the basis of HRESIMS and NMR spectroscopic data, and the relative stereochemistry was established by X-ray diffraction analysis. The absolute configuration of 1 was determined by electronic circular dichroism. Compound 1 suppressed luciferase reporter gene expression driven by the HBO1 (histone acetyltransferase binding to ORC1) gene promoter in human breast cancer MCF7 cells. Compound 1 also decreased the expression of endogenous HBO1 mRNA and protein, and inhibited proliferation of the cells. These results suggest that retusone A (1), which has a unique dimeric sesquiterpenoid structure with inhibitory activity against HBO1 expression, may contribute to the development of a novel therapeutic candidate for the treatment of breast cancer.

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.

SIRT2 inhibition activates hypoxia-inducible factor 1α signaling and mediates neuronal survival.

Sirtuins are deacetylases dependent on nicotine adenine dinucleotide (NAD) and take an important role in metabolism and aging. In mammals, there are seven sirtuins (SlRTl-7), and only SIRT2 is predominantly localized in cytoplasm. Under hypoxic environments, metazoan organisms must maintain oxygen homeostasis to survive. Hypoxia conditions induce reduction the ratio of NAD+/NADH, and aberrant increases or decreases in cellular O2 concentration induced excessive reactive oxygen species generation. Here, we report that inhibition of SIRT2 stabilizes hypoxia-inducible factor 1α (HIF-1α) protein levels and enhances hypoxia-responsive element-containing gene expression. We also show that the SIRT2 inhibitor AGK2 induces VEGF and HO-1 gene expression and protects neuronal viability from oxidative stress. Our findings suggest that SIRT2 negatively regulates HIF-1α signaling, indicating that SIRT2 inhibition may be a useful treatment strategy following ischemic injury.

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., SIRT5(iso1) and SIRT5(iso2) ) 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 SIRT5(iso2) was localized mainly in mitochondria, whereas the cleaved SIRT5(iso1) was localized in both mitochondria and cytoplasm. SIRT5ΔC, which is composed of only the common domain, showed the same mitochondrial localization as that of SIRT5(iso2) . These results suggest that the cytoplasmic localization of cleaved SIRT5(iso1) is dependent on the SIRT5(iso1) -specific C-terminus. Further analysis showed that the C-terminus of SIRT5(iso2) , 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 SIRT5(iso1) -specific C-terminus is necessary for maintaining the stability of SIRT5(iso1) . Moreover, genome sequence analysis from each organism examined indicated that SIRT5(iso2) 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.

Regulation of Hypoxic Signaling and Oxidative Stress via the MicroRNA-SIRT2 Axis and Its Relationship with Aging-Related Diseases.

The sirtuin family of nicotinamide adenine dinucleotide-dependent deacetylase and ADP-ribosyl transferases plays key roles in aging, metabolism, stress response, and aging-related diseases. SIRT2 is a unique sirtuin that is expressed in the cytosol and is abundant in neuronal cells. Various microRNAs were recently reported to regulate SIRT2 expression via its 3'-untranslated region (UTR), and single nucleotide polymorphisms in the miRNA-binding sites of SIRT2 3'-UTR were identified in patients with neurodegenerative diseases. The present review highlights recent studies into SIRT2-mediated regulation of the stress response, posttranscriptional regulation of SIRT2 by microRNAs, and the implications of the SIRT2-miRNA axis in aging-related diseases.

MITOL dysfunction causes dwarfism with anterior pituitary hypoplasia.

In mitochondrial disorders, short stature and growth failure are common symptoms, but their underlying mechanism remains unknown. In this study, we examined the cause of growth failure of mice induced by nestin promoter-driven knockout of the mitochondrial ubiquitin ligase MITOL (MARCH5), a key regulator of mitochondrial function. MITOL-knockout mice have congenital hypoplasia of the anterior pituitary caused by decreased expression of pituitary transcript factor 1 (Pit1). Consistently, both mRNA levels of growth hormone (GH) and prolactin levels were markedly decreased in the anterior pituitary of mutant mice. Growth failure of mutant mice was partly rescued by hypodermic injection of recombinant GH. To clarify whether this abnormality was induced by the primary effect of MITOL knockdown in the anterior pituitary or a secondary effect of other lesions, we performed lentiviral-mediated knockdown of MITOL on cultured rat pituitary GH3 cells, which secrete GH. GH production was severely compromised in MITOL-knockdown GH3 cells. In conclusion, MITOL plays a critical role in the development of the anterior pituitary; therefore, mice with MITOL dysfunction exhibited pituitary dwarfism caused by anterior pituitary hypoplasia. Our findings suggest that mitochondrial dysfunction is commonly involved in the unknown pathogenesis of pituitary dwarfism.

Mitochondrial Dynamics Regulation in Skin Fibroblasts from Mitochondrial Disease Patients.

Mitochondria are highly dynamic organelles that constantly fuse, divide, and move, and their function is regulated and maintained by their morphologic changes. Mitochondrial disease (MD) comprises a group of disorders involving mitochondrial dysfunction. However, it is not clear whether changes in mitochondrial morphology are related to MD. In this study, we examined mitochondrial morphology in fibroblasts from patients with MD (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and Leigh syndrome). We observed that MD fibroblasts exhibited significant mitochondrial fragmentation by upregulation of Drp1, which is responsible for mitochondrial fission. Interestingly, the inhibition of mitochondrial fragmentation by Drp1 knockdown enhanced cellular toxicity and led to cell death in MD fibroblasts. These results suggest that mitochondrial fission plays a critical role in the attenuation of mitochondrial damage in MD fibroblasts.

Critical role of CRAG, a splicing variant of centaurin-γ3/AGAP3, in ELK1-dependent SRF activation at PML bodies.

CRMP-5-associated GTPase (CRAG), a short splicing variant of centaurin-γ3/AGAP3, is predominantly expressed in the developing brain. We previously demonstrated that CRAG, but not centaurin-γ3, translocates to the nucleus and activates the serum response factor (SRF)-c-Fos pathway in cultured neuronal cells. However, the physiological relevance of CRAG in vivo is unknown. Here, we found that CRAG/centaurin-γ3-knockout mice showed intensively suppressed kainic acid-induced c-fos expression in the hippocampus. Analyses of molecular mechanisms underlying CRAG-mediated SRF activation revealed that CRAG has an essential role in GTPase activity, interacts with ELK1 (a co-activator of SRF), and activates SRF in an ELK1-dependent manner. Furthermore, CRAG and ELK1 interact with promyelocytic leukaemia bodies through SUMO-interacting motifs, which is required for SRF activation. These results suggest that CRAG plays a critical role in ELK1-dependent SRF-c-fos activation at promyelocytic leukaemia bodies in the developing brain.

MITOL deletion in the brain impairs mitochondrial structure and ER tethering leading to oxidative stress.

Mitochondrial abnormalities are associated with developmental disorders, although a causal relationship remains largely unknown. Here, we report that increased oxidative stress in neurons by deletion of mitochondrial ubiquitin ligase MITOL causes a potential neuroinflammation including aberrant astrogliosis and microglial activation, indicating that mitochondrial abnormalities might confer a risk for inflammatory diseases in brain such as psychiatric disorders. A role of MITOL in both mitochondrial dynamics and ER-mitochondria tethering prompted us to characterize three-dimensional structures of mitochondria in vivo. In MITOL-deficient neurons, we observed a significant reduction in the ER-mitochondria contact sites, which might lead to perturbation of phospholipids transfer, consequently reduce cardiolipin biogenesis. We also found that branched large mitochondria disappeared by deletion of MITOL. These morphological abnormalities of mitochondria resulted in enhanced oxidative stress in brain, which led to astrogliosis and microglial activation partly causing abnormal behavior. In conclusion, the reduced ER-mitochondria tethering and excessive mitochondrial fission may trigger neuroinflammation through oxidative stress.

Fanconi anemia protein FANCD2 promotes immunoglobulin gene conversion and DNA repair through a mechanism related to homologous recombination.

Recent studies show overlap between Fanconi anemia (FA) proteins and those involved in DNA repair mediated by homologous recombination (HR). However, the mechanism by which FA proteins affect HR is unclear. FA proteins (FancA/C/E/F/G/L) form a multiprotein complex, which is responsible for DNA damage-induced FancD2 monoubiquitination, a key event for cellular resistance to DNA damage. Here, we show that FANCD2-disrupted DT40 chicken B-cell line is defective in HR-mediated DNA double-strand break (DSB) repair, as well as gene conversion at the immunoglobulin light-chain locus, an event also mediated by HR. Gene conversions occurring in mutant cells were associated with decreased nontemplated mutations. In contrast to these defects, we also found increased spontaneous sister chromatid exchange (SCE) and intact Rad51 foci formation after DNA damage. Thus, we propose that FancD2 promotes a subpathway of HR that normally mediates gene conversion by a mechanism that avoids crossing over and hence SCEs.

Induction of Short NFATc1/αA Isoform Interferes with Peripheral B Cell Differentiation.

In lymphocytes, immune receptor signals induce the rapid nuclear translocation of preformed cytosolic NFAT proteins. Along with co-stimulatory signals, persistent immune receptor signals lead to high levels of NFATc1/αA, a short NFATc1 isoform, in effector lymphocytes. Whereas NFATc1 is not expressed in plasma cells, in germinal centers numerous centrocytic B cells express nuclear NFATc1/αA. When overexpressed in chicken DT40 B cells or murine WEHI 231 B cells, NFATc1/αA suppressed their cell death induced by B cell receptor signals and affected the expression of genes controlling the germinal center reaction and plasma cell formation. Among those is the Prdm1 gene encoding Blimp-1, a key factor of plasma cell formation. By binding to a regulatory DNA element within exon 1 of the Prdm1 gene, NFATc1/αA suppresses Blimp-1 expression. Since expression of a constitutive active version of NFATc1/αA interfered with Prdm1 RNA expression, LPS-mediated differentiation of splenic B cells to plasmablasts in vitro and reduced immunoglobulin production in vivo, one may conclude that NFATc1/αA plays an important role in controlling plasmablast/plasma cell formation.

DNA cross-link repair protein SNM1A interacts with PIAS1 in nuclear focus formation.

The yeast SNM1/PSO2 gene specifically functions in DNA interstrand cross-link (ICL) repair, and its role has been suggested to be separate from other DNA repair pathways. In vertebrates, there are three homologs of SNM1 (SNM1A, SNM1B, and SNM1C/Artemis; SNM1 family proteins) whose functions are largely unknown. We disrupted each of the SNM1 family genes in the chicken B-cell line DT40. Both SNM1A- and SNM1B-deficient cells were sensitive to cisplatin but not to X-rays, whereas SNM1C/Artemis-deficient cells exhibited sensitivity to X-rays but not to cisplatin. SNM1A was nonepistatic with XRCC3 (homologous recombination), RAD18 (translesion synthesis), FANCC (Fanconi anemia), and SNM1B in ICL repair. SNM1A protein formed punctate nuclear foci depending on the conserved SNM1 (metallo-beta-lactamase) domain. PIAS1 was found to physically interact with SNM1A, and they colocalized at nuclear foci. Point mutations in the SNM1 domain, which disrupted the interaction with PIAS1, led to mislocalization of SNM1A in the nucleus and loss of complementation of snm1a cells. These results suggest that interaction between SNM1A and PIAS1 is required for ICL repair.

Fanconi anemia FANCG protein in mitigating radiation- and enzyme-induced DNA double-strand breaks by homologous recombination in vertebrate cells.

The rare hereditary disorder Fanconi anemia (FA) is characterized by progressive bone marrow failure, congenital skeletal abnormality, elevated susceptibility to cancer, and cellular hypersensitivity to DNA cross-linking chemicals and sometimes other DNA-damaging agents. Molecular cloning identified six causative genes (FANCA, -C, -D2, -E, -F, and -G) encoding a multiprotein complex whose precise biochemical function remains elusive. Recent studies implicate this complex in DNA damage responses that are linked to the breast cancer susceptibility proteins BRCA1 and BRCA2. Mutations in BRCA2, which participates in homologous recombination (HR), are the underlying cause in some FA patients. To elucidate the roles of FA genes in HR, we disrupted the FANCG/XRCC9 locus in the chicken B-cell line DT40. FANCG-deficient DT40 cells resemble mammalian fancg mutants in that they are sensitive to killing by cisplatin and mitomycin C (MMC) and exhibit increased MMC and radiation-induced chromosome breakage. We find that the repair of I-SceI-induced chromosomal double-strand breaks (DSBs) by HR is decreased approximately 9-fold in fancg cells compared with the parental and FANCG-complemented cells. In addition, the efficiency of gene targeting is mildly decreased in FANCG-deficient cells, but depends on the specific locus. We conclude that FANCG is required for efficient HR-mediated repair of at least some types of DSBs.

The Fanconi anemia pathway promotes homologous recombination repair in DT40 cell line.

Fanconi anemia (FA) is a rare hereditary disorder characterized by bone marrow failure, compromised genome stability, and increased incidence of cancer. FA is caused by abnormalities that occur in components of the FA core complex, a key factor FancD2, breast cancer susceptibility protein BRCA2/FancD1, or BRIP1/FancJ. These proteins are proposed to function in a common biochemical process (FA pathway), however, its precise role is still unclear. In this chapter, we will summarize our genetic analysis on the FA pathway using DT40 cells line. Our data revealed that (1) FA pathway promotes DNA repair mediated by homologous recombination, and likely regulates translesion synthesis, thereby protecting cells against stalled replication forks; (2) BLM helicase can be regarded as an effector molecule of the FA pathway, since its subnuclear localization is regulated by FA pathway; (3) the FA core complex has multiple roles in the activation, relocalization, and DNA repair function of FANCD2.

Regulation of B cell differentiation by the ubiquitin-binding protein TAX1BP1.

Tax1-binding protein 1 (TAX1BP1) is a ubiquitin-binding protein that restricts nuclear factor-κB (NF-κB) activation and facilitates the termination of aberrant inflammation. However, its roles in B-cell activation and differentiation are poorly understood. To evaluate the function of TAX1BP1 in B cells, we established TAX1BP1-deficient DT40 B cells that are hyper-responsive to CD40-induced extracellular signal-regulated kinase (ERK) activation signaling, exhibit prolonged and exaggerated ERK phosphorylation and show enhanced B lymphocyte-induced maturation protein 1 (Blimp-1; a transcription factor inducing plasma cell differentiation) expression that is ERK-dependent. Furthermore, TAX1BP1-deficient cells exhibit significantly decreased surface IgM expression and increased IgM secretion. Moreover, TAX1BP1-deficient mice display reduced germinal center formation and antigen-specific antibody production. These findings show that TAX1BP1 restricts ERK activation and Blimp-1 expression and regulates germinal center formation.

Modularized functions of the Fanconi anemia core complex.

The Fanconi anemia (FA) core complex provides the essential E3 ligase function for spatially defined FANCD2 ubiquitination and FA pathway activation. Of the seven FA gene products forming the core complex, FANCL possesses a RING domain with demonstrated E3 ligase activity. The other six components do not have clearly defined roles. Through epistasis analyses, we identify three functional modules in the FA core complex: a catalytic module consisting of FANCL, FANCB, and FAAP100 is absolutely required for the E3 ligase function, and the FANCA-FANCG-FAAP20 and the FANCC-FANCE-FANCF modules provide nonredundant and ancillary functions that help the catalytic module bind chromatin or sites of DNA damage. Disruption of the catalytic module causes complete loss of the core complex function, whereas loss of any ancillary module component does not. Our work reveals the roles of several FA gene products with previously undefined functions and a modularized assembly of the FA core complex.

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.