Author Correction: NEMF mutations that impair ribosome-associated quality control are associated with neuromuscular disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

NEMF mutations that impair ribosome-associated quality control are associated with neuromuscular disease.

A hallmark of neurodegeneration is defective protein quality control. The E3 ligase Listerin (LTN1/Ltn1) acts in a specialized protein quality control pathway-Ribosome-associated Quality Control (RQC)-by mediating proteolytic targeting of incomplete polypeptides produced by ribosome stalling, and Ltn1 mutation leads to neurodegeneration in mice. Whether neurodegeneration results from defective RQC and whether defective RQC contributes to human disease have remained unknown. Here we show that three independently-generated mouse models with mutations in a different component of the RQC complex, NEMF/Rqc2, develop progressive motor neuron degeneration. Equivalent mutations in yeast Rqc2 selectively interfere with its ability to modify aberrant translation products with C-terminal tails which assist with RQC-mediated protein degradation, suggesting a pathomechanism. Finally, we identify NEMF mutations expected to interfere with function in patients from seven families presenting juvenile neuromuscular disease. These uncover NEMF's role in translational homeostasis in the nervous system and implicate RQC dysfunction in causing neurodegeneration.

Pyruvate Dehydrogenase PDH-E1ß Controls Tumor Progression by Altering the Metabolic Status of Cancer Cells.

Downregulation of pyruvate dehydrogenase (PDH) is critical for the aberrant preferential activation of glycolysis in cancer cells under normoxic conditions. Phosphorylation-dependent inhibition of PDH is a relevant event in this process, but it is not durable as it relies on PDH kinases that are activated ordinarily under hypoxic conditions. Thus, it remains unclear how PDH is durably downregulated in cancer cells that are not hypoxic. Building on evidence that PDH activity depends on the stability of a multi-protein PDH complex, we found that the PDH-E1ß subunit of the PDH complex is downregulated to inhibit PDH activity under conditions of prolonged hypoxia. After restoration of normoxic conditions, reduced expression of PDH-E1ß was sustained such that glycolysis remained highly activated. Notably, PDH-E1ß silencing in cancer cells produced a metabolic state strongly resembling the Warburg effect, but inhibited tumor growth. Conversely, enforced exogenous expression of PDH-E1ß durably increased PDH activity and promoted the malignant growth of breast cancer cells in vivo Taken together, our results establish the specific mechanism through which PDH acts as an oncogenic factor by tuning glycolytic metabolism in cancer cells.Significance: This seminal study offers a mechanistic explanation for why glycolysis is aberrantly activated in normoxic cancer cells, offering insights into this long-standing hallmark of cancer termed the Warburg effect. Cancer Res; 78(7); 1592-603. ©2018 AACR.

The Rqc2/Tae2 subunit of the ribosome-associated quality control (RQC) complex marks ribosome-stalled nascent polypeptide chains for aggregation.

Ribosome stalling during translation can potentially be harmful, and is surveyed by a conserved quality control pathway that targets the associated mRNA and nascent polypeptide chain (NC). In this pathway, the ribosome-associated quality control (RQC) complex promotes the ubiquitylation and degradation of NCs remaining stalled in the 60S subunit. NC stalling is recognized by the Rqc2/Tae2 RQC subunit, which also stabilizes binding of the E3 ligase, Listerin/Ltn1. Additionally, Rqc2 modifies stalled NCs with a carboxy-terminal, Ala- and Thr-containing extension-the 'CAT tail'. However, the function of CAT tails and fate of CAT tail-modified ('CATylated') NCs has remained unknown. Here we show that CATylation mediates formation of detergent-insoluble NC aggregates. CATylation and aggregation of NCs could be observed either by inactivating Ltn1 or by analyzing NCs with limited ubiquitylation potential, suggesting that inefficient targeting by Ltn1 favors the Rqc2-mediated reaction. These findings uncover a translational stalling-dependent protein aggregation mechanism, and provide evidence that proteins can become specifically marked for aggregation.

Roles of mitochondrial ubiquitin ligase MITOL/MARCH5 in mitochondrial dynamics and diseases.

Accumulating evidence indicate physiological significance of mitochondrial dynamics such as mitochondrial fusion and division, the dynamic movement of mitochondria along microtubules and interaction of mitochondria with the endoplasmic reticulum. A disruption in mitochondrial dynamics leads to a functional deterioration of mitochondria, resulting in a variety of diseases including neurodegenerative disorders. We previously identified a mitochondrial ubiquitin ligase MITOL/MARCH5, which belongs to the membrane-associated RING-CH E3 ubiquitin ligase (MARCH) family (also called MARCH5). MITOL plays an important role in the regulation of mitochondrial dynamics including mitochondrial morphology, transport and interaction with ER, at least in part, through the ubiquitinations of mitochondrial fission factor Drp1, microtubule-associated protein 1B and mitofusin2, respectively. This review focuses on recent findings that show how MITOL regulates mitochondrial dynamics and which suggest physiological disorders resulting from a failure in such regulation.

Mitochondrial ubiquitin ligase MITOL blocks S-nitrosylated MAP1B-light chain 1-mediated mitochondrial dysfunction and neuronal cell death.

Nitric oxide (NO) is implicated in neuronal cell survival. However, excessive NO production mediates neuronal cell death, in part via mitochondrial dysfunction. Here, we report that the mitochondrial ubiquitin ligase, MITOL, protects neuronal cells from mitochondrial damage caused by accumulation of S-nitrosylated microtubule-associated protein 1B-light chain 1 (LC1). S-nitrosylation of LC1 induces a conformational change that serves both to activate LC1 and to promote its ubiquination by MITOL, indicating that microtubule stabilization by LC1 is regulated through its interaction with MITOL. Excessive NO production can inhibit MITOL, and MITOL inhibition resulted in accumulation of S-nitrosylated LC1 following stimulation of NO production by calcimycin and N-methyl-D-aspartate. LC1 accumulation under these conditions resulted in mitochondrial dysfunction and neuronal cell death. Thus, the balance between LC1 activation by S-nitrosylation and down-regulation by MITOL is critical for neuronal cell survival. Our findings may contribute significantly to an understanding of the mechanisms of neurological diseases caused by nitrosative stress-mediated mitochondrial dysfunction.

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.

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.

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.

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.