Mitochondria serve as platforms for innate immunity. The mitochondrial antiviral signalling (MAVS) protein forms aggregates that elicit robust type-I interferon induction on viral infection, but persistent MAVS signalling leads to host immunopathology; it remains unknown how these signalling aggregates are resolved. Here we identify the mitochondria-resident E3 ligase, MARCH5, as a negative regulator of MAVS aggregates. March5+/− mice and MARCH5-deficient immune cells exhibit low viral replication and elevated type-I interferon responses to RNA viruses. MARCH5 binds MAVS only during viral stimulation when MAVS forms aggregates, and these interactions require the RING domain of MARCH5 and the CARD domain of MAVS. MARCH5, but not its RING mutant (MARCH5H43W), reduces the level of MAVS aggregates. MARCH5 transfers ubiquitin to Lys7 and Lys500 of MAVS and promotes its proteasome-mediated degradation. Our results indicate that MARCH5 modulates MAVS-mediated antiviral signalling, preventing excessive immune reactions.

Infection of hepatitis B virus (HBV) increase the incidence of chronic liver disease and hepatocellular carcinoma (HCC). The hepatitis B viral x (HBx) protein encoded by the HBV genome contributes to the pathogenesis of HCC and thus, negative regulation of HBx is beneficial for the alleviation of the disease pathogenesis. MARCH5 is a mitochondrial E3 ubiquitin ligase and here, we show that high MARCH5 expression levels are correlated with improved survival in HCC patients. MARCH5 interacts with HBx protein mainly accumulated in mitochondria and targets it for degradation. The N-terminal RING domain of MARCH5 was required for the interaction with HBx, and MARCH5H43W lacking E3 ligase activity failed to reduce HBx protein levels. High expression of HBx results in the formation of protein aggregates in semi-denaturing detergent agarose gels and MARCH5 mediates the elimination of protein aggregates through the proteasome pathway. HBx-induced ROS production, mitophagy, and cyclooxygenase-2 gene expression were suppressed in the presence of high MARCH5 expression. These results suggest MARCH5 as a target for alleviating HBV-mediated liver disease.

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We have previously shown that prolonged mitochondrial elongation triggers cellular senescence. Here, we report that enforced mitochondrial elongation by hFis1 depletion caused a severe defect in cell cycle progression through G2/M phase (~3-fold reduction in mitotic index; p < 0.01). Reintroduction of Myc-hFis1 to these cells induced mitochondrial fragmentation and restored the cell cycle, indicating that morphodynamic changes of mitochondria closely link to the cell cycle. In hFis1-knockdown cells, cell cycle regulators governing the G2/M phase, including cyclin A, cyclin B1, cyclin-dependent kinase1 (Cdk1), polo-like kinase1 (Plk1), aurora kinase A and Mad2, were significantly suppressed (2- to 10-fold). Notably, however, when mitochondrial fragmentation was induced by double knockdown of hFis1 and Opa1, the cells regained their ability to enter mitosis, and cell cycle regulators were rebounded. Reconstitution of the cyclin B1/Cdk1 complex, a major regulator of the G2/M transition, failed to restore mitotic entry in hFis1-depleted cells. In contrast, expression of Plk1, an upstream regulator of the cyclin B1/Cdk1 complex, or FoxM1 (forkhead box M1), a master transcriptional factor for the cell cycle regulators of G2/M phase, restored the cell cycle in these cells. Our findings suggest that mitochondrial fission molecule hFis1 ensures the proper cell division by interplay with the cell cycle machinery.

Genetic instability is intimately associated with tumour development. In particular, liver cancers associated with hepatitis B virus (HBV) exhibit high genetic instability; however, our understanding of the underlying molecular mechanisms remains limited. In this study, we found that c-H2AX, a marker of DNA double-strand breaks (DSBs), and the levels of phospho-Chk2 (p-Chk2, the activated form) were significantly elevated in HBV-associated hepatocellular carcinomas and neighbouring regenerating nodules. Likewise, introduction of the pHBV or pMyc-HBx plasmids into cells induced accumulation of c-H2AX foci and increased the p-Chk2 level. In these cells, inhibitory phosphorylation of Cdc25C phosphatase (Ser 216 ) and CDK1(Tyr 15 ) was elevated; consequently, cell-cycle progression was delayed at G 2 /M phase, suggesting that activation of the ATM-Chk2 pathway by the HBV X protein (HBx) induces cell-cycle delay. Accordingly, inhibition of ataxia telangiectasia mutated (ATM) by caffeine or siRNA abolished the increase in the p-Chk2 level and restored the delayed CDK1 kinase activity in ChangX cells. We also found that cytoplasmic HBx, but not nuclear HBx, induced reactive oxygen species (ROS) production and led to the accumulation of c-H2AX foci and the increased p-Chk2 level. Together, these data indicate that HBx-induced ROS accumulation induces DNA damage that activates the ATM-Chk2 pathway. Our findings provide insight into the mechanisms of HBV pathogenesis.

Accumulation of PLK1 at kinetochores is essential for chromosome alignment and segregation; however, the mechanism underlying PLK1 recruitment to kinetochores remains unresolved. The chromatin remodeller RSF1 tightly associates with centromere proteins, but its mitotic function is unknown. Here we show that RSF1 localizes at mitotic kinetochores and directly binds PLK1. RSF1 depletion disrupts localization of PLK1 at kinetochores; the C-terminal fragment of RSF1, which can bind PLK1, is sufficient to restore PLK1 localization. Moreover, CDK1 phosphorylates RSF1 at Ser1375, and this phosphorylation is necessary for PLK1 recruitment. Subsequently, PLK1 phosphorylates RSF1 at Ser1359, stabilizing PLK1 deposition. Importantly, RSF1 depletion mimicks the chromosome misalignment phenotype resulting from PLK1 knockdown; these defects are rescued by RSF1 S1375D or RSF1 S1359D but not RSF1 S1375A, showing a functional link between phosphorylation of RSF1 and chromosome alignment. Together, these data show that RSF1 is an essential centromeric component that recruits PLK1 to kinetochores and plays a crucial role in faithful cell division.

, and has been proposed to play a role in mitochondria quality control. However, it remains unclear how mutant MARCH5 found in cancer tissues is removed from cells. Here, we show that mutation in the MARCH5 ligase domain increased its half-life fourfold, resulting in a drastic increase in its protein level. Abnormal accumulation of the E3 ligase-defective MARCH5 mutants MARCH5H43W and MARCH5 C65/68S was diminished by overexpression of active MARCH5WT ; the mutant proteins were degraded through the ubiquitin-proteasome pathway. Coimmunoprecipitation revealed that MARCH5 forms homodimers, and that substitution of Gly to Leu at the first putative GxxxG dimerization motif, but not the second, resulted in a loss of dimeric interaction. Moreover, overexpression of the dimerization-defective mutant MARCH5 4GL could not decrease the level of accumulated MARCH5 H43W , suggesting that dimerization of MARCH5 is necessary for self-clearance. Abnormal accumulation of MARCH5 H43W and mitochondrial hyperfusion led to NF-KB activation, which was suppressed by overexpression of MARCH5 WT . Together, the data reveal a self-protective mechanism involving MARCH5, which can target its own dysfunctional mutant for degradation in order to maintain mitochondrial homeostasis.

The NLRP3 inflammasome plays a key role in responding to pathogens, and endogenous damage and mitochondria are intensively involved in inflammasome activation. The NLRP3 inflammasome forms multiprotein complexes and its sequential assembly is important for its activation. Here, we show that NLRP3 is ubiquitinated by the mitochondria‐associated E3 ligase, MARCH5. Myeloid cell‐specific March5 conditional knockout (March5 cKO) mice failed to secrete IL‐1β and IL‐18 and exhibited an attenuated mortality rate upon LPS or Pseudomonas aeruginosa challenge. Macrophages derived from March5 cKO mice also did not produce IL‐1β and IL‐18 after microbial infection. Mechanistically, MARCH5 interacts with the NACHT domain of NLRP3 and promotes K27‐linked polyubiquitination on K324 and K430 residues of NLRP3. Ubiquitination‐defective NLRP3 mutants on K324 and K430 residues are not able to bind to NEK7, nor form NLRP3 oligomers leading to abortive ASC speck formation and diminished IL‐1β production. Thus, MARCH5‐dependent NLRP3 ubiquitination on the mitochondria is required for NLRP3‐NEK7 complex formation and NLRP3 oligomerization. We propose that the E3 ligase MARCH5 is a regulator of NLRP3 inflammasome activation on the mitochondria.