PRKN/parkin activation through phosphorylation of its ubiquitin and ubiquitin-like domain by PINK1 is critical in mitophagy induction for eliminating the damaged mitochondria. Deubiquitinating enzymes (DUBs) functionally reversing PRKN ubiquitination are critical in controlling the magnitude of PRKNmediated mitophagy process. However, potential DUBs that directly target PRKN and antagonize its promitophagy effect remains to be identified and characterized. Here, we demonstrated that USP33/VDU1 is localized at the outer membrane of mitochondria and serves as a PRKN DUB through their interaction. Cellular and in vitro assays illustrated that USP33 deubiquitinates PRKN in a DUB activity-dependent manner. USP33 prefers to remove K6, K11, K48 and K63-linked ubiquitin conjugates from PRKN, and deubiquitinates PRKN mainly at Lys435. Mutation of this site leads to a significantly decreased level of K63-, but not K48-linked PRKN ubiquitination. USP33 deficiency enhanced both K48-and K63-linked PRKN ubiquitination, but only K63-linked PRKN ubiquitination was significantly increased under mitochondrial depolarization. Further, USP33 knockdown increased both PRKN protein stabilization and its translocation to depolarized mitochondria leading to the enhancement of mitophagy. Moreover, USP33 silencing protects SH-SY5Y human neuroblastoma cells from the neurotoxin MPTP-induced apoptotic cell death. Our findings convincingly demonstrate that USP33 is a novel PRKN deubiquitinase antagonizing its regulatory roles in mitophagy and SH-SY5Y neuron-like cell survival. Thus, USP33 inhibition may represents an attractive new therapeutic strategy for PD patients.
Human RecQL4 helicase plays critical roles in the maintenance of genomic stability. Mutations in RecQL4 helicase results in three clinically related autosomal recessive disorders: Rothmund–Thomson syndrome (RTS), RAPADILINO, and Baller–Gerold syndrome. In addition to several premature aging features, RTS patients are characterized by aneuploidy involving either loss or gain of a single chromosome. Chromosome mosaicism and isochromosomes involving chromosomes 2, 7, and 8 have been reported in RecQL4-deficient RTS patients, but the precise role of RecQL4 in chromosome segregation/stability remains to be elucidated. Here, we demonstrate that RecQL4 physically and functionally interacts with Aurora B kinase (AURKB) and stabilizes its expression by inhibiting its ubiquitination process. Our study indicates that the N-terminus of RecQL4 interacts with the catalytic domain of AURKB. Strikingly, RecQL4 suppression reduces the expression of AURKB leading to mitotic irregularities and apoptotic cell death. RecQL4 suppression increases the proportion of cells at the G2/M phase followed by an extensive cell death, presumably owing to the accumulation of mitotic irregularities. Both these defects (accumulation of cells at G2/M phase and an improper mitotic exit to sub-G1) are complemented by the ectopic expression of AURKB. Finally, evidence is provided for the requirement of both human telomerase reverse transcriptase and RecQL4 for stable immortalization and longevity of RTS fibroblasts. Collectively, our study suggests that the RecQL4–AURKB axis is essential for cellular proliferation, cell cycle progression, and mitotic stability in human cells.
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