USP33 deubiquitinates PRKN/parkin and antagonizes its role in mitophagy
USP33 deubiquitinates PRKN/parkin and antagonizes its role in mitophagy
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USP33 去泛素化 PRKN/parkin 并拮抗其在线粒体自噬中的作用
DOI：
10.1080/15548627.2019.1656957
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发表时间：
2019-08-28
期刊：
影响因子：
13.3
通讯作者：
Zhao, Yongliang
中科院分区：
文献类型：
--
作者：
Niu, Kaifeng;Fang, Hongbo;Zhao, Yongliang
摘要PRKN/Parkin通过PINK1对其泛素和泛素样结构域的磷酸化来激活PRKN/Parkin，在消除受损线粒体的有丝分裂诱导中起着至关重要的作用。功能上逆转PRKN泛素化的脱泛素化酶(DUBS)在控制PRKN介导的有丝分裂过程中起着关键作用。然而，直接靶向PRKN并拮抗其促有丝分裂效应的潜在DUBS仍有待鉴定和表征。在这里，我们证明了USP33/VDU1定位于线粒体的外膜，并通过它们的相互作用作为PRKN DUB。细胞和体外实验表明，USP33去泛素化PRKN的方式与活性有关。USP33倾向于从PRKN中去除K6、K11、K48和K63连接的泛素结合物，并主要在Lys435上去泛素化PRKN。该位点的突变导致K63-水平显著降低，但不会导致K48连接的PRKN泛素化水平显著降低。USP33缺乏可促进K48和K63连接的PRKN泛素化，但在线粒体去极化条件下，只有K63连接的PRKN泛素化显著增加。此外，USP33基因敲除增加了PRKN蛋白的稳定性和它对去极化线粒体的易位，从而增强了有丝分裂的吞噬作用。此外，USP33沉默保护SH-SY5Y人神经母细胞瘤细胞免受神经毒素MPTP诱导的细胞凋亡的影响。我们的发现令人信服地证明了USP33是一种新的PRKN去泛素酶，它拮抗了它在有丝分裂和SH-SY5Y神经元样细胞存活中的调节作用。因此，抑制USP33可能为PD患者提供了一种有吸引力的新治疗策略。缩写：CCCP：3-氯苯肼；DUB：脱泛素酶；MPTP：1-甲基-4-苯基-1，2，3，6-四氢吡啶；OMM：线粒体膜外膜；PD：帕金森病；PINK1：PTEN诱导的激酶1；PRKN/PARK2：Parkin RBR E3泛素蛋白连接酶；ROS：活性氧物种；TM：跨膜；Ub：泛素；UBA1：泛素样修饰物激活酶1；UBE2L3/UbcH7：泛素结合酶L3；USP33：泛素特异肽酶33；WT：野生型。
ABSTRACT 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 PRKN-mediated mitophagy process. However, potential DUBs that directly target PRKN and antagonize its pro-mitophagy 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. Abbreviations: CCCP: carbonyl cyanide 3-chlorophenylhydrazone; DUB: deubiquitinating enzymes; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; OMM: outer mitochondrial membrane; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; TM: transmembrane; Ub: ubiquitin; UBA1: ubiquitin like modifier activating enzyme 1; UBE2L3/UbcH7: ubiquitin conjugating enzyme E2 L3; USP33: ubiquitin specific peptidase 33; WT: wild type.