PHB2 (prohibitin 2) promotes PINK1-PRKN/Parkin-dependent mitophagy by the PARL-PGAM5-PINK1 axis

Mitophagy, which is a conserved cellular process for selectively removing damaged or unwanted mitochondria, is critical for mitochondrial quality control and the maintenance of normal cellular physiology. However, the precise mechanisms underlying mitophagy remain largely unknown. Prior studies on mitophagy focused on the events in the mitochondrial outer membrane. PHB2 (prohibitin 2), which is a highly conserved membrane scaffold protein, was recently identified as a novel inner membrane mitophagy receptor that mediates mitophagy. Here, we report a new signaling pathway for PHB2-mediated mitophagy. Upon mitochondrial membrane depolarization or misfolded protein aggregation, PHB2 depletion destabilizes PINK1 in the mitochondria, which blocks the mitochondrial recruitment of PRKN/Parkin, ubiquitin and OPTN (optineurin), leading to an inhibition of mitophagy. In addition, PHB2 overexpression directly induces PRKN recruitment to the mitochondria. Moreover, PHB2-mediated mitophagy is dependent on the mitochondrial inner membrane protease PARL, which interacts with PHB2 and is activated upon PHB2 depletion. Furthermore, PGAM5, which is processed by PARL, participates in PHB2-mediated PINK1 stabilization. Finally, a ligand of PHB proteins that we synthesized, called FL3, was found to strongly inhibit PHB2-mediated mitophagy and to effectively block cancer cell growth and energy production at nanomolar concentrations. Thus, our findings reveal that the PHB2-PARL-PGAM5-PINK1 axis is a novel pathway of PHB2-mediated mitophagy and that targeting PHB2 with the chemical compound FL3 is a promising strategy for cancer therapy. AIFM1: apoptosis inducing factor mitochondria associated 1; ATP5F1A/ATP5A1: ATP synthase F1 subunit alpha; BAF: bafilomycin A₁; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: chemical reagent carbonyl cyanide m-chlorophenyl hydrazine; FL3: flavaglines compound 3; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; LC3B/MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryo fibroblasts; MPP: mitochondrial-processing peptidase; MT-CO2/COX2: mitochondrially encoded cytochrome c oxidase II; MTS: mitochondrial targeting sequence; OA: oligomycin and antimycin A; OPTN: optineurin; OTC: ornithine carbamoyltransferase; PARL: presenilin associated rhomboid like; PBS: phosphate-buffered saline; PGAM5: PGAM family member 5, mitochondrial serine/threonine protein phosphatase; PHB: prohibitin; PHB2: prohibitin 2; PINK1: PTEN induced kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; Roc-A: rocaglamide A; TOMM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin beta class I.

线粒体自噬（Mitophagy）是一类保守的细胞过程，可选择性清除受损或多余的线粒体，对线粒体质量控制及正常细胞生理功能的维持至关重要。然而，线粒体自噬的具体分子机制仍知之甚少。既往关于线粒体自噬的研究多聚焦于线粒体外膜相关事件。近期研究发现，高度保守的膜支架蛋白抑制素2（PHB2，prohibitin 2）是一种新型线粒体内膜线粒体自噬受体，可介导线粒体自噬。本研究报道了一条PHB2介导的线粒体自噬新信号通路。当发生线粒体膜去极化或错误折叠蛋白聚集时，PHB2缺失会使线粒体中的PTEN诱导激酶1（PINK1，PTEN induced kinase 1）稳定性下降，进而阻断帕金RBR E3泛素蛋白连接酶（PRKN/Parkin，parkin RBR E3 ubiquitin protein ligase）、泛素及视神经蛋白（OPTN，optineurin）向线粒体的招募，最终抑制线粒体自噬。此外，PHB2过表达可直接诱导PRKN向线粒体招募。进一步研究表明，PHB2介导的线粒体自噬依赖于线粒体内膜蛋白酶早老素相关菱形样蛋白（PARL，presenilin associated rhomboid like）：PARL可与PHB2相互作用，并在PHB2缺失时被激活。经PARL加工的PGAM家族成员5（线粒体丝氨酸/苏氨酸蛋白磷酸酶，PGAM5，PGAM family member 5, mitochondrial serine/threonine protein phosphatase）可参与PHB2介导的PINK1稳定过程。最后，本团队合成的PHB蛋白配体FL3（黄皮林类化合物3，flavaglines compound 3）被证实可强烈抑制PHB2介导的线粒体自噬，并在纳摩尔浓度下有效阻断癌细胞的生长与能量产生。综上，本研究揭示PHB2-PARL-PGAM5-PINK1轴是PHB2介导的线粒体自噬的全新通路，而利用化合物FL3靶向PHB2是一种极具前景的癌症治疗策略。

附术语对照表：
凋亡诱导因子线粒体相关1（AIFM1，apoptosis inducing factor mitochondria associated 1）；ATP合酶F1亚基α（ATP5F1A/ATP5A1，ATP synthase F1 subunit alpha）；巴弗洛霉素A₁（BAF，bafilomycin A₁）；钙结合与卷曲螺旋结构域2（CALCOCO2/NDP52，calcium binding and coiled-coil domain 2）；羰基氰化物间氯苯腙（CCCP，chemical reagent carbonyl cyanide m-chlorophenyl hydrazine）；黄皮林类化合物3（FL3，flavaglines compound 3）；热休克蛋白家族D（Hsp60）成员1（HSPD1/HSP60，heat shock protein family D (Hsp60) member 1）；微管相关蛋白1轻链3β（LC3B/MAP1LC3B，microtubule associated protein 1 light chain 3 beta）；小鼠胚胎成纤维细胞（MEF，mouse embryo fibroblasts）；线粒体加工肽酶（MPP，mitochondrial-processing peptidase）；线粒体编码的细胞色素c氧化酶II（MT-CO2/COX2，mitochondrially encoded cytochrome c oxidase II）；线粒体靶向序列（MTS，mitochondrial targeting sequence）；寡霉素与抗霉素A（OA，oligomycin and antimycin A）；视神经蛋白（OPTN，optineurin）；鸟氨酸氨甲酰转移酶（OTC，ornithine carbamoyltransferase）；早老素相关菱形样蛋白（PARL，presenilin associated rhomboid like）；磷酸盐缓冲盐水（PBS，phosphate-buffered saline）；PGAM家族成员5（线粒体丝氨酸/苏氨酸蛋白磷酸酶，PGAM5，PGAM family member 5, mitochondrial serine/threonine protein phosphatase）；抑制素（PHB，prohibitin）；抑制素2（PHB2，prohibitin 2）；PTEN诱导激酶1（PINK1，PTEN induced kinase 1）；帕金RBR E3泛素蛋白连接酶（PRKN/Parkin，parkin RBR E3 ubiquitin protein ligase）；罗卡胺A（Roc-A，rocaglamide A）；线粒体外膜转位酶20（TOMM20，translocase of outer mitochondrial membrane 20）；βI类微管蛋白（TUBB，tubulin beta class I）。