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A Specific Subset of E2 Ubiquitin-Conjugating Enzymes

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A Specific Subset of E2 Ubiquitin-Conjugating Enzymes ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 3488–3504 doi:10.1242/jcs.147520 RESEARCH ARTICLE A specific subset of E2 ubiquitin-conjugating enzymes regulate Parkin activation and mitophagy differently Fabienne C. Fiesel1, Elisabeth L. Moussaud-Lamodie`re1, Maya Ando1 and Wolfdieter Springer1,2,* ABSTRACT 2010; Meissner et al., 2011; Greene et al., 2012) and rapidly degraded (Yamano and Youle, 2013). The gene product of Loss-of-function mutations in the genes encoding PINK1 and Parkin PARKIN is a cytosolic E3 ligase that attaches the small modifier (also known as PARK2) are the most common causes of recessive protein ubiquitin to substrate proteins. FBXO7 encodes a putative Parkinson’s disease. Both together mediate the selective substrate recognition component of a multi-protein E3 ubiquitin degradation of mitochondrial proteins and whole organelles via the ligase complex but also has ubiquitin-independent functions proteasome and the autophagy-lysosome pathway (mitophagy). The (Nelson et al., 2013). Strikingly, PINK1, Parkin and FBXO7 mitochondrial kinase PINK1 activates and recruits the E3 ubiquitin physically associate and functionally cooperate to identify, ligase Parkin to de-energized mitochondria. However, the cognate E2 label and target damaged mitochondria for selective degradation. co-enzymes of Parkin in this ubiquitin-dependent pathway have not Mutations in either gene disrupt this protective pathway; been investigated. Here, we discovered a total of four E2s that either however, they affect distinct steps of a sequential process. positively or negatively regulate the activation, translocation and Upon mitochondrial dysfunction, PINK1 protein is stabilized enzymatic functions of Parkin during mitochondrial quality control. on de-energized organelles. PINK1 accumulation on damaged UBE2D family members and UBE2L3 redundantly charged the mitochondria and its kinase activity are prerequisites for RING-HECT hybrid ligase Parkin with ubiquitin, resulting in its initial the translocation of Parkin from the cytosol. Once localized to activation and translocation to mitochondria. UBE2N, however, mitochondria, Parkin ubiquitylates numerous mitochondrial primarily operated through a different mechanism in order to substrate proteins to facilitate the degradation of individual mediate the proper clustering of mitochondria, a prerequisite for proteins by the 26S proteasome or of whole organelles by the degradation. Strikingly, in contrast to UBE2D, UBE2L3 and UBE2N, autophagy-lysosomal system (Chan et al., 2011; Sarraf et al., depletion of UBE2R1 resulted in enhanced Parkin translocation and 2013). Upon ubiquitin modification of mitochondria, adaptor clustering upon mitochondrial uncoupling. Our study uncovered proteins, such as VCP/p97 (Kim et al., 2013), HDAC6 (Lee et al., redundant, cooperative or antagonistic functions of distinct E2 2010) or p62/SQSTM1 (Geisler et al., 2010), are co-recruited to decode respective ubiquitin tags and facilitate the removal of enzymes in the regulation of Parkin and mitophagy that might substrates. In either case, the E3 ubiquitin ligase activities of Parkin suggest a putative role in Parkinson’s disease pathogenesis. are crucially involved. KEY WORDS: Parkin, PINK1, Mitochondria, Ubiquitin, E2 enzymes, Parkin is known as a broadly neuroprotective, multipurpose Proteasome, Autophagy, Mitophagy E3 ligase that is tightly controlled and modifies numerous unrelated substrate proteins (Walden and Martinez-Torres, 2012). Moreover, Parkin has been shown to catalyze the formation of INTRODUCTION various ubiquitin modifications ranging from (multi-) mono- Parkinson’s disease is the most common neurodegenerative ubiquitin to poly-ubiquitin chains with distinct characteristics movement disorder. Symptoms arise from the selective loss of (Sandebring and Cedazo-Mı´nguez, 2012). Ubiquitin itself dopamine-producing neurons in the substantia nigra. The contains seven internal lysine residues that all can be used to molecular mechanisms for this distinctive neuronal degeneration generate ubiquitin chains of unique topologies and biological are poorly understood. Although most Parkinson’s disease cases functions (Komander and Rape, 2012). In addition, ubiquitin can are sporadic, rare familial forms allow insights into potential form linear chains by intermolecular linkage between its C- and pathogenic mechanisms, such as failure of protein degradation N-termini. Parkin has long been regarded as a RING-type E3 pathways and mitochondrial dysfunctions (Corti et al., 2011). To ubiquitin ligase that utilizes E2 ubiquitin conjugating enzymes to date, three recessive parkinsonism genes, PINK1 (Valente et al., mediate the direct transfer of ubiquitin from the E2 to a substrate 2004), PARKIN (also known as PARK2; Kitada et al., 1998) and protein. Thereby, E2 enzymes bound to the RING finger domain FBXO7 (Di Fonzo et al., 2009), have been linked into a single of an E3 ligase denominate the ubiquitin chain linkages molecular pathway for mitochondrial quality control (Geisler et al., formed. However, recent data has challenged the ubiquitin 2010; Matsuda et al., 2010; Narendra et al., 2010b; Vives-Bauza transfer mechanism for Parkin and other members of the et al., 2010; Burchell et al., 2013). PINK1 encodes a mitochondrial RING-between-RING (RBR) family (Wenzel et al., 2011). Ser/Thr kinase that is cleaved in healthy mitochondria (Jin et al., Similar to HECT-type E3 ubiquitin ligases, Parkin has been shown to accept ubiquitin from an E2 enzyme in a thioester 1Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA. 2Mayo intermediate on its recently discovered active site C431 before Graduate School, Neurobiology of Disease, Jacksonville, FL 32224, USA. transfer onto a lysine residue of a substrate protein. In this case, *Author for correspondence ([email protected]) the E3 ligase itself dictates the linkage type of the growing poly- ubiquitin chain, largely independent of the E2 enzymes (Sheng Received 4 December 2013; Accepted 13 May 2014 et al., 2012). In fact, K48-, K63- and K27-linked ubiquitin chains Journal of Cell Science 3488 RESEARCH ARTICLE Journal of Cell Science (2014) 127, 3488–3504 doi:10.1242/jcs.147520 appear to be successively formed during mitochondrial quality ratio of the intensity of cytoplasmic and nuclear GFP (Cyto:Nuc) control and might facilitate certain aspects along the course (Fig. 1A) upon treatment with the uncoupler carbonylcyanide m- (Geisler et al., 2010; Chan et al., 2011; Birsa et al., 2014). The chlorophenylhydrazone (CCCP). To calculate the percentage of crystal structures of Parkin (Riley et al., 2013; Spratt et al., 2013; Parkin-translocation-positive cells, we used a cut-off at 2.5. With Trempe et al., 2013; Wauer and Komander, 2013), and other RBR- this threshold, approximately 50% of the control-silenced cells type E3 ubiquitin ligases (Duda et al., 2013), have been recently showed Parkin translocation after 2 h of treatment with CCCP, resolved and show an auto-inhibited, ‘closed’ conformation, which allowed us to determine positive and negative modifiers consistent with their generally very low enzymatic activity. alike (Fig. 1B). Recent studies suggest that the activation of Parkin through Combined knockdown of the redundant UBE2D family ‘ubiquitin charging’ is coupled to its enzymatic activity(ies) and its members UBE2D2 and UBE2D3 with a single siRNA that mitochondrial translocation (Iguchi et al., 2013; Lazarou et al., targeted both genes (abbreviated hereafter as UBE2D2/3) 2013; Zheng and Hunter, 2013). Accordingly, Parkin must receive significantly reduced the percentage of Parkin-translocation- a ubiquitin moiety from an E2 enzyme and pass this onto a positive cells. Similarly, knockdown of UBE2L3 or UBE2N each substrate, which might include itself, in order to localize to decreased the percentage by about 50% after 2 h of treatment with mitochondria. Besides a recent study that suggests UBE2A is CCCP (Fig. 1B). Of note, transfection with an siRNA against crucially involved (Haddad et al., 2013), the roles of E2 co- UBE2R1 resulted in significantly more cells with Parkin enzymes for Parkin activation, translocation to and enzymatic translocation compared with control-silenced cells, indicative of actions on mitochondria have not been investigated. an accelerated recruitment to mitochondria. Analyses of the In this study, we aimed to identify E2 co-enzymes that regulate average GFP Cyto:Nuc ratio (Fig. 1C) and distribution frequency the activation and mitochondrial translocation of Parkin upon (Fig. 1D) among the individual cells corroborated the inhibitory uncoupling of the mitochondrial membrane potential. In total, we effects of siRNAs against UBE2D2/3, UBE2L3 and UBE2N, as have previously analyzed 11 out of 35 human, active E2 enzymes well as the accelerated Parkin translocation upon UBE2R1 (van Wijk and Timmers, 2010). Strikingly, we identified four knockdown. Consistent across all measurements, knockdown of E2 cofactors that redundantly, cooperatively or antagonistically UBE2A, UBE2S or UBE2T showed no significant difference regulate the activation and mitochondrial translocation of compared with control-silenced cells. Parkin. We demonstrate that members of the UBE2D family Knockdown efficiency for all E2 enzymes was confirmed and UBE2L3 are able to ‘charge’ Parkin with ubiquitin and are by using western blot analysis and/or quantitative reverse essential for its initial activation. UBE2N,
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