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Loss-of-function mutations in the human PINK1 kinase (hPINK1) are causative of early-onset Parkinson’s disease (PD). Activation of hPINK1 induces phosphorylated ubiquitin to initiate removal of damaged mitochondria by autophagy. Previously we solved the structure of the insect PINK1 orthologue, Tribolium castaneum PINK1, and showed that autophosphorylation of Ser205 was critical for ubiquitin interaction and phosphorylation (Kumar, Tamjar, Waddell et al., 2017). In this advance, we report new findings on the regulation of hPINK1 by phosphorylation. We reconstitute E. coli expressed hPINK1 activity in vitro by direct incorporation of phosphoserine at the equivalent site Serine 228 (pSer228), providing direct evidence for a role for Ser228 phosphorylation in hPINK1 activation. Furthermore, using mass spectrometry, we identify six novel Ser/Thr autophosphorylation sites including regulatory Serine167 phosphorylation (pSer167), which in addition to pSer228 is required for ubiquitin recognition and phosphorylation. Strikingly, we also detect phosphorylation of a conserved Cysteine412 (pCys412) residue in the hPINK1 activation segment. Structural modelling suggests that pCys412 inhibits ubiquitin recognition and we demonstrate that mutation of Cys412 to Ala renders hPINK1 more active towards ubiquitin when expressed in human cells. These results outline new insights into hPINK1 activation by pSer167 and pSer228 and a novel inhibitory mechanism mediated by pCys412. These findings will aid in the development of small molecule activators of hPINK1. Description of files: Figure 1 Figure 1c OPA1 Coomassie: First gel: +Phos Tag Lane 1-2: WT, Lane 3-4 pS228 recombinant PINK1 Second gel: -Phos Tag Lane 1-2: WT, Lane 3-4 pS228 recombinant PINK1 Figure 1c blot: Lane 1-2: WT, Lane 3-4 pS228 recombinant PINK1 Figure 1d Coomassie: Recombinant PINK1: Lane 1-2: WT, Lane 3-4: KI, Lane 5-6 pS228, Lane 7-8: pS228DIns3, Lane 9-10: pS228G309D Figure 1d autorad: Recombinant PINK1: Lane 1-2: WT, Lane 3-4: KI, Lane 5-6 pS228, Lane 7-8: pS228DIns3, Lane 9-10: pS228G309D Figure 1f FLAG: Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP) Figure 1f HSP60: Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP) Figure 1f OPA1: Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP) Figure 1f pS228: Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP) Figure 1f PINK1 pS228 FLAG merge: Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP) Figure 1g PINK1 pS228 PINK1 multiplex: Lane 1: Marker, Lane 2-3: WT SK-OV-3 (-O/A), Lane 4-5: WT SK-OV-3 (+O/A), Lane 6-7: KO PINK1 SK-OV-3 (-O/A), Lane 8-9: KO PINK1 SK-OV-3 (+O/A) Figure 1g pS228: Lane 1: Marker, Lane 2-3: WT SK-OV-3 (-O/A), Lane 4-5: WT SK-OV-3 (+O/A), Lane 6-7: KO PINK1 SK-OV-3 (-O/A), Lane 8-9: KO PINK1 SK-OV-3 (+O/A) Figure 1g PINK1: Lane 1: Marker, Lane 2-3: WT SK-OV-3 (-O/A), Lane 4-5: WT SK-OV-3 (+O/A), Lane 6-7: KO PINK1 SK-OV-3 (-O/A), Lane 8-9: KO PINK1 SK-OV-3 (+O/A) Figure 1g Tom20: Lane 1: Marker, Lane 2-3: WT SK-OV-3 (-O/A), Lane 4-5: WT SK-OV-3 (+O/A), Lane 6-7: KO PINK1 SK-OV-3 (-O/A), Lane 8-9: KO PINK1 SK-OV-3 (+O/A) Figure 1h HSP60: Upper blot Lane 1: Marker, Lane 2-3: emp FLAG (-CCCP), Lane 4-5: emp FLAG (+CCCP), Lane 6-7: WT PINK1-FLAG (-CCCP), Lane 8-9: WT PINK1-FLAG (+CCCP), Lane10-11: KI PINK1-FLAG (-CCCP), Lane 12-13: KI PINK1-FLAG (+CCCP), Lane14-15: S228A PINK1-FLAG (-CCCP), Lane 16-17: S228A PINK1-FLAG (+CCCP) Lower blot Lane 1: Marker, Lane 2-3: S228N PINK1 FLAG (-CCCP), Lane 4-5: S228N PINK1 FLAG (+CCCP), Lane 6-7: S230A PINK1-FLAG (-CCCP), Lane 8-9: S230A PINK1-FLAG (+CCCP), Lane10-11: DIns3 PINK1-FLAG (-CCCP), Lane 12-13: DIns3 PINK1-FLAG (+CCCP), Rest of the lanes: Extra samples not included in the paper. Figure 1h OPA1: Upper blot Lane 1: Marker, Lane 2-3: emp FLAG (-CCCP), Lane 4-5: emp FLAG (+CCCP), Lane 6-7: WT PINK1-FLAG (-CCCP), Lane 8-9: WT PINK1-FLAG (+CCCP), Lane10-11: KI PINK1-FLAG (-CCCP), Lane 12-13: KI PINK1-FLAG (+CCCP), Lane14-15: S228A PINK1-FLAG (-CCCP), Lane 16-17: S228A PINK1-FLAG (+CCCP) Lower blot Lane 1: Marker, Lane 2-3: S228N PINK1 FLAG (-CCCP), Lane 4-5: S228N PINK1 FLAG (+CCCP), Lane 6-7: S230A PINK1-FLAG (-CCCP), Lane 8-9: S230A PINK1-FLAG (+CCCP), Lane10-11: DIns3 PINK1-FLAG (-CCCP), Lane 12-13: DIns3 PINK1-FLAG (+CCCP) Figure 1h pS228 PINK1: Upper blot Lane 1: Marker, Lane 2-3: emp FLAG (-CCCP), Lane 4-5: emp FLAG (+CCCP), Lane 6-7: WT PINK1-FLAG (-CCCP), Lane 8-9: WT PINK1-FLAG (+CCCP), Lane10-11: KI PINK1-FLAG (-CCCP), Lane 12-13: KI PINK1-FLAG (+CCCP), Lane14-15: S228A PINK1-FLAG (-CCCP), Lane 16-17: S228A PINK1-FLAG (+CCCP) Lower blot Lane 1: Marker, Lane 2-3: S228N PINK1 FLAG (-CCCP), Lane 4-5: S228N PINK1 FLAG (+CCCP), Lane 6-7: S230A PINK1-FLAG (-CCCP), Lane 8-9: S230A PINK1-FLAG (+CCCP), Lane10-11: DIns3 PINK1-FLAG (-CCCP), Lane 12-13: DIns3 PINK1-FLAG (+CCCP) Figure 1h pUb: Upper blot Lane 1: Marker, Lane 2-3: emp FLAG (-CCCP), Lane 4-5: emp FLAG (+CCCP), Lane 6-7: WT PINK1-FLAG (-CCCP), Lane 8-9: WT PINK1-FLAG (+CCCP), Lane10-11: KI PINK1-FLAG (-CCCP), Lane 12-13: KI PINK1-FLAG (+CCCP), Lane14-15: S228A PINK1-FLAG (-CCCP), Lane 16-17: S228A PINK1-FLAG (+CCCP) Lower blot Lane 1: Marker, Lane 2-3: S228N PINK1 FLAG (-CCCP), Lane 4-5: S228N PINK1 FLAG (+CCCP), Lane 6-7: S230A PINK1-FLAG (-CCCP), Lane 8-9: S230A PINK1-FLAG (+CCCP), Lane10-11: DIns3 PINK1-FLAG (-CCCP), Lane 12-13: DIns3 PINK1-FLAG (+CCCP) Figure 1- Figure Supplement 2b: Recombinant jason chin post-purification. 2 lanes loaded per construct, one for amylose purification and another for the subsequent 6His purification. Lane1 and Lane5 are included in the paper. Lane 1 - WT PINK1, amylose (included in paper), Lane 2 - WT PINK1, 6His, Lane 3 - KI PINK1, amylose, Lane 4 - KI PINK1, 6His, Lane 5 - pSer228 PINK1, amylose (included in paper), Lane 6 - pSer228 PINK1, 6His, Lane 7 - pSer284 PINK1, amylose, Lane 8 - pSer284 PINK1, 6His, Lane 9 - pSer402 PINK1, amylose, Lane 10 - pSer402 PINK1, 6His Figure 1- Figure Supplement 2c: Lane10 and Lane11 are included in the paper. Lane 1 - 2mg/ml BSA, Lane 2 - 1mg/ml BSA, Lane 3 - 0.5mg/ml BSA, Lane 4 - 0.25mg/ml BSA, Lane 5 - 0.125 mg/ml BSA, Lane 6 – ladder, Lane 7 - unrelated PINK1 construct (Ser228A), Lane 8 - unrelated PINK1 construct (Ser284A), Lane 9 - KI PINK1, Lane 10 - WT PINK1, Lane 11 - pSer228 PINK1, 6His, Lane 12 - pSer284 PINK1, 6His Figure 1- Figure Supplement 2d (+ phos tag): Lane 1-2: WT recombinant PINK1, Lane 3-4: KI recombinant PINK1, Lane 5-6: pS228 recombinant PINK1, Lane 7-8: pSer284 recombinant PINK1 (Not included in the paper) Figure 1- Figure Supplement 2d (no phos tag): Lane 1-2: WT recombinant PINK1, Lane 3-4: KI recombinant PINK1, Lane 5-6: pS228 recombinant PINK1, Lane 7-8: pSer284 recombinant PINK1 (Not included in the paper) Figure 1- Figure Supplement 2f Coomassie: Recombinant PINK1: Lane 1-2: WT, Lane 3-4: KI, Lane 5-6 pS228, Lane 7-8: pS228DIns3, Lane 9-10: pS228G309D Figure 1 - Figure Supplement 2f autorad: Recombinant PINK1: Lane 1-2: WT, Lane 3-4: KI, Lane 5-6 pS228, Lane 7-8: pS228DIns3, Lane 9-10: pS228G309D Figure 1 - Figure Supplement 3: Immunofluorescence images as mentioned in the paper Figure 2 Figure 2a FLAG: Lane 1: Marker, Lane3-4: WT PINK1 3FLAG (-CCCP), Lane 5-6: WT PINK1 3FLAG (+CCCP), Lane 7-8: KI PINK1 3FLAG (-CCCP), Lane 9-10: KI PINK1 3FLAG (+CCCP) Figure 2a HSP60: Lane 1: Marker, Lane3-4: WT PINK1 3FLAG (-CCCP), Lane 5-6: WT PINK1 3FLAG (+CCCP), Lane 7-8: KI PINK1 3FLAG (-CCCP), Lane 9-10: KI PINK1 3FLAG (+CCCP), rest of the lanes: Extra samples not included in the paper Figure 2a OPA1: Lane 1: Marker, Lane3-4: WT PINK1 3FLAG (-CCCP), Lane 5-6: WT PINK1 3FLAG (+CCCP), Lane 7-8: KI PINK1 3FLAG (-CCCP), Lane 9-10: KI PINK1 3FLAG (+CCCP) Figure 2a Phostag: Lane 1: Marker, Lane3-4: WT PINK1 3FLAG (-CCCP), Lane 5-6: WT PINK1 3FLAG (+CCCP), Lane 7-8: KI PINK1 3FLAG (-CCCP), Lane 9-10: KI PINK1 3FLAG (+CCCP) Figure 2- Figure Supplement 1a: Lane 1: Marker, Lane 3: emp 3-FLAG (-CCCP), Lane 4: emp 3-FLAG (+CCCP), Lane 5: KI PINK1 3-FLAG (-CCCP), Lane 6: KI PINK1 3-FLAG (+CCCP), Lane 7: WT PINK1 3-FLAG (-CCCP), Lane 8: WT PINK1 3-FLAG (+CCCP), Rest of the lanes: Not included in the paper Figure 2- Figure Supplement 1b: Lane 1: Marker, Lane3-5: 1% TX-100 (Total, soluble, insoluble), Lane 6-8: 1% SDS (Total, soluble, insoluble), Lane 10-12: 2% SDS (Total, soluble, insoluble) Figure 2- Figure Supplement 1c: Lane 1: Marker, Lane3-5: WT DMSO (Input, SN, IP), Lane 6-8: WT CCCP (Input, SN, IP), Lane 10-12: KI DMSO (Input, SN, IP), Lane 14-16: KI CCCP (Input, SN, IP) Figure 2- Figure Supplement 1d DCP: Upper blot Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP) Lower blot Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d ps228: Upper blot Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP) Lower blot Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d mix: Upper blot Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP) Lower blot Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d HSP60 KO: Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d HSP60 WT: Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d ACSL1 HSP60 KO: HSP60 blot (Lower band) re-probed for ACSL1 (Upper band); Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d ACSL1 HSP60 WT: HSP60 blot (Lower band) re-probed for ACSL1 (Upper band); Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1e: Lane 1: Marker, Lane 3: WT Trypsin (-CCCP), Lane 5: WT chymotrypsin (-CCCP), Lane 7: WT Trypsin (+CCCP), Lane 9: WT chymotrypsin (+CCCP), Lane 11: KI Trypsin (-CCCP), Lane 13: KI chymotrypsin (-CCCP), Lane 15: KI Trypsin (+CCCP), Lane 17: KI chymotrypsin (+CCCP) Figure 3 Figure 3c OPA1 PINK1: Upper blot: OPA1 Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A) Lower blot: PINK1 Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A) Figure 3c ps228 PINK1: Upper blot: pS228 PINK1 Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A) Lower blot: HSP60 Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A) Figure 3c pUb: Blot: pS65 Ub Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A) Figure 4 Figure 4e PINK1: Full blot (cut according to kDa before probing with antibody); Lane 1: Marker, Lane 3: emp 3FLAG (-CCCP), Lane4: emp 3FLAG (+CCCP for 180 min), Lane 5: KI PINK 3FLAG (-CCCP), Lane6: KI PINK 3FLAG (+CCCP for 180 min), Lane 7: WT PINK 3FLAG (-CCCP), Lane 8: WT PINK 3FLAG (+CCCP for 15 min), Lane 9: WT PINK 3FLAG (+CCCP for 30 min), Lane 10: WT PINK 3FLAG (+CCCP for 45 min), Lane 11: WT PINK 3FLAG (+CCCP for 60 min), Lane 12: WT PINK 3FLAG (+CCCP for 120 min), Lane 13: WT PINK 3FLAG (+CCCP for 180 min), Lane 14: C412A PINK 3FLAG (-CCCP), Lane 15: C412A PINK 3FLAG (+CCCP for 15 min), Lane 16: C412A PINK 3FLAG (+CCCP for 30 min), Lane 17: C412A PINK 3FLAG (+CCCP for 45 min), Lane 18: C412A PINK 3FLAG (+CCCP for 60 min), Lane 19: C412A PINK 3FLAG (+CCCP for 120 min), Lane 20: C412A PINK 3FLAG (+CCCP for 180 min) Figure 4e GAPDH OPA: Upper bands: OPA1, Lower bands: GAPDH; Lane 1: Marker, Lane 3: emp 3FLAG (-CCCP), Lane4: emp 3FLAG (+CCCP for 180 min), Lane 5: KI PINK 3FLAG (-CCCP), Lane6: KI PINK 3FLAG (+CCCP for 180 min), Lane 7: WT PINK 3FLAG (-CCCP), Lane 8: WT PINK 3FLAG (+CCCP for 15 min), Lane 9: WT PINK 3FLAG (+CCCP for 30 min), Lane 10: WT PINK 3FLAG (+CCCP for 45 min), Lane 11: WT PINK 3FLAG (+CCCP for 60 min), Lane 12: WT PINK 3FLAG (+CCCP for 120 min), Lane 13: WT PINK 3FLAG (+C

Loss of function mutations of the PINK1 kinase cause familial early-onset Parkinson’s disease. PINK1 is activated upon mitochondrial damage to phosphorylate Ubiquitin and Parkin to trigger removal of damaged mitochondria by autophagy (mitophagy). PINK1 also indirectly phosphorylates a subset of Rab GTPases including Rab8A. We have performed a siRNA screen of all human Ser/Thr kinases in HeLa cells and discovered the integrated stress response kinase EIF2AK1 (HRI) negatively regulates PINK1 following mitochondrial damage. We demonstrate that EIF2AK1 knockout cells enhance mitochondrial depolarization-induced stabilization of PINK1 and increased phosphorylation of Ubiquitin and Rab8A. We confirm our findings in multiple human cell lines including SK-OV-3, U2OS and ARPE-19 cells. Knockdown of upstream components of the recently described mitochondrial-cytosol relay pathway, OMA1 and DELE1, enhanced PINK1 stabilisation and activation similar to EIF2AK1. Using themito-QC mitophagy reporter in human cells, we observe that EIF2AK1 knockdown moderately increases PINK1-dependent mitophagy. Our findings indicate that EIF2AK1 is a negative regulator of PINK1 and suggest that inhibitors of EIF2AK1 could have therapeutic benefits in Parkinson’s disease and related disorders of ageing and mitophagy.

Dissection of the function of two Parkinson's disease-linked genes encoding the protein kinase, PTEN-induced kinase 1 (PINK1) and ubiquitin E3 ligase, Parkin, has illuminated a highly conserved mitochondrial quality control pathway found in nearly every cell type including neurons. Mitochondrial damage-induced activation of PINK1 stimulates phosphorylation-dependent activation of Parkin and ubiquitin-dependent elimination of mitochondria by autophagy (mitophagy). Structural, cell biological and neuronal studies are unravelling the key steps of PINK1/Parkin-dependent mitophagy and uncovering new insights into how the pathway is regulated. The emerging role for aberrant immune activation as a driver of dopaminergic neuron degeneration after loss of PINK1 and Parkin poses new exciting questions on cell-autonomous and noncell-autonomous mechanisms of PINK1/Parkin signalling in vivo.

Loss of function mutations in the PTEN-induced kinase 1 (PINK1) kinase are causal for autosomal recessive Parkinson's disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies, we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor and GTPase activating protein. In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2 in vitro. Strikingly, we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 of recombinant nucleotide-bound Rab8A in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in transfected HEK293 cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and nuclear magnetic resonance structure of Ser111-phosphorylated Rab1B. The structures reveal that the phosphorylated SF3 motif does not induce any major changes, but may interfere with effector-Switch II interactions through intramolecular H-bond formation and/or charge effects with Arg79. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A indicating a potential cross-talk between PINK1-regulated mitochondrial homeostasis and LRRK2 signalling that requires further investigation in vivo.

Parkinson’s disease (PD) is a progressive neurological disorder that manifests clinically as alterations in movement (bradykinesia, postural instability, loss of balance, and resting tremors) as well as multiple non-motor symptoms including but not limited to cognitive and autonomic abnormalities. Mitochondrial dysfunction has been linked to sporadic PD and loss-of-function mutations in genes encoding the ubiquitin E3 ligase Parkin and protein kinase, PTEN-induced kinase 1 (PINK1), that regulate mitophagy, are causal for familial and juvenile PD1-3. Among several therapeutic approaches being explored to treat or improve PD patient’s prognosis, the use of small molecules able to reinstate or boost Parkin activity represents a potential pharmacological treatment strategy4. A major barrier is the lack of high throughput platforms based on robust and accurate quantification of Parkin activity in vitro. Here we present two different and complementary Matrix Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (MALDI-TOF MS) based approaches for the quantification of Parkin E3 ligase activity in vitro. These methods recapitulate distinct aspects of ubiquitin conjugation: Parkin auto-ubiquitylation and Parkin-catalysed discharge on lysine residues. Both approaches are scalable for high-throughput primary screening to facilitate the identification of Parkin modulators.

Human autosomal recessive mutations in the Parkin gene are causal for Parkinson’s disease (PD). Parkin encodes a ubiquitin E3 ligase that functions together with the PD associated kinase, PINK1, in a mitochondrial quality control pathway. Structural studies reveal that Parkin exists in an inactive conformation mediated by multiple autoinhibitory domain interfaces. Here we have performed comprehensive mutational analysis of both human and rat Parkin to unbiasedly determine Parkin activating mutations across all major autoinhibitory interfaces. Out of 31 mutations tested, we identify 11 activating mutations clustered near the RING0:RING2 or REP:RING1 interfaces, which reduce the thermal stability of Parkin. Of these, we demonstrate that three mutations, V393D, A401D, and W403A located at the REP:RING1 interface were able to completely rescue a Parkin S65A mutant, defective in mitophagy, in cell-based studies. Overall our data extends previous analysis of Parkin activation mutants and suggests that small molecules that mimic REP:RING1 destabilisation offer therapeutic potential for PD patients harbouring select Parkin mutations.Summary blurbParkin, an E3 ubiquitin ligase involved in Parkinson’s disease, is inactive in the basal state and is activated by PINK1 to mediate mitophagy. Here we characterized 31 mutations and discovered three that activate Parkin and rescue loss of PINK1 phosphorylation.

Description, Identification of neuronal targets of PD-linked enzyme Parkin by quantitative ubiquitin-based proteomics., How activation of PINK1 and Parkin leads to elimination of damaged mitochondria by mitophagy is largely based on cell lines with few studies in neurons. Here, we have undertaken proteomic analysis of mitochondria from mouse neurons to identify ubiquitylated substrates of endogenous Parkin. Comparative analysis with human iNeuron datasets revealed a subset of 49 PINK1 activation–dependent diGLY sites in 22 proteins conserved across mouse and human systems. We use reconstitution assays to demonstrate direct ubiquitylation by Parkin in vitro. We also identified a subset of cytoplasmic proteins recruited to mitochondria that undergo PINK1 and Parkin independent ubiquitylation, indicating the presence of alternate ubiquitin E3 ligase pathways that are activated by mitochondrial depolarization in neurons. Last, we have developed an online resource to search for ubiquitin sites and enzymes in mitochondria of neurons, MitoNUb. These findings will aid future studies to understand Parkin activation in neuronal subtypes.

SUMMARYAutosomal recessive mutations in PINK1 and Parkin cause Parkinson’s disease. How activation of PINK1 and Parkin leads to elimination of damaged mitochondria by mitophagy is largely based on cell culture studies with few molecular studies in neurons. Herein we have undertaken a global proteomic-analysis of mitochondria from mouse neurons to identify ubiquitylated substrates of endogenous Parkin activation. Comparative analysis with human iNeuron datasets revealed a subset of 49 PINK1-dependent diGLY sites upregulated upon mitochondrial depolarisation in 22 proteins conserved across mouse and human systems. These proteins were exclusively localised at the mitochondrial outer membrane (MOM) including, CISD1, CPT1α, ACSL1, and FAM213A. We demonstrate that these proteins can be directly ubiquitylated by Parkin in vitro. We also provide evidence for a subset of cytoplasmic proteins recruited to mitochondria that undergo PINK1 and Parkin independent ubiquitylation including SNX3, CAMK2α and CAMK2β indicating the presence of alternate ubiquitin E3 ligase pathways that are activated by mitochondrial depolarisation in neurons. Finally we have developed an online resource to visualise mitochondrial ubiquitin sites in neurons and search for ubiquitin components recruited to mitochondria upon mitochondrial depolarisation, MitoNUb. This analysis will aid in future studies to understand Parkin activation in neuronal subtypes. Our findings also suggest that monitoring ubiquitylation status of the 22 identified MOM proteins may represent robust biomarkers for PINK1 and Parkin activity in vivo.

Parkinson's disease (PD) is a leading cause of neurodegeneration that is defined by the selective loss of dopaminergic neurons and the accumulation of protein aggregates called Lewy bodies (LBs). The unequivocal identification of Mendelian inherited mutations in 13 genes in PD has provided transforming insights into the pathogenesis of this disease. The mechanistic analysis of several PD genes, including α-synuclein (α-syn), leucine-rich repeat kinase 2 (LRRK2), PTEN-induced kinase 1 (PINK1), and Parkin, has revealed central roles for protein aggregation, mitochondrial damage, and defects in endolysosomal trafficking in PD neurodegeneration. In this review, we outline recent advances in our understanding of these gene pathways with a focus on the emergent role of Rab (Ras analog in brain) GTPases and vesicular trafficking as a common mechanism that underpins how mutations in PD genes lead to neuronal loss. These advances have led to previously distinct genes such as vacuolar protein–sorting-associated protein 35 (VPS35) and LRRK2 being implicated in a common signaling pathway. A greater understanding of these common nodes of vesicular trafficking will be crucial for linking other PD genes and improving patient stratification in clinical trials underway against α-syn and LRRK2 targets.

Loss of function mutations in the PINK1 kinase are causal for autosomal recessive Parkinson disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2. Strikingly we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and NMR structure of Ser111-phosphorylated Rab1B that does not demonstrate any major changes suggesting that the phosphorylated SF3 motif may disrupt effector-Switch II interactions. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A suggesting that small molecule activators of PINK1 may have therapeutic potential in patients harbouring LRRK2 mutations.

The discovery of rare familial monogenic forms of early-onset Parkinson's disease has led to the identification of a mitochondrial quality control process as a key player in this disease. Loss-of-function mutations in the genes encoding PINK1 or Parkin result in insufficient removal of dysfunctional mitochondria through autophagy, a process termed mitophagy. Understanding the mechanism of this process and the function of its two key players, PINK1 and Parkin, has led to the discovery of new therapeutic approaches. Small molecule activators of mitophagy, either activating PINK1 or Parkin directly or inhibiting Parkin's counterplayer, the ubiquitin-specific protease USP30, are in preclinical development. To enable clinical success of future small molecule mitophagy enhancers, biomarkers for mitochondrial integrity and mitophagy are being developed. Only a few years after the discovery of mitophagy deficits in Parkinson's disease, research of the underlying mechanisms, drug discovery of modulators for this mechanism and identification of biomarkers provide new avenues towards the development of disease-modifying therapies.

International audience; Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser 65) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embry-onic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser 111) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser 111 of each of the Rabs, we demonstrate that Rab Ser 111 phosphoryla-tion occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolari-sation. We provide evidence that Rab8A GTPase Ser 111 phosphory-lation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser 111 phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser 65. We further show mechanistically that phosphorylation at Ser 111 significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/ 8B/13 at Ser 111 may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.

Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and in vitro studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation in vivo in mammals remains unknown. To address this, we generated a Parkin Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in ParkinS65A/S65A neurons. Phenotypically, ParkinS65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (PARK2) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the ParkinS65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.

Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and in vitro studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation in vivo in mammals remains unknown. To address this, we generated a Parkin Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in ParkinS65A/S65A neurons. Phenotypically, ParkinS65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (PARK2) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the ParkinS65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.

Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and in vitro studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation in vivo in mammals remains unknown. To address this, we generated a Parkin Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in ParkinS65A/S65A neurons. Phenotypically, ParkinS65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (PARK2) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the ParkinS65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.

Mutations in PINK1, which impair its catalytic kinase activity, are causal for autosomal recessive early‐onset Parkinson's disease (PD). Various studies have indicated that the activation of PINK1 could be a useful strategy in treating neurodegenerative diseases, such as PD. Herein, it is shown that the anthelmintic drug niclosamide and its analogues are capable of activating PINK1 in cells through the reversible impairment of the mitochondrial membrane potential. With these compounds, for the first time, it is demonstrated that the PINK1 pathway is active and detectable in primary neurons. These findings suggest that niclosamide and its analogues are robust compounds for the study of the PINK1 pathway and may hold promise as a therapeutic strategy in PD and related disorders.

The discovery of mutations in genes encoding protein kinase PTEN-induced kinase 1 (PINK1) and E3 ubiquitin ligase Parkin in familial Parkinson's disease and their association with mitochondria provides compelling evidence that mitochondrial dysfunction is a major contributor to neurodegeneration in Parkinson's disease. In recent years, tremendous progress has been made in the understanding of how PINK1 and Parkin enzymes are regulated and how they influence downstream mitochondrial signalling processes. We provide a critical overview of the key advances in the field and also discuss the outstanding questions, including novel ways in which this knowledge could be exploited to develop therapies against Parkinson's disease.

We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser65 within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser65 that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser65 in ubiquitin lies in a similar motif to Ser65 in the Ubl domain of Parkin. Remarkably, PINK1 directly phosphorylates Ser65 of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser65-phosphorylated ubiquitin (ubiquitinPhospho−Ser65) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser65 (ΔUbl-Parkin) is robustly activated by ubiquitinPhospho−Ser65, but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser65 (UblPhospho−Ser65) can also activate ΔUbl-Parkin similarly to ubiquitinPhospho−Ser65. Thirdly, we establish that ubiquitinPhospho−Ser65, but not non-phosphorylated ubiquitin or UblPhospho−Ser65, activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser65 and ubiquitin at Ser65, since only mutation of both proteins at Ser65 completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser65, but also by phosphorylating ubiquitin at Ser65. We propose that phosphorylation of Parkin at Ser65 serves to prime the E3 ligase enzyme for activation by ubiquitinPhospho−Ser65, suggesting that small molecules that mimic ubiquitinPhospho−Ser65 could hold promise as novel therapies for Parkinson's disease., We describe a novel and unexpected mechanism by which PINK1 protein kinase activates Parkin E3 ligase. We show that PINK1 phosphorylates ubiquitin at Ser65 and that phosphorylated ubiquitin acts as a direct activator of Parkin.

Whether mitophagy occurs within specific cellular subtypes in vivo is unclear. McWilliams et al. present “mito-QC,” a transgenic mouse containing a pH-sensitive fluorescent mitochondrial signal, allowing in vivo detection of mitophagy and mitochondrial morphology at single-cell resolution., Autophagic turnover of mitochondria, termed mitophagy, is proposed to be an essential quality-control (QC) mechanism of pathophysiological relevance in mammals. However, if and how mitophagy proceeds within specific cellular subtypes in vivo remains unclear, largely because of a lack of tractable tools and models. To address this, we have developed “mito-QC,” a transgenic mouse with a pH-sensitive fluorescent mitochondrial signal. This allows the assessment of mitophagy and mitochondrial architecture in vivo. Using confocal microscopy, we demonstrate that mito-QC is compatible with classical and contemporary techniques in histochemistry and allows unambiguous in vivo detection of mitophagy and mitochondrial morphology at single-cell resolution within multiple organ systems. Strikingly, our model uncovers highly enriched and differential zones of mitophagy in the developing heart and within specific cells of the adult kidney. mito-QC is an experimentally advantageous tool of broad relevance to cell biology researchers within both discovery-based and translational research communities.

Objective: We performed a mutation screen of NR4A2 (also known as NURR1) in 409 Parkinson's disease (PD) patients. We identified a novel single base substitution in the 5'UTR of the NR4A2 (also known as NURR1) gene (c.-309C > T). Results: We have performed expression studies in neuronal cell lines showing that the c.-309C > T mutation reduces NR4A2 mRNA expression in vitro. We have confirmed this finding in vivo by performing allele specific real-time PCR from brain tissue harbouring the 309C > T mutation and show a 3.48 +/- 1.62 fold reduction in mRNA expression of the mutant allele compared to wild-type. In addition we have undertaken genome wide expression analysis of the mutant NR4A2 brain and shown underexpressed genes were significantly enriched for gene ontology categories in nervous system development and synaptic transmission and overexpressed genes were enriched for unfolded protein response and morphogenesis. Lastly we have shown that the c.-309C > T mutation abrogates the protective effect of wild-type NR4A2 against apoptopic stress. Conclusions: Our findings indicate the c.-309C > T mutation reduces NR4A2 expression resulting in the downregulation of genes involved in the development and maintenance of the nervous system and synaptic transmission. These downregulated pathways contained genes known to be transactivated by NR4A2 and were not disrupted in idiopathic PD brain suggesting causality of the mutation. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Background: Oculopharyngeal muscular dystrophy (OPMD) is caused by expansions of the poly (A) binding protein 2 ( PABP2 ) gene. Previous histological analyses have revealed mitochondrial abnormalities in the muscles of OPMD patients but their significance remains uncertain. Objective: We had the rare opportunity to study monozygotic twins with identical expansions of the PABP2 gene but with markedly different severities of OPMD. Both had histological features of mitochondrial myopathy. We determined whether mitochondrial DNA abnormalities underlay these changes. Methods: Clinical information was obtained by history and examination. Muscle biopsies were obtained from each subject and genetic analysis was performed using long-range PCR and Southern blotting. Results: We demonstrate, for the first time, the presence of mitochondrial DNA (mtDNA) deletions by Southern blotting in individuals with OPMD. This correlates with the presence of mitochondrial myopathy in both twins. Moreover, both twins had different mtDNA deletions, which might explain their phenotypic differences. Conclusion: We hypothesise that mitochondrial dysfunction may occur as a consequence of PABP2 gene mutations, and that this dysfunction may affect the phenotypic manifestations of OPMD.

E3 ligases represent an important class of enzymes, yet there are currently no chemical probes for profiling their activity. We develop a new class of activity-based probe by re-engineering a ubiquitin-charged E2 conjugating enzyme and demonstrate the utility of these probes by profiling the transthiolation activity of the RING-in-between-RING (RBR) E3 ligase parkin in vitro and in cellular extracts. Our study provides valuable insight into the roles, and cellular hierarchy, of distinct phosphorylation events in parkin activation. We also profile parkin mutations associated with patients with Parkinson's disease and demonstrate that they mediate their effect largely by altering transthiolation activity. Furthermore, our probes enable direct and quantitative measurement of endogenous parkin activity, revealing that endogenous parkin is activated in neuronal cell lines (≥75%) in response to mitochondrial depolarization. This new technology also holds promise as a novel biomarker of PINK1-parkin signaling, as demonstrated by its compatibility with samples derived from individuals with Parkinson's disease.

Ubiquitin phosphorylation is emerging as an important regulatory layer in the ubiquitin system. This is exemplified by the phosphorylation of ubiquitin on Ser65 by the Parkinson's disease-associated kinase PINK1, which mediates the activation of the E3 ligase Parkin. Additional phosphorylation sites on ubiquitin might also have important cellular roles. Here we report a versatile strategy for preparing phosphorylated ubiquitin. We biochemically and structurally characterise semisynthetic phospho-Ser65-ubiquitin. Unexpectedly, we observed disulfide bond formation between ubiquitin molecules, and hence a novel crystal form. The method outlined provides a direct approach to study the combinatorial effects of phosphorylation on ubiquitin function. Our analysis also suggests that disulfide engineering of ubiquitin could be a useful strategy for obtaining alternative crystal forms of ubiquitin species thereby facilitating structural validation.

Objective To investigate the significance of PINK1 mutations in sporadic Parkinson's disease (PD). Methods We determined the frequency of PINK1 mutations by direct sequencing in a large series of PD patients with apparently sporadic disease (n = 768). Results Twelve heterozygous mutations were identified, nine in PD patients and three in control subjects. Interpretation Given the difficulty in interpreting the pathogenic significance of the heterozygous mutations that have already been reported in parkin and DJ-1, we first determined the frequency of heterozygous PINK1 mutations in the general population by sequencing a large number of control subjects (n = 768), then subsequently assessed their functional significance by examining their effects on stress-induced alterations to the mitochondrial membrane potential (ΔΨm). We demonstrate an enrichment of heterozygous mutations in sporadic PD patients compared with matched control subjects (1.2% in PD vs 0.4% in control subjects). Furthermore, we show that they adversely affect ΔΨm in a similar way to the familial G309D mutation. Although it remains difficult to conclusively demonstrate the pathogenicity of heterozygous mutations, the results of this study and the previously reported subclinical nigrostriatal dysfunction in carriers of heterozygous PINK1 mutations suggest the possibility that these heterozygous mutations are a significant risk factor in the development of later onset PD. Ann Neurol 2006

Parkinson's disease (PD) is one of the most common neurodegenerative disorders. Gene mutations have been found in rare familial forms of PD, with mutations in parkin being the most common cause. Oxidative stresses have also been implicated as an important contributing factor in the pathogenesis of PD. Currently, there is accumulating evidence that parkin may play a role in maintaining mitochondrial function and preventing oxidative stress. We demonstrated here that parkin is up-regulated when SH-SY5Y dopaminergic neuroblastoma cells are exposed to the oxidant dopamine. The up-regulation of parkin appeared to be due to transcriptional activation as luciferase assays confirmed that specific parkin promoter constructs could confer enhanced transcriptional activation in response to dopamine. Moreover, this effect was also seen when SH-SY5Y cells were subjected to another oxidative stress, 1-methyl-4-phenylpyridinium. In parallel with these studies, we also observed similar transcriptional activation of the parkin coregulated gene by oxidative stress. This is the first demonstration that parkin expression is up-regulated by oxidative stresses and may suggest that this might be a general neuroprotective response of parkin to oxidative stresses.

Following our identification of PTEN-induced putative kinase 1 (PINK1) gene mutations in PARK6-linked Parkinson's disease (PD), we have recently reported that PINK1 protein localizes to Lewy bodies (LBs) in PD brains. We have used a cellular model system of LBs, namely induction of aggresomes, to determine how a mitochondrial protein, such as PINK1, can localize to aggregates. Using specific polyclonal antibodies, we firstly demonstrated that human PINK1 was cleaved and localized to mitochondria. We demonstrated that, on proteasome inhibition with MG-132, PINK1 and other mitochondrial proteins localized to aggresomes. Ultrastructural studies revealed that the mechanism was linked to the recruitment of intact mitochondria to the aggresome. Fractionation studies of lysates showed that PINK1 cleavage was enhanced by proteasomal stress in vitro and correlated with increased expression of the processed PINK1 protein in PD brain. These observations provide valuable insights into the mechanisms of LB formation in PD that should lead to a better understanding of PD pathogenesis.

Parkinson's disease is a common incurable neurodegenerative disease whose molecular aetiology remains unclear. The identification of Mendelian genes causing rare familial forms of Parkinson's disease has revealed novel proteins and pathways that are likely to be relevant in the pathogenesis of sporadic Parkinson's disease. Recently, mutations in a novel gene, PINK1, encoding a 581 amino acid protein with both mitochondrial targeting and serine/threonine kinase domains, were identified as a cause of autosomal recessive parkinsonism. This provided important evidence for the role of the mitochondrial dysfunction and kinase pathways in neurodegeneration. In this study, we report the first characterization of the PINK1 protein in normal human and sporadic Parkinson's brains, in addition to Parkinson's cases with heterozygous PINK1 mutations. The possible role of the PINK1 protein was also assessed in a number of neurodegenerative diseases characterized by proteinaceous inclusions. For these studies, rabbit polyclonal antibodies were raised against two peptide sequences within the N-terminal hydrophilic loops of PINK1 protein. Using immunohistochemistry and western blotting we were able to demonstrate that PINK1 is a ubiquitous protein expressed throughout the human brain and it is found in all cell types showing a punctate cytoplasmic staining pattern consistent with mitochondrial localization. Fractionation studies of human and rat brain confirm that PINK1 is localized to the mitochondrial membranes. In addition, we show that PINK1 is detected in a proportion of Lewy bodies in cases of sporadic Parkinson's disease and Parkinson's disease associated with heterozygous mutations in the PINK1 gene, which are clinically and pathologically indistinguishable from the sporadic cases. PINK1 was absent in cortical Lewy bodies, in neurofibrillary tangles in Alzheimer's disease, progressive supranuclear palsy and corticobasal degeneration, and in the glial and neuronal alpha-synuclein positive inclusions in multiple system atrophy. These studies provide for the first time in vivo morphological and biochemical evidence to support a mitochondrial localization of PINK1 and underpin the significance of mitochondrial dysfunction in the pathogenesis of nigral cell degeneration in Parkinson's disease.

The NR4A2 gene, which may cause autosomal dominant Parkinson's disease (PD), has also been reported to be a susceptibility factor for sporadic PD. Here, we use a haplotype-tagging approach in 802 PD patients and 784 controls and demonstrate that common genetic variation, including NR4A2 haplotypes, does not influence the risk of PD in the Caucasian population.

Lewy bodies (LBs) are the characteristic inclusions of Parkinson's disease brain but the mechanism responsible for their formation is obscure. Lewy bodies (LBs) are composed of a number of proteins of which alpha-synuclein (alpha-SYN) is a major constituent. In this study, we have investigated the distribution patterns of synphilin-1 and parkin proteins in control and sporadic PD brain tissue by immunohistochemistry (IH), immunoblotting, and immunoelectron microscopy (IEM). We demonstrate the presence of synphilin-1 and parkin in the central core of a majority of LBs using IH and IEM. Using IH, we show an overlapping distribution profile of the two proteins in central neurons. Additionally, we show sensitivity of both endogenous synphilin-1 and parkin to proteolytic dysfunction and their co-localization in aggresomes formed in response to the proteasome inhibitor MG-132. We confirm that synphilin-1 and parkin are components of majority of LBs in Parkinson's disease and that both proteins are susceptible to proteasomal degradation.

Mutations in the parkin gene give rise to a familial form of Parkinson's disease, autosomal recessive juvenile Parkinsonism (AR-JP). Although the exact mechanisms are unclear, it is thought that these 'loss-of-function' mutations contribute to the pathological process by interfering with parkin's E3 ubiquitin ligase activity. In order to mimic the in vivo loss-of-function, we produced tet-inducible glial cell lines that, in the presence of doxycycline, were able either to under- or to over-express the parkin protein. Using this cell-culture system, we found that the induced alteration of parkin levels in glial cell lines caused different responses compared with their un-induced counterparts under conditions of stress (staurosporine, hydrogen peroxide and dopamine). In particular, reduction in the levels of parkin within the transfected cells rendered them more susceptible to both apoptotic and necrotic cell death. Interestingly, blocking the cell death pathway with caspase inhibitors rescued the cells under-expressing parkin from only some of the stress-induced death. These findings implicate a pathogenic role of glial cells in the pathogenesis of AR-JP caused by mutations in the parkin gene.

Mutations in the PINK1 gene (PARK6), a putative serine-threonine kinase, cause autosomal recessive Parkinson's disease. PINK1 functions as a protein kinase and confers protective effects in the mitochondria, where it is primarily located. We assessed in a population of European ancestry whether common genetic variation in this novel gene influences nonmendelian forms of Parkinson's disease. We defined the linkage disequilibrium structure of PINK1 and used this to identify a set of tagging single nucleotide polymorphisms that we estimate will efficiently represent all of the common DNA variation in the entire gene. Genotyping these tags in a set of 576 Parkinson's disease patients and 514 controls did not demonstrate a case-control partition for allele or for haplotype and thus provides evidence against the existence of a common functional variants in PINK1 that has a strong influence on PD risk.

A newly engineered phosphoserine synthetase/tRNA pair allows quantitative insertion of phosphoserine or, when coupled with metabolic rewiring, a non-hydrolyzable analog into protein sequences, leading to high yields of modified constructs for functional analysis. Serine phosphorylation is a key post-translational modification that regulates diverse biological processes. Powerful analytical methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair that directs the efficient incorporation of phosphoserine (pSer (1)) into recombinant proteins in Escherichia coli. Moreover, combining the orthogonal pair with a metabolically engineered E. coli enables the site-specific incorporation of a nonhydrolyzable analog of pSer. Our approach enables quantitative decoding of the amber stop codon as pSer, and we purify, with yields of several milligrams per liter of culture, proteins bearing biologically relevant phosphorylations that were previously challenging or impossible to access—including phosphorylated ubiquitin and the kinase Nek7, which is synthetically activated by a genetically encoded phosphorylation in its activation loop.

Neurological and psychiatric disorders continue to intrigue and perplex the clinical and scientific world. Neurodegenerative conditions follow a relentless course, causing increasing disability as they progress, and ultimately, early demise. Despite intensive basic and clinical research, progress has been painfully slow, with not a single treatment developed to slow or limit the progression of any of the many neurodegenerative disorders. This includes some of the most common diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD), as well as other equally debilitating conditions such as amyotrophic lateral sclerosis (ALS). So why has progress been so slow? There is no denying that neurodegenerative disorders are complex, but the discovery of several genes causing rare familial forms should have provided vital clues to jumpstart our understanding of neuronal dysfunction and disease pathogenesis, and hence to develop therapies. Each new discovery of a gene linked to neurodegenerative disease is hailed as a major breakthrough » Each new discovery of a gene linked to neurodegenerative disease is hailed as a major breakthrough. « and widely anticipated to lead to improved treatment. However, subsequent advances have generally been disappointingly slow. For example, mutations in genes such as the amyloid precursor protein, causing AD (Goate et al, 1991); Cu/Zn superoxide dismutase (SOD1) in ALS (Rosen et al, 1993); and Huntington in Huntington's disease (HD) (Huntington's disease collaborative research group, 1993) were uncovered more than 15 years ago. Since then, despite several thousands of research articles, these advances are yet to be translated into therapies of any proven benefit to patients. What is holding up progress? » What is holding up progress? «The reasons are multifold. Many of the disease‐causing genes so far unearthed are largely uncharacterized; their functions are obscure. The laboratories making these discoveries have often lacked the expertise to uncover the functions …

The etiology of the selective neuronal death that occurs in Huntington's disease (HD) is unknown. Several lines of evidence implicate the involvement of energetic defects and oxidative damage in the disease process, including a recent study that demonstrated an interaction between huntingtin protein and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using spectrophotometric assays in postmortem brain tissue, we found evidence of impaired oxidative phosphorylation enzyme activities restricted to the basal ganglia in HD brain, while enzyme activities were unaltered in three regions relatively spared by HD pathology (frontal cortex, parietal cortex, and cerebellum). Citrate synthase-corrected complex II-III activity was markedly reduced in both HD caudate (-29%) and putamen (-67%), and complex IV activity was reduced in HD putamen (-62%). Complex I and GAPDH activities were unaltered in all regions examined. We also measured levels of the oxidative damage product 8-hydroxydeoxyguanosine (OH8dG) in nuclear DNA, and superoxide dismutase (SOD) activity. OH8dG levels were significantly increased in HD caudate. Cytosolic SOD activity was slightly reduced in HD parietal cortex and cerebellum, whereas particulate SOD activity was unaltered in these regions. These results further support a role for metabolic dysfunction and oxidative damage in the pathogenesis of HD.

Frontotemporal lobar degeneration (FTLD) consists of a group of neurodegenerative diseases characterized by behavioural and executive impairment, language disorders and motor dysfunction. About 20–30 % of cases are inherited in a dominant manner. Mutations in the microtubule-associated protein tau gene (MAPT) cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17T). Here we report a novel MAPT mutation (K298E) in exon 10 in a patient with FTDP-17T. Neuropathological studies of post-mortem brain showed widespread neuronal loss and gliosis and abundant deposition of hyperphosphorylated tau in neurons and glia. Molecular studies demonstrated that the K298E mutation affects both protein function and alternative mRNA splicing. Fibroblasts from a skin biopsy of the proband taken at post-mortem were directly induced into neurons (iNs) and expressed both 3-repeat and 4-repeat tau isoforms. As well as contributing new knowledge on MAPT mutations in FTDP-17T, this is the first example of the successful generation of iNs from skin cells retrieved post-mortem.

Summary Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser 65 . We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser 65 . We further show that phosphorylation of Parkin at Ser 65 leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser 65 or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr 257 , which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser 65 and/or PINK1 at Thr 257 represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo . Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive condition caused by mutations in the nuclear gene ECGF1 coding for thymidine phosphorylase (TP).1,2 Clinical features include gastrointestinal dysmotility, peripheral sensorimotor polyneuropathy, progressive external ophthalmoplegia, and hepatopathy. In vitro evidence and the improvement following stem cell transplantation (alloSCT) in one patient suggest the pathogenesis centers on elevated systemic levels of the TP substrates, thymidine (Thd) and deoxyuridine (dUrd).3,4 ### Case report. In September 2005, a 21-year-old woman presented after a 4-week history of progressive bilateral distal lower limb numbness and foot drop. She had bouts of unexplained gastrointestinal symptoms and weight loss since age 6 years and at 19 developed an acute abdomen leading to a laparotomy that revealed gross small bowel distension (thought to be caused by an elevated ligament of Treitz) and hepatosplenomegaly. A gastrojejunostomy was performed. On neurologic examination, the positive findings were subtle pigmentary retinopathy, partial ptosis, slight reduction of eye abduction, markedly slow horizontal saccades, severe weakness of ankle movements, absent muscle stretch reflexes and plantar reflexes, and stocking diminution for light touch and pain (all findings bilateral and symmetric). Of note on systemic examination: underweight, gross hepatosplenomegaly, tachycardia, and hyperdynamic cardiac apex. Her parents were not related. She left higher education in autumn 2005 due to her illness. After the laparotomy, investigations including hepatitis serology and serum copper were normal; a liver biopsy revealed steatohepatitis. A brain MRI was normal except equivocal diffuse high T2 signal in the centrum semiovale bilaterally. Following the initial presentation, abnormal results included normochromic normocytic anemia; serum lactate 3.72 mmol/L; CSF protein 1.95 g/L; CSF lactate 5.6 mmol/L; plasma Thd 13 μmol/L and …

Parkinson disease is a devastating neurodegenerative disorder with no known cure. Impairment in mitochondrial dysfunction is thought to play a major role in the pathogenesis. Recent genetic advances suggest that mitochondrial dysfunction may be the primary defect. Drugs that target the mitochondria may therefore represent the best hope for disease-modifying therapies in Parkinson disease.

The quest to disentangle the aetiopathogenesis of Parkinson's disease has been heavily influenced by the genes associated with the disease. The alpha-synuclein-centric theory of protein aggregation with the adjunct of parkin-driven proteasome deregulation has, in recent years, been complemented by the discovery and increasing knowledge of the functions of DJ1, PINK1 and OMI/HTRA2, which are all associated with the mitochondria and have been implicated in cellular protection against oxidative damage. We critically review how these genes fit into and enhance our understanding of the role of mitochondrial dysfunction in Parkinson's disease, and consider how oxidative stress might be a potential unifying factor in the aetiopathogenesis of the disease.

OBJECTIVE: The UCHL-1 gene is widely cited as a susceptibility factor for sporadic Parkinson's disease (PD). The strongest evidence comes from a meta-analysis of small studies that reported the S18Y polymorphism as protective against PD, after pooling studies of white and Asian subjects. Here, we present data that challenge this association. METHODS: In a new large case-control study in white individuals (3,023 subjects), the S18Y variant was not protective against PD under any genetic model of inheritance. Similarly, a more powerful haplotype-tagging approach did not detect other associated variants. RESULTS: Finally, in an updated S18Y-PD meta-analysis (6,594 subjects), no significant association was observed under additive, recessive, or dominant models (odds ratio = 1.00 [95% confidence interval: 0.74-1.33]; odds ratio = 1.01 [95% confidence interval: 0.76-1.35]; and odds ratio = 0.96 [95% confidence interval: 0.86-1.08], respectively), and a cumulative meta-analysis showed a trend toward a null effect. INTERPRETATION: Based on the current evidence, the UCHL-1 gene does not exhibit a protective effect in PD.

Autosomal recessive spinal muscular atrophy (SMA) shows substantial phenotypic variability, presenting at a variety of ages from infancy to adult life. Diagnostic difficulties may arise because SMA sometimes produces a dystrophic or myopathic phenotype rather than classical neurogenic abnormalities. Two brothers are described who illustrate this principle and highlight the increasing importance of molecular genetics in investigating patients with neuromuscular diseases. The findings are discussed in the light of recent observations in a mouse model of SMA.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra. We previously mapped a locus for a rare familial form of PD to chromosome 1p36 (PARK6). Here we show that mutations in PINK1 (PTEN-induced kinase 1) are associated with PARK6. We have identified two homozygous mutations affecting the PINK1 kinase domain in three consanguineous PARK6 families: a truncating nonsense mutation and a missense mutation at a highly conserved amino acid. Cell culture studies suggest that PINK1 is mitochondrially located and may exert a protective effect on the cell that is abrogated by the mutations, resulting in increased susceptibility to cellular stress. These data provide a direct molecular link between mitochondria and the pathogenesis of PD.