The origin of esterase activity of Parkinson's disease causative factor DJ-1 implied by evolutionary trace analysis of its prokaryotic homolog HchA.

DJ-1, a causative gene for hereditary recessive Parkinsonism, is evolutionarily conserved across eukaryotes and prokaryotes. Structural analyses of DJ-1 and its homologs suggested the 106th Cys is a nucleophilic cysteine functioning as the catalytic center of hydratase or hydrolase activity. Indeed, DJ-1 and its homologs can convert highly electrophilic α-oxoaldehydes such as methylglyoxal into α-hydroxy acids as hydratase in vitro, and oxidation-dependent ester hydrolase (esterase) activity has also been reported for DJ-1. The mechanism underlying such plural activities, however, has not been fully characterized. To address this knowledge gap, we conducted a series of biochemical assays assessing the enzymatic activity of DJ-1 and its homologs. We found no evidence for esterase activity in any of the Escherichia coli DJ-1 homologs. Furthermore, contrary to previous reports, we found that oxidation inactivated rather than facilitated DJ-1 esterase activity. The E. coli DJ-1 homolog HchA possesses phenylglyoxalase and methylglyoxalase activities but lacks esterase activity. Since evolutionary trace analysis identified the 186th H as a candidate residue involved in functional differentiation between HchA and DJ-1, we focused on H186 of HchA and found that an esterase activity was acquired by H186A mutation. Introduction of reverse mutations into the equivalent position in DJ-1 (A107H) selectively eliminated its esterase activity without compromising α-oxoaldehyde hydratase activity. The obtained results suggest that differences in the amino acid sequences near the active site contributed to acquisition of esterase activity in vitro, and provide an important clue to the origin and significance of DJ-1 esterase activity.

HKDC1, a target of TFEB, is essential to maintain both mitochondrial and lysosomal homeostasis, preventing cellular senescence.

Mitochondrial and lysosomal functions are intimately linked and are critical for cellular homeostasis, as evidenced by the fact that cellular senescence, aging, and multiple prominent diseases are associated with concomitant dysfunction of both organelles. However, it is not well understood how the two important organelles are regulated. Transcription factor EB (TFEB) is the master regulator of lysosomal function and is also implicated in regulating mitochondrial function; however, the mechanism underlying the maintenance of both organelles remains to be fully elucidated. Here, by comprehensive transcriptome analysis and subsequent chromatin immunoprecipitation-qPCR, we identified hexokinase domain containing 1 (HKDC1), which is known to function in the glycolysis pathway as a direct TFEB target. Moreover, HKDC1 was upregulated in both mitochondrial and lysosomal stress in a TFEB-dependent manner, and its function was critical for the maintenance of both organelles under stress conditions. Mechanistically, the TFEB-HKDC1 axis was essential for PINK1 (PTEN-induced kinase 1)/Parkin-dependent mitophagy via its initial step, PINK1 stabilization. In addition, the functions of HKDC1 and voltage-dependent anion channels, with which HKDC1 interacts, were essential for the clearance of damaged lysosomes and maintaining mitochondria-lysosome contact. Interestingly, HKDC1 regulated mitophagy and lysosomal repair independently of its prospective function in glycolysis. Furthermore, loss function of HKDC1 accelerated DNA damage-induced cellular senescence with the accumulation of hyperfused mitochondria and damaged lysosomes. Our results show that HKDC1, a factor downstream of TFEB, maintains both mitochondrial and lysosomal homeostasis, which is critical to prevent cellular senescence.

Optineurin provides a mitophagy contact site for TBK1 activation.

Tank-binding kinase 1 (TBK1) is a Ser/Thr kinase that is involved in many intracellular processes, such as innate immunity, cell cycle, and apoptosis. TBK1 is also important for phosphorylating the autophagy adaptors that mediate the selective autophagic removal of damaged mitochondria. However, the mechanism by which PINK1-Parkin-mediated mitophagy activates TBK1 remains largely unknown. Here, we show that the autophagy adaptor optineurin (OPTN) provides a unique platform for TBK1 activation. Both the OPTN-ubiquitin and the OPTN-pre-autophagosomal structure (PAS) interaction axes facilitate assembly of the OPTN-TBK1 complex at a contact sites between damaged mitochondria and the autophagosome formation sites. At this assembly point, a positive feedback loop for TBK1 activation is initiated that accelerates hetero-autophosphorylation of the protein. Expression of monobodies engineered here to bind OPTN impaired OPTN accumulation at contact sites, as well as the subsequent activation of TBK1, thereby inhibiting mitochondrial degradation. Taken together, these data show that a positive and reciprocal relationship between OPTN and TBK1 initiates autophagosome biogenesis on damaged mitochondria.

Mitochondrial quality control via organelle and protein degradation.

Mitochondria are essential eukaryotic organelles that produce ATP as well as synthesize various macromolecules. They also participate in signalling pathways such as the innate immune response and apoptosis. These diverse functions are performed by >1,000 different mitochondrial proteins. Although mitochondria are continuously exposed to potentially damaging conditions such as reactive oxygen species, proteases/peptidases localized in different mitochondrial subcompartments, termed mitoproteases, maintain mitochondrial quality and integrity. In addition to processing incoming precursors and degrading damaged proteins, mitoproteases also regulate metabolic reactions, mitochondrial protein half-lives and gene transcription. Impaired mitoprotease function is associated with various pathologies. In this review, we highlight recent advances in our understanding of mitochondrial quality control regulated by autophagy, ubiquitin-proteasomes and mitoproteases.

Mitochondrial lipid dynamics regulated by MITOL-mediated ubiquitination.

Mitochondria-endoplasmic reticulum (ER) contact sites in mammals provide platforms for various reactions, such as calcium signaling, lipid metabolism, organelle dynamics and autophagy. To fulfill these tasks, a number of proteins assemble at the contact sites including MITOL/MARCHF5, a critical mitochondrial ubiquitin ligase. How MITOL regulates mitochondrial function from the contact site, however, has been largely unresolved. Recently, a new role for MITOL in the active transport of phosphatidic acid from the ER to mitochondria was reported. In this commentary, we briefly summarize our current understanding of mitochondria-ER contact sites and discuss the recently elucidated mechanism of MITOL fine-tuning phospholipid transfer activity through ubiquitination.

Integrated proteomics identifies p62-dependent selective autophagy of the supramolecular vault complex.

In addition to membranous organelles, autophagy selectively degrades biomolecular condensates, in particular p62/SQSTM1 bodies, to prevent diseases including cancer. Evidence is growing regarding the mechanisms by which autophagy degrades p62 bodies, but little is known about their constituents. Here, we established a fluorescence-activated-particle-sorting-based purification method for p62 bodies using human cell lines and determined their constituents by mass spectrometry. Combined with mass spectrometry of selective-autophagy-defective mouse tissues, we identified vault, a large supramolecular complex, as a cargo within p62 bodies. Mechanistically, major vault protein directly interacts with NBR1, a p62-interacting protein, to recruit vault into p62 bodies for efficient degradation. This process, named vault-phagy, regulates homeostatic vault levels in vivo, and its impairment may be associated with non-alcoholic-steatohepatitis-derived hepatocellular carcinoma. Our study provides an approach to identifying phase-separation-mediated selective autophagy cargoes, expanding our understanding of the role of phase separation in proteostasis.

Elucidation of ubiquitin-conjugating enzymes that interact with RBR-type ubiquitin ligases using a liquid-liquid phase separation-based method.

RING-between RING (RBR)-type ubiquitin (Ub) ligases (E3s) such as Parkin receive Ub from Ub-conjugating enzymes (E2s) in response to ligase activation. However, the specific E2s that transfer Ub to each RBR-type ligase are largely unknown because of insufficient methods for monitoring their interaction. To address this problem, we have developed a method that detects intracellular interactions between E2s and activated Parkin. Fluorescent homotetramer Azami-Green fused with E2 and oligomeric Ash (Assembly helper) fused with Parkin form a liquid-liquid phase separation (LLPS) in cells only when E2 and Parkin interact. Using this method, we identified multiple E2s interacting with activated Parkin on damaged mitochondria during mitophagy. Combined with in vitro ubiquitination assays and bioinformatics, these findings revealed an underlying consensus sequence for E2 interactions with activated Parkin. Application of this method to other RBR-type E3s including HOIP, HHARI, and TRIAD1 revealed that HOIP forms an LLPS with its substrate NEMO in response to a proinflammatory cytokine and that HHARI and TRIAD1 form a cytosolic LLPS independent of Ub-like protein NEDD8. Since an E2-E3 interaction is a prerequisite for RBR-type E3 activation and subsequent substrate ubiquitination, the method we have established here can be an in-cell tool to elucidate the potentially novel mechanisms involved in RBR-type E3s.

Unfolding is the driving force for mitochondrial import and degradation of the Parkinson's disease-related protein DJ-1.

Diverse genes associated with familial Parkinson's disease (familial Parkinsonism) have been implicated in mitochondrial quality control. One such gene, PARK7 encodes the protein DJ-1, pathogenic mutations of which trigger its translocation from the cytosol to the mitochondrial matrix. The translocation of steady-state cytosolic proteins like DJ-1 to the mitochondrial matrix upon missense mutations is rare, and the underlying mechanism remains to be elucidated. Here, we show that the protein unfolding associated with various DJ-1 mutations drives its import into the mitochondrial matrix. Increasing the structural stability of these DJ-1 mutants restores cytosolic localization. Mechanistically, we show that a reduction in the structural stability of DJ-1 exposes a cryptic N-terminal mitochondrial-targeting signal (MTS), including Leu10, which promotes DJ-1 import into the mitochondrial matrix for subsequent degradation. Our work describes a novel cellular mechanism for targeting a destabilized cytosolic protein to the mitochondria for degradation.

Molecular functions of autophagy adaptors upon ubiquitin-driven mitophagy.

BACKGROUND: Perturbations in organellar health can lead to an accumulation of unwanted and/or damaged organelles that are toxic to the cell and which can contribute to the onset of neurodegenerative diseases such as Parkinson's disease. Mitochondrial health is particularly critical given the indispensable role the organelle has not only in adenosine triphosphate production but also other metabolic processes. Byproducts of oxidative respiration, such as reactive oxygen species, however, can negatively impact mitochondrial fitness. Consequently, selective degradation of damaged mitochondria, which occurs via a specific autophagic process termed mitophagy, is essential for normal cell maintenance. SCOPE OF REVIEW: Recent accumulating evidence has shown that autophagy adaptors (also referred to as autophagy receptors) play critical roles in connecting ubiquitinated mitochondria with the autophagic machinery of the autophagy-lysosome pathway that is required for degradation. In this review, we focus on our current understanding of the autophagy adaptor mechanisms underlying PINK1/Parkin-driven mitophagy. MAJOR CONCLUSIONS: Although autophagy adaptors are canonically defined as proteins that possess ubiquitin-binding domains and ATG8s-binding motifs, the recent identification of novel binding partners has contributed to the development of a more sophisticated model for how autophagy adaptors contribute to the molecular hub that organizes autophagic cargo-degradation. GENERAL SIGNIFICANCE: Although mitophagy is recognized as one of the selective autophagy pathways that removes dysfunctional mitochondria, a more nuanced understanding of the interactions connecting autophagy adaptors and their associated proteins is needed to gain deeper insights into the fundamental biological processes underlying human diseases, including neurodegenerative disorders. This review is part of a Special Issue entitled Mitophagy.

Molecular mechanisms and physiological functions of mitophagy.

Degradation of mitochondria via a selective form of autophagy, named mitophagy, is a fundamental mechanism conserved from yeast to humans that regulates mitochondrial quality and quantity control. Mitophagy is promoted via specific mitochondrial outer membrane receptors, or ubiquitin molecules conjugated to proteins on the mitochondrial surface leading to the formation of autophagosomes surrounding mitochondria. Mitophagy-mediated elimination of mitochondria plays an important role in many processes including early embryonic development, cell differentiation, inflammation, and apoptosis. Recent advances in analyzing mitophagy in vivo also reveal high rates of steady-state mitochondrial turnover in diverse cell types, highlighting the intracellular housekeeping role of mitophagy. Defects in mitophagy are associated with various pathological conditions such as neurodegeneration, heart failure, cancer, and aging, further underscoring the biological relevance. Here, we review our current molecular understanding of mitophagy, and its physiological implications, and discuss how multiple mitophagy pathways coordinately modulate mitochondrial fitness and populations.

Mammalian BCAS3 and C16orf70 associate with the phagophore assembly site in response to selective and non-selective autophagy.

Macroautophagy/autophagy is an intracellular degradation process that delivers cytosolic materials and/or damaged organelles to lysosomes. De novo synthesis of the autophagosome membrane occurs within a phosphatidylinositol-3-phosphate-rich region of the endoplasmic reticulum, and subsequent expansion is critical for cargo encapsulation. This process is complex, especially in mammals, with many regulatory factors. In this study, by utilizing PRKN (parkin RBR E3 ubiquitin protein ligase)-mediated mitochondria autophagy (mitophagy)-inducing conditions in conjunction with chemical crosslinking and mass spectrometry, we identified human BCAS3 (BCAS3 microtubule associated cell migration factor) and C16orf70 (chromosome 16 open reading frame 70) as novel proteins that associate with the autophagosome formation site during both non-selective and selective autophagy. We demonstrate that BCAS3 and C16orf70 form a complex and that their association with the phagophore assembly site requires both proteins. In silico structural modeling, mutational analyses in cells and in vitro phosphoinositide-binding assays indicate that the WD40 repeat domain in human BCAS3 directly binds phosphatidylinositol-3-phosphate. Furthermore, overexpression of the BCAS3-C16orf70 complex affects the recruitment of several core autophagy proteins to the phagophore assembly site. This study demonstrates regulatory roles for human BCAS3 and C16orf70 in autophagic activity.Abbreviations: AO: antimycin A and oligomycin; Ash: assembly helper; ATG: autophagy-related; BCAS3: BCAS3 microtubule associated cell migration factor; C16orf70: chromosome 16 open reading frame 70; DAPI: 4',6-diamidino-2-phenylindole; DKO: double knockout; DMSO: dimethyl sulfoxide; ER: endoplasmic reticulum; fluoppi: fluorescent-based technology detecting protein-protein interactions; FIS1: fission, mitochondrial 1; FKBP: FKBP prolyl isomerase family member 1C; FRB: FKBP-rapamycin binding; hAG: humanized azami-green; IP: immunoprecipitation; IRES: internal ribosome entry site; KO: knockout; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MFN2: mitofusin 2; MS: mass spectrometry; MT-CO2: mitochondrially encoded cytochrome c oxidase II; mtDNA: mitochondrial DNA; OPTN: optineurin; PFA: paraformaldehyde; PE: phosphatidylethanolamine; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol-3,5-bisphosphate; PINK1: PTEN induced kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; PROPPIN: ß-propellers that bind polyphosphoinositides; RB1CC1/FIP200: RB1 inducible coiled-coil 1; TOMM20: translocase of outer mitochondrial membrane 20; ULK1: unc-51 like autophagy activating kinase 1; WDR45B/WIPI3: WD repeat domain 45B; WDR45/WIPI4: WD repeat domain 45; WIPI: WD repeat domain, phosphoinositide interacting; WT: wild type; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.

Two different axes CALCOCO2-RB1CC1 and OPTN-ATG9A initiate PRKN-mediated mitophagy.

PINK1 and PRKN, proteins mutated in Parkinson disease, selectively amplify ubiquitin signals on damaged mitochondria for elimination via mitophagy. Because all five macroautophagy/autophagy receptors in mammals possess domains binding to ubiquitin and Atg8-family proteins, they were thought to recruit Atg8-family protein labeled phagophores from a cytosolic pool. However, our recent findings show that, in addition to Atg8-family protein binding, two of the receptors CALCOCO2 and OPTN interact with RB1CC1 and ATG9A, respectively, indicating that two different axes, CALCOCO2-RB1CC1 and OPTN-ATG9A, can initiate de novo biogenesis of autophagic membranes on ubiquitin-coated damaged mitochondria. These results explain the critical roles of the autophagy receptors CALCOCO2 and OPTN in mitochondrial degradation, and their abilities to simultaneously bind multiple autophagy core proteins propose a new function, i.e. a scaffold to build multivalent interactions for the orchestrated assembly of autophagy proteins near the ubiquitinated cargo. ABBREVIATIONS: ATG: autophagy-related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CRABP2: cellular retinoic acid binding protein 2; LIR: MAP1LC3/LC3-interacting region; MAP1LC3: microtubule associated protein 1 light chain 3; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SNIPER: specific and nongenetic IAP-dependent protein eraser; SQSTM1/p62: sequestosome 1; ULK: unc-51 like autophagy activating kinase.

Critical role of mitochondrial ubiquitination and the OPTN-ATG9A axis in mitophagy.

Damaged mitochondria are selectively eliminated in a process called mitophagy. Parkin and PINK1, proteins mutated in Parkinson's disease, amplify ubiquitin signals on damaged mitochondria with the subsequent activation of autophagic machinery. Autophagy adaptors are thought to link ubiquitinated mitochondria and autophagy through ATG8 protein binding. Here, we establish methods for inducing mitophagy by mitochondria-targeted ubiquitin chains and chemical-induced mitochondrial ubiquitination. Using these tools, we reveal that the ubiquitin signal is sufficient for mitophagy and that PINK1 and Parkin are unnecessary for autophagy activation per se. Furthermore, using phase-separated fluorescent foci, we show that the critical autophagy adaptor OPTN forms a complex with ATG9A vesicles. Disruption of OPTN-ATG9A interactions does not induce mitophagy. Therefore, in addition to binding ATG8 proteins, the critical autophagy adaptors also bind the autophagy core units that contribute to the formation of multivalent interactions in the de novo synthesis of autophagosomal membranes near ubiquitinated mitochondria.

Two sides of a coin: Physiological significance and molecular mechanisms for damage-induced mitochondrial localization of PINK1 and Parkin.

In 1998, PARKIN was reported as a causal gene for hereditary recessive Parkinsonism by Kitada, Mizuno, Hattori, and Shimizu et al. Later in 2004, PINK1 was also reported as a causal gene for hereditary recessive Parkinsonism by Valente, Auburger, and Wood et al. Although many unsolved mysteries still remain, our knowledge of PINK1 and Parkin function has increased dramatically since then. Despite a number of milestone studies that advanced the PINK1 and Parkin research field, a critical turning point was undoubtedly the determination that their genuine subcellular localization was on depolarized mitochondria. In this review, we outline the key studies that have contributed to our current model for mitochondrial localization of PINK1 and Parkin. Interestingly, like two sides of a coin, our attempts to elucidate the mechanisms underlying the localization of PINK1 and Parkin were inextricably tied to the identification of the PINK1 substrate and molecular dissection of the Parkin activation mechanism.

Parkin-mediated ubiquitylation redistributes MITOL/March5 from mitochondria to peroxisomes.

Ubiquitylation of outer mitochondrial membrane (OMM) proteins is closely related to the onset of familial Parkinson's disease. Typically, a reduction in the mitochondrial membrane potential results in Parkin-mediated ubiquitylation of OMM proteins, which are then targeted for proteasomal and mitophagic degradation. The role of ubiquitylation of OMM proteins with non-degradative fates, however, remains poorly understood. In this study, we find that the mitochondrial E3 ubiquitin ligase MITOL/March5 translocates from depolarized mitochondria to peroxisomes following mitophagy stimulation. This unusual redistribution is mediated by peroxins (peroxisomal biogenesis factors) Pex3/16 and requires the E3 ligase activity of Parkin, which ubiquitylates K268 in the MITOL C-terminus, essential for p97/VCP-dependent mitochondrial extraction of MITOL. These findings imply that ubiquitylation directs peroxisomal translocation of MITOL upon mitophagy stimulation and reveal a novel role for ubiquitin as a sorting signal that allows certain specialized proteins to escape from damaged mitochondria.

Parkin recruitment to impaired mitochondria for nonselective ubiquitylation is facilitated by MITOL.

PINK1 (PARK6) and PARKIN (PARK2) are causal genes of recessive familial Parkinson's disease. Parkin is a ubiquitin ligase E3 that conjugates ubiquitin to impaired mitochondrial proteins for organelle degradation. PINK1, a Ser/Thr kinase that accumulates only on impaired mitochondria, phosphorylates two authentic substrates, the ubiquitin-like domain of Parkin and ubiquitin. Our group and others have revealed that both the subcellular localization and ligase activity of Parkin are regulated through interactions with phosphorylated ubiquitin. Once PINK1 localizes on impaired mitochondria, PINK1-catalyzed phosphoubiquitin recruits and activates Parkin. Parkin then supplies a ubiquitin chain to PINK1 for phosphorylation. The amplified ubiquitin functions as a signal for the sequestration and degradation of the damaged mitochondria. Although a bewildering variety of Parkin substrates have been reported, the basis for Parkin substrate specificity remains poorly understood. Moreover, the mechanism underlying initial activation and translocation of Parkin onto mitochondria remains unclear, because the presence of ubiquitin on impaired mitochondria is thought to be a prerequisite for the initial PINK1 phosphorylation process. Here, we show that artificial mitochondria-targeted proteins are ubiquitylated by Parkin, suggesting that substrate specificity of Parkin is not determined by its amino acid sequence. Moreover, recruitment and activation of Parkin are delayed following depletion of the mitochondrial E3, MITOL/March5. We propose a model in which the initial step in Parkin recruitment and activation requires protein ubiquitylation by MITOL/March5 with subsequent PINK1-mediated phosphorylation. Because PINK1 and Parkin amplify the ubiquitin signal via a positive feedback loop, the low substrate specificity of Parkin might facilitate this amplification process.

YoungMito 2018: Report on the 1st International Mitochondria Meeting for Young Scientists.

The 1st International Mitochondria Meeting for Young Scientists (International YoungMito 2018) was held at Hotel Co-op Inn Kyoto in Kyoto, Japan, from 20 to 22 April 2018. The meeting was attended by 130 mitochondrial researchers from 15 countries. International YoungMito 2018 was the first international mitochondria meeting held in Japan organized by and for young mitochondrial researchers. Over the 3-day period, there were 28 oral presentations including two keynote lectures, 20 presentations from invited speakers, and six short talks selected from abstract submissions. Many different topics were covered including quality control pathways acting against mitochondrial stresses, mitochondrial dynamics, protein/lipid transport, cristae organization, respiration/ATP synthesis, mtDNA maintenance, mitochondrial disease models, and pharmacological approaches. In addition, we had 64 posters, a number which represented almost half of all attendees. Thanks to the cutting-edge information and high-quality unpublished data that were presented, there were many lively discussions during oral and poster sessions that continued into the coffee breaks, lunchtime, and nighttime discussions. The 1st international YoungMito meeting was successful in promoting intellectual exchange among all participants, facilitating collaborations beyond national boundaries, and closed with great success. It was a great pleasure that many participants were looking forward to the next YoungMito meeting.

Structural insights into ubiquitin phosphorylation by PINK1.

Mutations of PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin (Ub) ligase parkin can cause familial parkinsonism. These two proteins are essential for ubiquitylation of damaged mitochondria and subsequent degradation. PINK1 phosphorylates Ser65 of Ub and the Ub-like (UBL) domain of parkin to allosterically relieve the autoinhibition of parkin. To understand the structural mechanism of the Ub/UBL-specific phosphorylation by PINK1, we determined the crystal structure of Tribolium castaneum PINK1 kinase domain (TcPINK1) in complex with a nonhydrolyzable ATP analogue at 2.5 Å resolution. TcPINK1 consists of the N- and C-terminal lobes with the PINK1-specific extension. The ATP analogue is bound in the cleft between the N- and C-terminal lobes. The adenine ring of the ATP analogue is bound to a hydrophobic pocket, whereas the triphosphate group of the ATP analogue and two coordinated Mg ions interact with the catalytic hydrophilic residues. Comparison with protein kinases A and C (PKA and PKC, respectively) unveils a putative Ub/UBL-binding groove, which is wider than the peptide-binding groove of PKA or PKC to accommodate the globular head of Ub or UBL. Further crosslinking analyses suggested a PINK1-interacting surface of Ub. Structure-guided mutational analyses support the findings from the present structural analysis of PINK1.

Endosomal Rab cycles regulate Parkin-mediated mitophagy.

Damaged mitochondria are selectively eliminated by mitophagy. Parkin and PINK1, gene products mutated in familial Parkinson's disease, play essential roles in mitophagy through ubiquitination of mitochondria. Cargo ubiquitination by E3 ubiquitin ligase Parkin is important to trigger selective autophagy. Although autophagy receptors recruit LC3-labeled autophagic membranes onto damaged mitochondria, how other essential autophagy units such as ATG9A-integrated vesicles are recruited remains unclear. Here, using mammalian cultured cells, we demonstrate that RABGEF1, the upstream factor of the endosomal Rab GTPase cascade, is recruited to damaged mitochondria via ubiquitin binding downstream of Parkin. RABGEF1 directs the downstream Rab proteins, RAB5 and RAB7A, to damaged mitochondria, whose associations are further regulated by mitochondrial Rab-GAPs. Furthermore, depletion of RAB7A inhibited ATG9A vesicle assembly and subsequent encapsulation of the mitochondria by autophagic membranes. These results strongly suggest that endosomal Rab cycles on damaged mitochondria are a crucial regulator of mitophagy through assembling ATG9A vesicles.

Parkinson's disease-related DJ-1 functions in thiol quality control against aldehyde attack in vitro.

DJ-1 (also known as PARK7) has been identified as a causal gene for hereditary recessive Parkinson's disease (PD). Consequently, the full elucidation of DJ-1 function will help decipher the molecular mechanisms underlying PD pathogenesis. However, because various, and sometimes inconsistent, roles for DJ-1 have been reported, the molecular function of DJ-1 remains controversial. Recently, a number of papers have suggested that DJ-1 family proteins are involved in aldehyde detoxification. We found that DJ-1 indeed converts methylglyoxal (pyruvaldehyde)-adducted glutathione (GSH) to intact GSH and lactate. Based on evidence that DJ-1 functions in mitochondrial homeostasis, we focused on the possibility that DJ-1 protects co-enzyme A (CoA) and its precursor in the CoA synthetic pathway from aldehyde attack. Here, we show that intact CoA and ß-alanine, an intermediate in CoA synthesis, are recovered from methylglyoxal-adducts by recombinant DJ-1 purified from E. coli. In this process, methylglyoxal is converted to L-lactate rather than the D-lactate produced by a conventional glyoxalase. PD-related pathogenic mutations of DJ-1 (L10P, M26I, A104T, D149A, and L166P) impair or abolish detoxification activity, suggesting a pathological significance. We infer that a key to understanding the biological function of DJ-1 resides in its methylglyoxal-adduct hydrolase activity, which protects low-molecular thiols, including CoA, from aldehydes.