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.

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.

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.

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.

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.

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.

In vivo protein-interaction mapping of a mitochondrial translocator protein Tom22 at work.

Mitochondrial protein import requires cooperation of the machineries called translocators in the outer and inner mitochondrial membranes. Here we analyze the interactions of Tom22, a multifunctional subunit of the outer membrane translocator TOM40 complex, with other translocator subunits such as Tom20, Tom40, and Tim50 and with substrate precursor proteins at a spatial resolution of the amino acid residue by in vivo and in organello site-specific photocross-linking. Changes in cross-linking patterns caused by excess substrate precursor proteins or presequence peptides indicate how the cytosolic receptor domain of Tom22 accepts substrate proteins and how the intermembrane space domain of Tom22 transfers them to Tim50 of the inner-membrane translocator.

Transport of proteins across or into the mitochondrial outer membrane.

Mitochondria are surrounded by two biological membranes. The outer mitochondrial membrane contains two major translocators, the TOM40 (TOM) and TOB/SAM complexes for protein translocation across and/or insertion into the outer membrane. The TOM40 complex functions as an entry gate for most mitochondrial proteins, and the TOB/SAM complex as a specialized insertion machinery for beta-barrel membrane proteins. In order to handle loosely folded or unfolded precursor polypeptides, those translocators cooperate with chaperones in the cytosol and intermembrane space, and also exhibit chaperone-like functions on their own. Several alpha-helical membrane proteins take 'non-standard' routes to be inserted into the outer membrane. Here we review the current view on a remarkable variety of mechanisms of protein transport taking place at the mitochondrial outer membrane.

Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space.

The mitochondrial intermembrane space (IMS) contains many small cysteine-bearing proteins, and their passage across the outer membrane and subsequent folding require recognition and disulfide bond transfer by an oxidative translocator Tim40/Mia40 in the inner membrane facing the IMS. Here we determined the crystal structure of the core domain of yeast Mia40 (Mia40C4) as a fusion protein with maltose-binding protein at a resolution of 3 A. The overall structure of Mia40C4 is a fruit-dish-like shape with a hydrophobic concave region, which accommodates a linker segment of the fusion protein in a helical conformation, likely mimicking a bound substrate. Replacement of the hydrophobic residues in this region resulted in growth defects and impaired assembly of a substrate protein. The Cys296-Cys298 disulfide bond is close to the hydrophobic concave region or possible substrate-binding site, so that it can mediate disulfide bond transfer to substrate proteins. These results are consistent with the growth phenotypes of Mia40 mutant cells containing Ser replacement of the conserved cysteine residues.

Step-size analyses of the mitochondrial Hsp70 import motor reveal the Brownian ratchet in operation.

Newly synthesized mitochondrial precursor proteins have to become unfolded by the mitochondrial Hsp70 (mtHsp70) import motor to cross the mitochondrial membranes. To assess the mechanism of unfolding of precursor proteins by mtHsp70, we designed a system to measure step sizes of the mtHsp70 import motor, which are distances at which the motor system moves along polypeptide chains during a single turnover of ATP. We made a series of fusion proteins consisting of a mitochondrial presequence containing the first mtHsp70 binding site, a spacer sequence containing an Hsp70 avoidance segment followed by the second mtHsp70 binding site, and different folded mature domains. Analyses of the dependence of the import rates of those fusion proteins on the lengths of Hsp70 avoidance segments allowed us to estimate the step sizes, which differ for different mature domains and different lengths of the spacers. These results suggest that the mtHsp70 import motor functions at least as a molecular Brownian ratchet to unfold mitochondrial precursor proteins.

Tom20 and Tom22 share the common signal recognition pathway in mitochondrial protein import.

Precise targeting of mitochondrial precursor proteins to mitochondria requires receptor functions of Tom20, Tom22, and Tom70 on the mitochondrial surface. Tom20 is a major import receptor that recognizes preferentially mitochondrial presequences, and Tom70 is a specialized receptor that recognizes presequence-less inner membrane proteins. The cytosolic domain of Tom22 appears to function as a receptor in cooperation with Tom20, but how its substrate specificity differs from that of Tom20 remains unclear. To reveal possible differences in substrate specificities between Tom20 and Tom22, if any, we deleted the receptor domain of Tom20 or Tom22 in mitochondria in vitro by introducing cleavage sites for a tobacco etch virus protease between the receptor domains and transmembrane segments of Tom20 and Tom22. Then mitochondria without the receptor domain of Tom20 or Tom22 were analyzed for their abilities to import various mitochondrial precursor proteins targeted to different mitochondrial subcompartments in vitro. The effects of deletion of the receptor domains on the import of different mitochondrial proteins for different import pathways were quite similar between Tom20 and Tom22. Therefore Tom20 and Tom22 are apparently involved in the same step or sequential steps along the same pathway of targeting signal recognition in import.