building and decoding ubiquitin chains for mitophagy

Building and decoding ubiquitin chains for mitophagyMitochondria play a central role in the life of cells and organisms by serving as a centre for energy gener ation, in the form of ATP via oxidative phosphoryl ation, and by organizing metabolic machineries, such as the citric acid cycle, that drive many cellular functions. The spatial organization of mitochondria is key to their function, wherein oxidative phosphorylation occurs via the respir atory chain in the mitochondrial inner membrane, and other metabolic reactions are organized in the mitochondrial matrix.
Mitochondrial proteins are encoded by the nuclear and mitochondrial genomes and require the precise orchestration of protein import through the outer and inner mitochondrial membranes, folding and assembly into protein complexes to ultimately form a functional spatially organized organelle. Errors in these processes can result in damaged mitochondria that are detri- mental to cellular physiology. For example, defects in respir atory chain function promote the production of damaging reactive oxygen species and the loss of membrane potential that is crucial for mitochondrial function. Moreover, defects in protein folding in the matrix promote the mitochondrial unfolded protein response (mtUPR), which controls both protein synthesis in the mitochondrial matrix and production of mitochondrial chaperones1–4. Although homeostatic mechanisms such as the mtUPR may be sufficient to repair mitochondria when damage is transient, prolonged or unrepairable
damage can lead to elimination of mitochondria via a process known as mitophagy5–7. Mitophagy is a form of selective autophagy, during which mitochondria are decorated with polyubi quitin chains, engulfed by autophagosomes and degraded following lysosomal fusion (FIG. 1).
Mitophagy was first visualized in electron micro- graphs of cultured cells8, and work over the past two decades has revealed the fundamental biochemical steps involved in targeting of mitochondria to the autophagy system through both ubiquitin-dependent and ubiquitin- independent pathways. Our understanding of ubiquitin-dependent mitophagy has been driven largely through analysis of two genes that were found to be mutated in familial forms of Parkinson disease — the E3 ubiquitin-protein ligase parkin (PRKN)9 and its activating kinase PTEN-induced putative kinase 1 (PINK1), which is present on damaged mitochondria10–12. Early studies in Drosophila melanogaster revealed a genetic pathway in which PINK1 functioned upstream of parkin and overexpression of parkin could bypass defects in PINK1 (REFS 13,14). We now know that these proteins operate in a common pathway to catalyse the assembly of ubiquitin chains on mitochondrial outer membrane (MOM) proteins13–15. These ubiquitin chains bind autophagic cargo receptors such as optineurin (OPTN) and sequestosome 1 (SQSTM1, also known as p62), which act in concert with the general autophagy machinery to capture
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.
Correspondence to J.W.H. wade_harper@ hms.harvard.edu
doi:10.1038/nrm.2017.129 Published online 23 Jan 2018
Ubiquitin A 76-amino-acid protein that can be covalently conjugated to lysine residues in other proteins to specify several protein fates. Poly-ubiquitin chains can be generated using seven internal lysine residues in ubiquitin or its first methionine. Lys11-linked or Lys48-linked chains usually target proteins for degradation, whereas other chains, such as Lys63-linked or Met1-linked chains, have signalling roles.
Building and decoding ubiquitin chains for mitophagy J. Wade Harper, Alban Ordureau and JinMi Heo
Abstract | Mitochondria produce energy in the form of ATP via oxidative phosphorylation. As defects in oxidative phosphorylation can generate harmful reactive oxygen species, it is important that damaged mitochondria are efficiently removed via a selective form of autophagy known as mitophagy. Owing to a combination of cell biological, structural and proteomic approaches, we are beginning to understand the mechanisms by which ubiquitin-dependent signals mark damaged mitochondria for mitophagy. This Review discusses the biochemical steps and regulatory mechanisms that promote the conjugation of ubiquitin to damaged mitochondria via the PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin-protein ligase parkin and how ubiquitin chains promote autophagosomal capture. Recently discovered roles for parkin and PINK1 in the suppression of mitochondrial antigen presentation provide alternative models for how this pathway promotes the survival of neurons. A deeper understanding of these processes has major implications for neurodegenerative diseases, including Parkinson disease, where defects in mitophagy and other forms of selective autophagy are prominent.
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Parkinson disease A long-term disease of the central nervous system that primarily affects motor functions as a result of loss of dopaminergic neurons.
E3 ubiquitin-protein ligase A protein or protein complex that can facilitate the transfer of ubiquitin from an E2 conjugating enzyme to a substrate.
General autophagy machinery Composed of protein and lipid kinases that coordinate the formation of autophagic membranes and the ATG8 conjugation machinery, which is involved in maturation of autophagosomal membranes and fusion with lysosomes.
Amyotrophic lateral sclerosis (ALS). A progressive and fatal motor neuron disorder that affects the function of voluntary muscles, leading to an inability to move, swallow, speak and breathe.
Translocase of the outer membrane (TOM). The TOM complex is a multi-protein channel that functions to facilitate import of nuclear-encoded but mitochondrial-localized proteins into all intra- mitochondrial compartments. The only proteins that do not pass through the TOM complex during import are single-pass mitochondrial outer membrane proteins.
N-end rule ubiquitin ligase A subfamily of RING E3 ubiquitin ligases, including UBR1, UBR2 and UBR3, that use their N-terminal UBR domain to bind to substrates containing hydrophobic or arginine residues at their N-terminus.
damaged mitochondria in the autophagosomal double- membrane (FIG. 1). Fusion with lyso somes facilitates degradation of mitochondria via lysosomal hydrolases.
OPTN and SQSTM1, as well as their associated serine/threonine-protein kinase TBK1, have recently been genetically linked to sporadic and familial forms of amyotrophic lateral sclerosis (ALS)16–19. This genetic link has two major implications: first, it suggests that selective forms of autophagy underlie particular forms of ALS, and second, it suggests that understanding the biochemical mechanisms involved in marking autophagic cargo, including mitochondria, with ubiquitin and its capture by cargo receptors could pave the way for new approaches for therapeutic intervention in neurodegenerative diseases. In addition to these implications in disease, recent studies have shown that some forms of parkin-mediated mitophagy that remove apparently healthy mitochondria are important for several physiological processes, such as self-renewal of angiopoietin 1 receptor (TIE2)-positive haematopoietic stem cells, and paternal mitochondrial clearance during fertiliza- tion20–25 (BOX 1). Moreover, parkin has been implicated in the xenophagic removal of Mycobacterium tuberculosis, which may have parallels with mitophagy, but the underlying mechanisms are poorly understood26.
In this Review, we focus on the biochemical mechanisms that drive the assembly of ubiquitin chains on damaged mitochondria and on how ubiquitylation is decoded by the autophagy machinery to capture damaged organelles. Given that mitochondria are con- stantly engaged in fusion–fission cycles that may mix healthy and damaged organelles, it is important that mitophagy pathways are rapid and robust in their ability to selectively mark and degrade only damaged organelles. This selectivity is achieved through the use of two distinct and sequential feedforward loops, driven by the kinase PINK1 and ubiquitin on the surface of mitochondria that are predicted to create a switch-like behaviour for the detection and capture of only damaged organelles. We describe a generalizable framework for ubiquitin- dependent forms of mitophagy and discuss the substantial gaps that exist in our understanding of these crucial pathways.
Overview of the PINK1–parkin pathway It is thought that the assembly of ubiquitin chains on mitochondria is necessary for the recruitment of this autophagy machinery and the removal of damaged mitochondria by mitophagy6,7. In its simplest form, the ubiquitin chain assembly pathway can be described as containing three positively acting elements: a mitochondrial damage sensor (PINK1), a signal amplifier (parkin) and a signal effector (ubiquitin chains), which determines which mitochondria should be captured by the autophagy machinery (FIG. 1).
PINK1 contains an N-terminal mitochondrial targeting sequence and binds to the translocase of the outer membrane (TOM) complex10,27. When the mito chondrial targeting sequence and transmembrane segment of PINK1 reach the translocase of the inner membrane (TIM) complex and are laterally transferred to the inner
membrane, the transmembrane segment is proteo- lytically cleaved by the inner membrane- localized protease PARL (presenilins-associated rhomboid-like protein, mitochondrial). Cleavage results in a 52 kDa protein fragment containing the kinase domain that is probably still associated with the TOM complex28–30.
When mitochondria are healthy, this 52 kDa PINK1 fragment is released into the cytosol and is rapidly ubiquitylated by an N-end rule ubiquitin ligase, which targets it for degradation by the proteasome30. Thus, PINK1 levels are low in cells with healthy mitochondria. However, when mitochondria are damaged, PINK1 translocation and processing is blocked, leading to the accumulation of active PINK1 on the MOM31, where it can activate the E3 ubiquitin ligase activity of parkin via a multistep feed forward mechanism, as detailed below. Parkin-dependent ubiquitin chain assembly on the MOM then promotes recruitment of ubiquitin- binding mitophagy receptors to promote capture by the autophagosome32–35 (FIG. 1).
As with most signal transduction pathways, neg ative regulators help to ensure that damage signals are suffi- ciently strong such that healthy mitochondria are not inappropriately degraded. In this mitophagy pathway, deubiquitinating enzymes (DUBs), including the mitochondrially localized ubiquitin carboxyl-terminal hydrolase 30 (USP30), seem to antagonize the pathway by removing ubiquitin chains from mitochondria until parkin activation is sufficient to outpace ubiquitin chain removal by USP30 (REF. 36). Elucidating the biochemical mechanisms that determine the activation state of parkin has been a major focus of this research field.
Mechanism of parkin activation by PINK1 In cells with healthy mitochondria, parkin is localized diffusely in the cytoplasm in an autoinhibited form37–40. However, following mitochondrial damage and stabiliza- tion of PINK1 on the MOM, parkin can undergo a series of modifications (including phosphorylation, multiple conformational changes and association with Ser65-phosphorylated ubiquitin (pSer65-Ub)) initiated by PINK1 that promote its stable association with the MOM and activation of its E3 ubiquitin ligase activity. For simplicity, the two major steps of parkin activation — direct activation by phosphorylation of the parkin ubiquitin-like (UBL) domain and activation by binding to pSer65-Ub — are described separately, followed by a description of how these events work together on the MOM to generate a feedforward ubiquitylation process that promotes mitophagy. It should be noted that in many studies described below, the experi mental approach has been to overexpress parkin or PINK1 in model cell systems, which, given the involvement of feedback loops, could have unknown effects, and the results could be more difficult to interpret.
Intrinsic parkin activation by PINK1dependent UBL phosphorylation. The ubiquitin system employs a series of ubiquitin charging and transfer events culminat- ing in the transfer of the C-terminal Gly76 residue of ubiquitin to a lysine residue on the substrate (primary
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Isopeptide bond An amide bond formed between the amino group of a lysine side chain on a protein (substrate) and the C-terminus of another protein (ubiquitin).
Figure 1 | Overview of parkin-dependent mitophagy. In cells with healthy mitochondria, the PTEN-induced putative kinase 1 (PINK1) is rapidly degraded, and the E3 ubiquitin-protein ligase parkin is in an autoinhibited form in the cytoplasm. Upon mitochondrial damage, PINK1 is stabilized on the mitochondrial outer membrane (MOM) and can activate parkin through a feedforward mechanism involving parkin and ubiquitin phosphorylation. Parkin then assembles ubiquitin chains on numerous MOM proteins, which can recruit ubiquitin-binding autophagy receptors. In the canonical model of autophagy, ubiquitin-binding autophagy receptors function to recruit the ATG8-positive phagophore, which ultimately encases the damaged mitochondria and allows fusion with lysosomes, thereby promoting degradation of damaged mitochondria. The canonical autophagosome assembly pathway is composed of three major arms136–138: the phosphatidylinositol 3-kinase catalytic subunit type 3 (VPS34) arm responsible for production of phosphatidylinositol-3-phosphate (PtdIns3P) on donor membranes (step 1), the serine/threonine- protein kinase ULK1 arm that regulates phagophore initiation and expansion (step 2) and the ATG8 conjugation pathway involving ATG7 (E1), ATG3 (E2) and the ATG5/ATG12–ATG16 (E3) complex (where ‘/’ indicates an isopeptide bond). The conjugation pathway attaches ATG8 proteins to phosphatidylethanolamine (PE) on the growing autophagosomal membrane (step 3). ATG8 proteins are thought to function by interacting with cargo receptors and other regulators of the pathway. However, there is evidence of ATG8-conjugation-independent forms of autophagosome formation through non-canonical pathways101,102, which may also function in mitophagy100, possibly with reduced efficiency. AMBRA1, activating molecule in BECN1-regulated autophagy protein 1; AMPK, 5-AMP-activated protein kinase; BECN1, beclin 1; DFCP1, zinc-finger FYVE domain-containing protein 1; ER, endoplasmic reticulum; FIP200, RB1-inducible coiled-coil protein 1; TBK1, serine/threonine-protein kinase TBK1; ULK2, serine/threonine-protein kinase ULK2; VPS15, phosphoinositide 3-kinase regulatory subunit 4; WIPI2, WD repeat domain phosphoinositide-interacting protein 2.
Nature Reviews | Molecular Cell Biology
ER
Omegasome
ATG4 ATG7 E1
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ubiquitylation) or to lysine residues on ubiquitin itself to extend a ubiquitin chain. Ubiquitin is activated by an E1 ubiquitin-activating enzyme in the presence of ATP and is transferred to the active site cysteine residue in one of several E2 ubiquitin-conjugating enzymes to form a thioester bond. Such E2 enzymes interact with RING finger or HECT domains in E3 ubiquitin ligases to facilitate the transfer of ubiquitin to the substrate. Parkin is a RING-between-RING (RBR) E3 ubiquitin ligase; it contains an N-terminal UBL domain, a central RING1 domain that binds an E2 enzyme, an in-between RING (IBR) domain and a C-terminal RING2 domain containing the catalytic cysteine residue (FIG. 2a).
RBR E3s are analogous to HECT domain-containing proteins41 and use their RING1 domain to catalyse the transfer of ubiquitin from a charged E2 enzyme to the catalytic cysteine residue in RING2 (REF. 42). As with HECT E3s, this ubiquitin thioester is subsequently discharged onto a substrate lysine residue (FIG. 2a, left panel). Three mechanisms are involved in parkin auto inhibition and must be overcome to convert parkin into an active enzyme37–40,43 (FIG. 2a, right panel). First, the UBL domain of parkin rests against one of two core α-helical elements within RING1, blocking E2 access (FIG. 2b). Second, the repressive (REP) element docks with both the UBL domain and RING1,
Box 1 | Mitophagy during development and via parkin-independent mechanisms
There is an increasing appreciation that large alterations in the abundance of mitochondria are necessary during particular cellular transitions that occur during development and in normal physiology, in addition to the removal of defective mitochondria (see the figure).
Loss of paternal mitochondrial DNA during fertilization occurs through the process of mitophagy in Caenorhabditis elegans, Drosophila melanogaster and vertebrates89,121–124. In mice, the E3 ubiquitin-protein ligase parkin and the mitochondrial ubiquitin ligase activator of NFKB 1 (MUL1) function redundantly and in combination with sequestosome 1 (SQSTM1) and PTEN-induced putative kinase 1 (PINK1), indicating that some elements of the established PINK1–parkin pathway are used in this case89. By contrast, the analogous process in D. melanogaster requires autophagy machinery and SQSTM1 but not the parkin orthologue121.
For removal of mitochondria during reticulocyte maturation, autophagosome assembly for mitochondrial capture is orchestrated by an autophagy receptor called BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like (NIX; also known as BNIP3L), which is located on the mitochondrial outer membrane (MOM), contains LC3-interacting region (LIR) motifs used to associated with ATG8 proteins and is required for efficient mitochondrial clearance20,125. Recent data suggest that phosphorylation of NIX near the LIR motif increases binding to ATG8 and increases autophagosomal recruitment to mitochondria126. Additional MOM proteins containing LIR motifs, including the peptidyl-prolyl cis-trans isomerase FKBP8 and FUN14 domain-containing protein 1 (FUNDC1), have been implicated in direct interactions with ATG8 proteins to promote mitophagy127,128. In the absence of parkin, overexpression of FKBP8 and the ATG8 protein LC3A can promote mitophagy, although a physiological setting for this form of mitophagy is unknown. Similarly, overexpression of FUNDC1 can promote mitophagy in response to hypoxia128 in a manner that is regulated by the E3 ubiquitin-protein ligase MARCH5 (REF. 129). Understanding the physiological circumstances used to promote direct mitophagy by ATG8 recruitment remains a goal for future research.
CALCOCO2, calcium-binding and coiled-coil domain-containing protein 2; mtUPR, mitochondrial unfolded protein response; OPTN, optineurin; ROS, reactive oxygen species. Nature Reviews | Molecular Cell Biology
Mitophagy
Autolysosome
NIX
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Nanobody A type of single-chain antibody frequently used to stabilize weak interactions for structural biology.
further blocking E2 access (FIG. 2b). Third, the catalytic Cys431 residue in RING2 is shielded by the unique parkin domain (UPD; also known as RING0), located between the UBL domain and RING1, thereby blocking transfer of ubiquitin from the charged E2 to the catalytic cysteine residue of parkin (FIG. 2b). Thus, multiple conformational changes are necessary to remove these auto-inhibitory constraints.
Since the discovery that PINK1 functions upstream of parkin14,27, substantial effort has been focused on understanding how PINK1 directly regulates parkin activity. An early study found that PINK1 phosphorylates parkin in human SH-SY5Y cells and that PINK1- dependent phosphorylation promoted the ability of parkin to make Lys63-linked ubiquitin chains44. However, the phosphorylation sites in parkin and the activation mechanism were not reported. Key insights into parkin activation came from the finding that PINK1 phosphorylates Ser65 in the UBL domain of parkin45,46 and that phosphorylation of parkin at Ser65 (pSer65-parkin) promotes ubiquitin chain assembly by parkin in vitro45. Subsequent studies using parkin stoichio metrically phosphorylated on Ser65 in con- junction with quantitative measurements of ubiquitin chain formation by mass spectrometry revealed that pSer65-parkin increases the chain assembly activity in vitro by ~2,400-fold47. Thus, phosphorylation of parkin on its UBL domain dramatically activates its intrinsic ubiquitin ligase activity. UBL domain phosphorylation at Ser65 disrupts the UBL–RING1 interaction and is thought to allow movement of the UBL domain through its tether to the UPD43,48,49 (FIG. 2b–d). Interestingly, release of the UBL domain is mimicked by a mutation in parkin (Trp403Ala) that is thought to partially release the REP element from RING1 to facilitate binding of the E2 (REFS 39,43) (FIG. 2b–d). This model is consistent with molecular dynamics simu- lations, which suggested that UBL phosphorylation releases the UBL domain from its close interaction with RING1 and leads to small changes in the conformation of the REP element, providing access for the charged E2 to RING1 (REF. 50). Release of the UBL domain from the parkin core provides access to the hydrophobic Ile44 residue in the UBL domain known to be important for phosphoryl ation by PINK1 (REF. 48), which explains in part the need for UBL domain release before phosphorylation. Subsequent structural alterations are propagated to the RING2 domain, as indicated by the increased reactivity of the catalytic Cys431 residue (BOX 2).
Ubiquitin phosphorylation by PINK1 promotes parkin activation and mitochondrial retention. A second mode of PINK1 action on damaged mitochondria was identified when it was discovered that ubiquitin and ubiquitin chains are PINK1 substrates47,51–54 and that pSer65-Ub chains are linked to parkin activation and retention on mitochondria47,55. The UBL domain of parkin is ~30% identical (~50% similar) in amino acid sequence to ubiquitin, including substantial con- servation of the surface containing Ile44, which is near
Ser65 (FIG. 2e,f). Importantly, Ser65 in the parkin UBL domain was found to be conserved in ubi quitin, which led to the demonstration that PINK1 can directly phosphorylate ubiquitin on Ser65 (REFS 51,52). Quantitative phosphoproteomics of mitochondria during mitophagy induction47,53, unbiased identification of candidate PINK1 substrates47 and biochemical activation studies of parkin by PINK1 in the presence of ubiquitin54 also independently identified ubiquitin as a PINK1 target. Initial biochemical studies indicated that monomeric pSer65-Ub not only physically associates with parkin but can also partially activate its ubi quitin chain assembly activity independently of Ser65 phosphorylation on parkin47,51–53. Although it seems that in vivo parkin associates with pSer65-Ub in the form of ubiquitin chains on mitochondria (discussed below), monomeric pSer65-Ub has served as a useful tool for understanding the biochemical and structural basis of parkin activation and retention on the MOM. pSer65-Ub can bind to unphosphorylated parkin and pSer65-parkin in vitro, but binding to pSer65- parkin is ~20-fold stronger47,48,56. Moreover, while stoichiometric binding of pSer65-Ub to unphosphoryl ated parkin activates chain synthesis by ~1,000-fold, a complex of pSer65-parkin and pSer65-Ub displays ~4,400-fold higher chain synthesis activity than unphosphorylated parkin47,57.
Recently, insights into how PINK1 specifically recog- nizes ubiquitin were obtained through structural analy sis of a PINK1–ubiquitin complex stabilized via a nanobody. PINK1 is unique among protein kinases in that its N-terminal lobe contains three stretches of amino acid sequences, referred to as insertions, that are absent from other protein kinases58. The structure reveals that these insertions are stabilized by auto- phosphorylation of Ser202 and Ser204 in PINK1, creating a unique conformation of insertion 3 that enables the recognition of ubiquitin as a substrate. In addition, ubiquitin is in a unique C-terminally retracted conformation ( ubiquitin-CR) when bound to PINK1, which places its Ser65 residue in an extended loop near the catalytic centre of PINK1. The ubiquitin- CR conformation was thought to be unique to pSer65-Ub54, but it was recently reported that it is found at low abundance in unmodi- fied ubiquitin and is in rapid exchange with the con- ventional conformation of ubiquitin59. The structure of PINK1 lacking insertion 3 was also recently described60. These studies explain key features of PINK1, including why PINK1 is highly selective for ubiquitin and the parkin UBL domain as substrates and how many of the PINK1 mutations identified in patients with Parkinson disease disrupt either catalytic activity or substrate recognition58.
Structural and functional studies have shown that, by enabling important contacts within parkin, pSer65-Ub promotes the formation of a central α-helix (H3) linking RING1 and the IBR domain (FIG. 2a,d), and thus release of the UBL domain from the parkin core48,49,56,61,62 (FIG. 2d). The linchpin in the parkin– pSer65-Ub co-complex is a cluster of positively charged residues in parkin (Lys151 and His302) that bind to the phosphate in pSer65-Ub, the mutation of which
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4651
Ser65
PINK1-mediated UBL domain and ubiquitin phosphorylation
? (pUBL position unknown)
Phe146
Trp403
Asn273
e
Ser65 phosphorylation
0º 180º
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diGLY capture proteomics In this approach, di-Gly-Gly ubiquitin ‘remnants’ that remain on substrate lysine residues after trypsinization are captured using a specific antibody and identified using mass spectrometry.
ε-Amino group Refers to the NH3
+ group in a lysine side chain, which is often used as a recipient for ubiquitin transfer in proteins.
abolished binding to pSer65-Ub and in vitro parkin activation by pSer65-Ub43,48,63. Crystallographic analysis of pSer65-Ub bound to a form of parkin that is missing 59 residues of the UBL domain linker (FIG. 2a) led to an alternative model for activation involving a parkin dimer, wherein pSer65-Ub binding opens a surface on the IBR domain that then interacts with the donor ubiquitin of a charged ubiquitin~E2 thioester (where ~ indicates the thioester bond), with the E2 itself being bound to RING1 of the neighbouring parkin mol- ecule64. This model is supported by the finding that mutations in IBR residues that are predicted to directly interact with the donor ubiquitin reduce chain synthesis by parkin in vitro, although these mutants were not tested in vivo. Biophysical analysis of active, full-length pSer65-parkin in complex with pSer65-Ub revealed a monomeric 1:1 complex47,56. While it is conceivable that during the catalytic process, a pSer65-parkin dimer assembles transiently, the UBL domain in pSer65- parkin seems to be unable to promote dimer formation at least in the absence of charged UBCH7 at concentra- tions used for biophysical measurements. Biophysical and molecular dynamics measurements have also suggested that binding of parkin lacking its UBL domain to the ubiquitin~E2 triggers large-scale diffusional motion of the RING2 domain towards RING1, thereby facilitating ubiquitin transfer to Cys431 located in RING2 (REF. 65). Analysis of full-length pSer65-parkin– pSer65-Ub–ubiquitin~E2–substrate complexes in both the pre-transfer state and a state containing ubiquitin- charged RING2 is required to confirm which of these models is correct. It is also noteworthy that activation mechanisms involving allosteric UBL domains have been described for other RBR E3s64,66, suggesting conserved elements of activation mechanisms for RBR E3s.
Mitochondrial surface ubiquitylation Within minutes of mitochondrial damage, parkin is recruited and activated on the MOM and initiates ubi- quityl ation of local substrates10,46,67–70. Much effort has been focused on the identification of parkin substrates in response to mitochondrial damage. Early studies identified several targets of diverse function, including mitofusin 1 (MFN1), MFN2, voltage-dependent anion- selective channel (VDAC) proteins, mitochondrial fission 1 protein (FIS1), mitochondrial import receptor subunit TOM20 homologue (TOMM20) and CDGSH iron-sulfur domain-containing protein 1 (CISD1), which are all located on the MOM70–72. To identify parkin targets and primary ubiquitylation sites, quantitative diGLY capture proteomics following mitochondrial depolarization was performed73–75. Ubiquitylation sites were found on the cytoplasmic domain of numerous MOM proteins and on several cytoplasmic proteins that are recruited to mitochondria in response to parkin activation (see below).
Ubiquitylation on mitochondria in HeLa cells overexpressing parkin occurs in two phases: an initial phase (in the first two hours) during which the primary substrates are cytosolic domains of MOM proteins, followed by a second phase during which a cohort of proteins that were localized inside mitochondria become targeted for ubiquitylation75. Mitochondrial depolarization in the presence of overexpressed parkin can lead to rupture of the MOM, thereby potentially exposing inner membrane proteins to the action of parkin or other E3s70,76. However, it is unclear whether MOM rupture occurs at endogenous parkin levels in neurons and whether it has a specific role in mitophagy.
The diversity of parkin substrates on the MOM and the absence of an obvious substrate recognition element within parkin suggest that parkin lacks inherent substrate specificity73. Thus, it is possible that the iden- tity of substrates on the MOM is less important than the density of ubiquitin chains that are assembled on these substrates for specifying mitophagy. Indeed, as described below, mitophagy receptors have the ability to bind to particular types of ubiquitin chains on mitochondria. Ubiquitin chains can be assembled through the ε-amino group in each of the seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys63) on ubiquitin as well as through the α-amino group of its N-terminal methionine residue, and the chains can be either linear, branched or mixed. The different types of chain linkages can be distinguished by specific ubiquitin binding domains (UBDs), such as those found in mitophagy receptors77. We now know that in cells overexpressing parkin in the context of endogenous ubiquitin, mitochondrial depolarization leads to the formation of Lys6, Lys11, Lys48 and Lys63 chain linkages on the MOM, and parkin can catalyse the formation of these same linkages in vitro, but it is not known how such chains are distributed across different mitochondrial substrates, how long chains built on each type of substrate are or the extent to which there might be chains with mixed or branched chain topologies47,78. While poly-ubiquitylation, as opposed to

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mono-ubiquitylation, is presumed to be crucial for the recruitment of mitophagy receptors, it is also possible that mono-ubiquitylation plays an important role by serving as a target for phosphorylation by PINK1.
A model for parkin activation on mitochondria The mechanisms of ubiquitin and parkin phosphorylation described above have led to a model whereby mitochondrial damage promotes rapid ubiquitin chain polymerization as a result of two mechanisms acting in parallel that, together, generate a positive feedback loop43,47,51–53,55,57,63 (FIG. 3).
On one hand, the accumulation of pSer65-Ub on mitochondria as a result of PINK1 phosphorylating pre-existing ubiquitin molecules, or chains that are built by parkin, promotes the recruitment of cytosolic unphosphorylated parkin through direct inter action with pSer65-Ub48,55,63 (FIG. 3Ba). As many as 20% of ubiqui tin molecules on damaged mitochondria in HeLa cells are phosphorylated in a PINK1-dependent manner upon mitochondrial depolarization in the presence of catalytically active parkin47,57. The parkin–pSer65-Ub interaction has two major consequences: it partially activates the ubiquitin ligase activity of parkin by ~1,000-fold47,51–53, thereby contributing to ubiquitin chain assembly on the MOM57; and it greatly increases the rate at which PINK1 phosphorylates the parkin UBL domain, as was shown in vitro48. Similar results were obtained using a fluorescent ubiquitin probe, which also revealed that addition of mitochondrial
Rho GTPase (MIRO) as a parkin substrate can further increase rates of ubiquitin transfer and chain poly- meriza tion79. The binding of pSer65-Ub to pSer65- parkin is ~20-fold stronger than to unphosphoryl ated par kin, thus favouring the retention of fully active pSer65- parkin on damaged MOMs45,47,48,55,63. Moreover, because pSer65-parkin bound to pSer65-Ub is opti- mally active (~4,400-fold activation)57, its retention on the MOM promotes further ubiquitin chain assembly and provides additional ubiquitin molecules for phosphorylation by PINK1, creating the feedforward mechanism (FIG. 3).
On the other hand, parkin can be directly phosphorylated and activated by PINK1 on the MOM (independently of its initial encounter with pSer65-Ub) to locally generate ubiquitin chains (FIG. 3Bb) that become substrates for PINK1 to recruit more pSer65-parkin to the MOM, thereby serving as an initial amplification step47. The importance of ubiquitin chain phosphorylation in the feedforward process is highlighted by the observation that cells expressing a mutant form of ubiquitin (ubiquitin-Ser65Ala) that cannot be phosphorylated display decreased ubiquitin chain synthesis on MOM proteins, dramatically reduced recruitment of parkin to the MOM and reduced rates of mitophagy57. Ubiquitin conjugation is further reduced when parkin is mutated in its PINK1 phosphorylation site (parkin- Ser65Ala), which is consistent with the finding that the most active form of parkin is phosphorylated on its UBL domain and bound to pSer65-Ub57. The importance of ubiquitin phosphorylation for parkin recruitment was also shown by the ability of overexpressed Ser65 phosphomimetic linear tetrameric ubiquitin chains to promote parkin recruitment in the absence of PINK1 (REF. 55).
The relative importance of the two mechanisms that contribute to the feedforward loop is unclear. Parkin mutants that cannot bind pSer65-Ub failed to be recruited to mitochondria and promote ubiquitin chain assembly despite retaining catalytic activity when activ ated by UBL domain phosphorylation43,48,63. This finding suggests that recruitment and full parkin activation requires phosphorylation of pre- existing ubiquitin on mitochondria by PINK1 (FIG. 3). However, the catalytically defective parkin-Cys431Ser mutant is not detectably recruited to depolarized mito chon- dria39,47,80,81, suggesting that ubiquitin chain synthesis by parkin is necessary for sufficient pSer65-Ub to accumulate on mitochondria and recruit parkin to detectable levels, at least in the HeLa cell model system that was used. While it is clear that binding of parkin to pSer65-Ub accelerates PINK1-dependent phosphorylation of its UBL domain48,63, a parkin- His302Ala mutant that binds ~270-fold more weakly to pSer65-Ub can still be phosphorylated on its UBL domain in cells upon mitochondrial depolarization63,82. Moreover, several parkin mutants that were isolated from patients with Parkinson disease and are not stably recruited to depolarized mitochondria can nevertheless be phosphorylated by PINK1 to the same extent as wild-type parkin, indicating that PINK1-dependent
Box 2 | Parkin Cys431 reactivity as a tool for monitoring activation status
The E2 conjugating enzyme UBCH7 functions together with the RING1 domain of the E3 ubiquitin-protein ligase parkin to discharge ubiquitin onto the catalytic Cys431 residue. As such, the ability of Cys431 to form a thioester bond with ubiquitin, as well as formation of the more stable oxyester in the context of the parkin-Cys431Ser mutant, has been a useful tool for monitoring conformational changes that alleviate parkin autoinhibition. Indeed, phosphorylation of parkin on Ser65 is sufficient to allow discharge of ubiquitin from UBCH7~ubiquitin (where ~ indicates a thioester bond) to the catalytic residue and greatly increases modification of Cys431 in parkin in vitro by ubiquitin-vinyl sulfone, a reactive catalytic site probe47,62,81. Thus, parkin phosphorylation seems to render RING1 more accessible to charged E2s and the catalytic cysteine in RING2 more accessible to activated forms of ubiquitin. Interestingly, binding of pSer65-Ub to unphosphorylated parkin does not promote reactivity of Cys431 towards ubiquitin-vinyl sulfone in vitro, suggesting that this interaction alone is not sufficient to fully release autoinhibition of the RING2 domain and Cys431 (REFS 47,82). Similarly, parkin phosphorylation and binding to pSer65-Ub is required to facilitate optimal reactivity towards an activity-based ubiquitin~E2 transthiolation probe, and pSer65-Ub alone is only weakly supportive of reactivity with the ubiquitin~E2 probe82, consistent with the idea that pSer65-Ub binding is only partially able to activate unphosphorylated parkin57. Parkin-Cys431Ser has also been used to scan for defects in activation in cells in the context of a large set of parkin mutations in patients with Parkinson disease, revealing defects in activation for most patient mutations upon mitochondrial damage86. These defects largely correlate with the efficiency of recruitment of the parkin mutant to damaged mitochondria86. Importantly, phospho-mimetic mutants such as parkin-Ser65Glu and ubiquitin-Ser65Glu are poor mimics of activation54,57. While genetic studies in Drosophila melanogaster have suggested that parkin-Ser65Glu can rescue mutant mitochondrial phenotypes of PTEN-induced putative kinase 1 (PINK1)83, the underlying mechanisms are unclear at present. Moreover, ubiquitin-Ser65Glu is a poor mimic of pSer65-Ub in terms of parkin activation in vitro54,57, despite the fact that these mutants are extensively used in overexpression experiments to examine the role of phospho-ubiquitin.
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Piecemeal mitophagy A process through which subdomains of mitochondria harbouring misfolded matrix proteins are separated from the areas of mitochondria that are healthy before engulfment by autophagy.
phosphorylation can occur without stable association with mitochondria46,47,63. Considering all these data, the simplest model is that very small amounts of pSer65-Ub — generated either from ubiquitin already present on mitochondria before damage or from ubiquitin chains synthesized locally by pSer65-parkin — are necessary to initiate the feedforward mech anism but are not sufficient for full parkin activation. Given the mechanistic basis of the feedforward process, parkin or PINK1 overexpression could contribute to artificial activation of the pathway. This appears to be the case in experiments using parkin-Ser65Ala, as independent studies reported different levels of mitochondrial recruitment, possibly reflecting dif- ferences in expression levels43,50,52,53,57,83. Since parkin- Ser65Ala can bind to and be activated by pSer65-Ub in vitro47,51, high levels of parkin-Ser65Ala expression together with low levels of pSer65-Ub could promote a feedforward reaction artificially. Given the fundamental role of mitochondrial ubiquitin chains in promoting PINK1-dependent parkin activation, it is not
surprising that ubiquitin chain disassembly via DUBs could reduce available mitochondrial ubiquitin for initiation of the feedforward process36 (BOX 3). Recent studies have identified a role for the E3 ubiquitin- protein ligase HUWE1 in making Lys6 ubiquitin chains on mitochondria, and these types of chains on TOMM20 are removed by the mitochondrial DUB USP30 (REF. 84) (BOX 3). Thus, HUWE1 could control the level of basal ubiquitin on mitochondria that may participate in parkin activation (FIG. 3Ba).
A key question going forward concerns how mitophagy is controlled spatially. Insights into this question have come from a recent study85 that revealed ‘sub domains’ of mitochondria harbouring misfolded matrix proteins initiate piecemeal mitophagy via PINK1 activ ation and parkin recruitment to the damaged sub- domain. Interestingly, blocking mitochondrial fission in this context increased mitophagy while decreasing selectivity for damaged ‘domains’, suggesting a new model whereby fission protects healthy mitochondrial regions from unchecked PINK1–parkin activity85.
Figure 3 | Feedforward mechanism of parkin activation in response to mitochondrial depolarization. In healthy mitochondria, PTEN-induced putative kinase 1 (PINK1) is imported and rapidly degraded in a presenilins-associated rhomboid-like protein, mitochondrial (PARL)-dependent manner. When mitochondria are depolarized, PINK1 is stabilized on the translocase of the outer membrane (TOM) complex (step A), where it can access its substrates — ubiquitin chains near the TOM complex (step Ba) or the E3 ubiquitin-protein ligase parkin, which may come in contact with PINK1 through a diffusion-limited mechanism (step Bb). Phosphorylation of ubiquitin by PINK1 creates a binding site for parkin (step C), which can then increase parkin chain assembly activity by ~1,000-fold and facilitate phosphorylation of Ser65 on the ubiquitin-like domain of parkin by PINK1 (step Da). Direct phosphorylation of parkin by PINK1 leads to local activation of its E3 activity (~2,400-fold), providing additional ubiquitin for phosphorylation by PINK1 (steps Db–F). Parkin that is phosphorylated on Ser65 and is associated with Ser65-phosphorylated ubiquitin (pSer65-Ub) is maximally active in ubiquitin chain assembly (~4,400-fold) (step E). The combination of parkin activation, recruitment to pSer65-Ub chains, further ubiquitin chain synthesis and further PINK1-dependent phosphorylation of ubiquitin constitutes the feedforward mechanism (step F). DUB, deubiquitinating enzyme; MIM, mitochondrial inner membrane; MOM, mitochondrial outer membrane; USP30, ubiquitin carboxyl-terminal hydrolase 30.
MOM MIM
Local ubiquitylation of substrates and phosphorylation of ubiquitin by PINK1
Feedforward amplification of ubiquitin chain synthesis
Parkin makes Lys6, Lys11, Lys48 and Lys63 chains, and stochiometry of phosphorylation of ubiquitin on mitochondria can reach ~20%
Diffusion-limited encounter
Some parkin phosphorylation before significant ubiquitin chain assembly (Bb)
Further parkin recruitment and phosphorylation of both parkin and ubiquitin by PINK1
A
C
Phosphorylation of local ubiquitin by PINK1 recruits inactive parkin (Ba)
Opposing DUB (USP30) activity to reverse ubiquitin tagging of mitochondria
PINK1
Activation of parkin by ~1,000-fold (Da) or ~2,400-fold (Db)
Parkin phosphorylated and bound to pSer65-Ub: further activation (now ~4,400-fold) and retention on the MOM
Parkin binds to single phosphorylated ubiquitin molecules (Kd ~17 nM) and to phosphorylated ubiquitin chains
Phosphorylation Ubiquitylation
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Mutations in patients with Parkinson disease Numerous mutations have been identified in PRKN genes through sequencing of patient genomes, spanning all domains in the protein6. Functional analysis of such parkin mutant proteins provides important insights into the defective cellular mechanisms that underlie this form of Parkinson disease, and results are consistent with the hypothesis that multiple structural elements contribute to parkin activity37–39,48,86.
Perhaps the most distinguishing characteristic of parkin mutants is that most are defective in their recruitment to mitochondria. Given that parkin-dependent ubiquitin chain assembly is necessary for stable parkin recruitment to mitochondria, the inability of individual parkin mutants to be recruited could be caused by the absence of catalytic activity or the inability to be retained on the MOM, for example, through defects in binding to pSer65-Ub. For example, parkin-Lys161Asn and parkin-Lys211Asn mutants are strongly defective in intrinsic activation by phosphorylation on Ser65, pSer65-Ub-dependent activation and recruitment to damaged mitochondria despite being capable of binding to Lys63-linked pSer65-Ub chains in vitro47. Thus, these and other parkin mutants identified in patients may be primarily defective in steps that support the feedforward mechanism.
A major question in this medical research field is whether it is possible to design small molecules that target a mutant parkin protein and reinstate its catalytic activity by locking it in an active conformation. The finding that parkin undergoes multiple conformational
changes during activation (FIG. 2) provides several oppor- tunities for identifying small molecules that bind and stabil ize one or more active forms. It is also possible that small molecules that stabilize active forms of wild-type parkin downstream of PINK1 could facilitate the removal of damaged mitochondria in forms of Parkinson disease that are unlinked genetically to parkin and PINK1.
Decoding ubiquitin chains for mitophagy The assembly of ubiquitin chains by parkin on damaged mitochondria initiates the process of decoding by ubiquitin-chain-binding autophagy receptors (FIG. 4). These receptors, which include SQSTM1, next to BRCA1 gene 1 protein (NBR1), OPTN, calcium-binding and coiled-coil domain-containing protein 2 (CALCOCO2; also known as NDP52) and Tax1-binding protein 1 (TAX1BP1), contain a C-terminal UBD and a short hydrophobic sequence (known as an LC3-interacting region (LIR)) (FIG. 4a) that can bind to ATG8 proteins to potentially promote the recruitment of autophagosomal membranes via a canonical autophagy mechanism87 (FIGS 1,4b).
SQSTM1 is recruited to depolarized mitochondria in a parkin-dependent manner72, but it is not required for mitophagy in most cell lines examined thus far33,34,71. SQSTM1 is instead required for mitochondrial cluster- ing71. HeLa cells lacking OPTN, CALCOCO2 and TAX1BP1, but still expressing SQSTM1 and NBR1, are defective in mitophagy, with the most prominent defects shown in cells lacking OPTN32–35. These data suggest some level of functional redundancy between receptors, with the relative contributions of individual
Box 3 | Factors acting in opposition of ubiquitin chain assembly
Chain synthesis by ubiquitin ligases is often negatively regulated by deubiquitinating enzymes (DUBs) (see also FIG. 3). Several studies have examined the role of DUBs in parkin-dependent mitochondrial ubiquitylation and mitophagy36,47,54,74,78,130–134. Ubiquitin carboxyl-terminal hydrolase 30 (USP30), which is tethered to the mitochondrial outer membrane (MOM) via a single transmembrane domain, is perhaps the best understood DUB antagonizing parkin activity, although USP8 and USP15 have also been implicated36. Alteration in USP30 levels has a profound effect on PTEN-induced putative kinase 1 (PINK1)– parkin-dependent mitophagy in multiple systems. First, elevated USP30 expression blocks ubiquitin chain assembly by parkin on the MOM47,74, thereby reducing parkin recruitment74,132, while depletion of USP30 from postmitotic neurons increased rates of mitophagy74, consistent with USP30 actively antagonizing parkin function. Second, depletion of USP30 in neuronal cultures reduced basal mitochondrial oxidative stress74. Third, reduced USP30 expression substantially improved behavioural phenotypes in pink and park mutant Drosophila melanogaster, which is thought to be related to mitochondrial function74. Finally, depletion of USP30 specifically in dopaminergic neurons in D. melanogaster partially rescued paraquat-induced behavioural phenotypes thought to model mitochondrial damage74. Mechanistically, USP30 can remove ubiquitin chains from numerous parkin substrates on the MOM but, interestingly, is also a target of parkin-dependent mono-ubiquitylation, leading to proteasomal degradation of USP30 (REFS 74,133). Thus, degradation of USP30 may help enforce the feedforward mechanism for parkin function by eliminating the negative regulator in the system (FIG. 3). Indeed, the antagonistic relationship between parkin and USP30 suggests that the balance between the activities of these two enzymes (ubiquitin chain synthesis and ubiquitin chain disassembly) sets a threshold for mitophagic
flux that is dictated by the activity of PINK1 on the mitochondria, thereby controlling which mitochondria are detected as damaged. Although the precise activities differ, multiple studies78,133,134 indicate a preference of USP30 in vitro for Lys6 linkages over other chain types, although other chain types can also be hydrolysed. This selectivity can be explained by unique interactions revealed in the structure of USP30 bound to Lys6- di-ubiquitin133,134. Interestingly, ubiquitin phosphorylation within chains, but particularly when phosphorylated on the distal ubiquitin within a tetrameric chain, also reduces activity133,134. This result, along with the finding that PINK1 displays a kinetic preference for phosphorylation of distal ubiquitin moieties in Lys6 tetramers but less so in other chain types, suggests that PINK1 could ‘end protect’ Lys6 ubiquitin chains from disassembly by USP30. Consistent with this, depletion of USP30 specifically increased the abundance of Lys6 chains on mitochondrial import receptor subunit TOM20 homologue (TOMM20) but did not increase Lys6 chains on several other parkin targets133. Distinct models have emerged for other DUBs36. USP8 has been proposed to remove Lys6 ubiquitin chains from parkin itself to alter its ability to be recruited to mitochondria in cancer cell lines131. By contrast, USP15 overexpression does not block parkin recruitment to the MOM but does block MOM ubiquitylation, while its depletion in fibroblasts from patients with Parkinson disease induces mitophagy130. However, USP15 is thought to participate in mRNA splicing135, and it is currently unclear if there is a contribution of altered gene expression to the phenotypes observed, rather than a direct effect on mitochondrial ubiquitylation. Further studies are necessary to understand how these DUBs as well as a second mitochondrial DUB, USP35 (REF. 132), are linked with parkin function. Importantly, USP30 is a candidate target for small molecules that will increase mitophagy in patients with decreased parkin or PINK1 function36.
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receptors probably reflecting their relative abundance in individual cell types rather than intrinsically distinct activities33. Indeed, expression of several receptors is tissue-specific, which has implications for disease, as t hey may function in and affect only specific cell lineages33.
For example, there is clear evidence of an essential role for SQSTM1 in parkin-dependent mitophagy in mouse macrophages in vivo88 and in mouse embryonic fibroblasts undergoing mitophagy as a result of high levels of oxidative phosphorylation89.
Figure 4 | Principles of mitophagy receptor recruitment and activation. a | Domain structures of the major ubiquitin (Ub)-binding autophagy receptors in mammals. b | Scheme depicting how ubiquitin-binding autophagy receptors may function to recruit phagophores through ATG8-dependent (canonical) or independent (non-canonical) mechanisms. ‘?’ represents a hypothetical protein involved in non-canonical phagophore recruitment. c,d | Models for phosphorylation- dependent regulation of optineurin (OPTN)–ubiquitin binding. e | Mechanism of feedforward phosphorylation upon binding of OPTN to ubiquitin chains on damaged mitochondria. Cytosolic OPTN–serine/threonine-protein kinase TBK1 complexes are recruited to ubiquitin chains in response to mitochondrial activity of the E3 ubiquitin-protein ligase parkin and PTEN-induced putative kinase 1 (PINK1). Engagement of ubiquitin chains by the UBAN (ubiquitin binding domain in ABINs and NEMO) domain of OPTN leads to TBK1 phosphorylation on Ser172 by an unknown process. TBK1 activation promotes phosphorylation of OPTN on its LC3-interacting region (LIR) and UBAN domains, which increases both association of OPTN with ATG8 and binding of OPTN to ubiquitin chains. The feedforward process promotes accumulation of OPTN–TBK1 on ubiquitin chains on mitochondria. Note that the length of ubiquitin chains on individual substrates is unknown. AR, autophagy receptor; CALCOCO2, calcium-binding and coiled-coil domain-containing protein 2; CC, coiled-coil domain; PB, Phox and Bem1p domain; SKICH, skeletal muscle and kidney-enriched inositol phosphatase carboxyl homology domain; SQSTM1, sequestosome 1; TAX1BP1, Tax1-binding protein 1; UBA, ubiquitin-associated domain; UBZ, ubiquitin binding zinc-finger domain; UFD, ubiquitin fold domain; ZnF, zinc-finger domain.
PINK1-mediated and parkin- mediated ubiquitylation on mitochondrial outer membrane proteins
Kinase X
Phagophore membrane
Phagophore membrane
d Reduced OPTN binding to phosphorylated ubiquitin chains in vitro
b Canonical autophagy Non-canonical autophagy
LIR
Substrate
a Autophagy receptors
–
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Xenophagy The process by which intracellular bacteria are targeted for autophagy.
A major focus of research is on understanding which types of ubiquitin chains are recognized by autophagy receptors. Replacement of Lys6 or Lys63 in ubiquitin with arginine, which cannot be conjugated to ubi quitin, reduced mitophagy rates compared with Lys11 replacement, which led to only a minor reduction57. Overexpression of Lys6Arg or Lys48Arg and Lys63Arg ubiquitin mutants also inhibited mitophagy78. Moreover, overexpression of ubiquitin-Lys11Arg reduced mitophagy to the greatest extent among the mutants tested (~50% reduction)57. The reason for this discrepancy is unknown but might reflect indirect effects of ubiquitin- Lys11Arg on ubiquitin chain synthesis by parkin, a hypothesis that has not been examined. In vitro, SQSTM1, OPTN and CALCOCO2 bind more efficiently to Lys63 chains than to Lys48 chains34,57,90, consistent with a role for Lys63 chains in mitophagy, although the other chain types have not been tested systematically.
The finding that ~20% of ubiquitin molecules on mitochondria are phosphorylated upon depolarization in HeLa cells overexpressing parkin47,57 raises the question of whether this modification plays a direct role in the recruitment of mitophagy receptors. Indeed, overexpression of PINK1 artificially targeted to mitochondria promotes OPTN recruitment and mitophagy in the absence of parkin, albeit with an extensive delay and lower efficiency compared with when parkin activity is present33. Although these observations have led to the conclusion that pSer65-Ub is the receptor for OPTN and other autophagy receptors, other data suggest that unphosphorylated forms of ubiquitin conjugates on mitochondria function to recruit autophagy receptors (FIG. 4c,d). In particular, in vitro experiments indicate that OPTN, CALCOCO2 and SQSTM1 bind efficiently to unphosphorylated Lys63 (but not Lys48) ubiquitin chains, and phosphorylation of these chains on Ser65 (with a stoichio metry of ~0.7) largely abolishes direct interactions between autophagy receptors and ubiquitin chains34,47,57.
These findings are inconsistent with pSer65-Ub being directly involved in the decoding of ubiquitin conjugates to promote mitophagy. Moreover, quantitative proteomic experiments demonstrated recruitment of endogenous OPTN, SQSTM1, TAX1BP1 and CALCOCO2 to damaged mitochondria in the presence of wild-type PINK1 and parkin, but recruitment was absent in PINK1−/− HeLa cells or in cells expressing parkin-Ser65Ala (which failed to build ubiquitin chains on mitochondria but expressed active PINK1)75. Given that PINK1 should phosphorylate ubiquitin molecules that are present on the mitochondrial surface to promote OPTN recruitment, these data suggest that endogenous levels of PINK1 are not sufficient for receptor recruitment in the absence of ubiquitin chain assembly by parkin. Moreover, imaging studies have shown that pSer65-Ub signals uniformly cover damaged mitochondria, whereas mitophagy receptors are recruited to highly focal puncta that cover only a small part of the surface area of mitochondria34, indicating that pSer65-Ub conjugated to mitochondria is not sufficient to directly recruit autophagy receptors and that additional signals are necessary to direct receptors to these puncta.
Further studies are required to identify the signals that enable mitophagy receptors to decode ubiquitin chains. Based on structural data, a minimum of two ubiquitin molecules in a Lys63 chain are necessary for binding to the ubiquitin binding domain in ABINs and NEMO (UBAN) modules as found in OPTN91,92. How the ubiquitin chain length is optimized to provide sufficient sites for parkin recruitment to pSer65-Ub and sufficient conjugates of two or more ubiquitin molecules to support autophagy receptor recruitment is unknown.
TBK1 promotes mitophagy A common feature of autophagy receptors is their ability to interact with the kinase TBK1. Early studies examining Salmonella enterica turnover by xenophagy demonstrated that TBK1 can phosphorylate serine residues adjacent to the LIR motif in OPTN and that this phosphorylation promotes binding of OPTN to ATG8 proteins to increase xenophagy93. It is now known that TBK1 is required for multiple types of selective autophagy that rely on ubiquitin- binding autophagy receptors34,94–97 and it can phosphorylate OPTN, TAX1BP1, CALCOCO2 and SQSTM1 when overexpressed35.
The best understood role for TBK1 is in regulating OPTN during mitophagy and xenophagy. TBK1 phosphoryl ates residues within and adjacent to the UBAN motif in OPTN to increase its affinity for unphosphoryl ated Met1, Lys48 and Lys63 chains34,35,97 (FIG. 4c–e). Inter estingly, a positive feedback mechanism controls TBK1-dependent phosphorylation of OPTN (FIG. 4e). The ability of TBK1 to phosphorylate OPTN in response to mitochondrial depolarization depends on OPTN binding to ubiquitin chains (FIG. 4c,e). Inhibition of TBK1 activity with small molecule inhibitors or dele- tion of TBK1 reduces OPTN recruitment to damaged mitochondria, indicating that TBK1 activity is needed for OPTN binding to ubi quitin chains34,35. Importantly, mutation of serine residues in OPTN phosphorylated by TBK1 reduces its associ ation with damaged mitochondria and TBK1-dependent activation, which delays mitophagy. Reciprocally, OPTN binding to ubiquitin chains is required for TBK1 phos phorylation on Ser172, which activates its kinase activity34,35. These findings have revealed another crucial feedforward loop that is required for the efficient clearance of defective mitochondria through mitophagy (FIG. 4e).
Several questions remain to be addressed to fully understand this pathway. First, by what mechanism is TBK1 activated in response to the binding of OPTN to ubiquitin chains on mitochondria? Small molecule inhib itors of TBK1 that prevent trans-autoactivation fail to block its activation via phosphorylation on Ser172, suggesting that one or more additional kinases may be involved34. Second, it is unclear whether phosphoryl ation of the autophagy receptors CALCOCO2 or TAX1BP1 by TBK1 (REF. 35) also increases their affinity for ubi quitin chains. Phosphorylation on the UBDs of OPTN and SQSTM1 increases their affinity for ubiquitin chains34,35,98, suggesting that similar mechanisms regulate other receptors. Third, it is not clear whether TBK1 has other functions in addition to receptor phosphorylation. In this
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regard, it has been shown that fusion of TBK1 lacking its C-terminal autophagy receptor binding domain to UBDs from OPTN and other proteins can rescue the clearing of Salmonella enterica in TBK1-null cells95. However, the role of TBK1 kinase activity in the context of Salmonella- infected cells remains unknown and could involve phosphorylation of proteins other than ubiquitin-binding cargo receptors.
Finally, TBK1 and OPTN, but not parkin or PINK1, can be mutated in patients with ALS and frontal temporal dementia17–19, and these mutations affect the TBK1–OPTN interaction18,97. This suggests that forms of TBK1–OPTN-driven selective autophagy, possibly involving cargo other than damaged mitochondria, may be important for the health of motor neurons and possibly other neuronal cell types19.
Evolving roles of ATG8 proteins in mitophagy Canonical models for mitophagy posit that recruitment of autophagy receptors to ubiquitylated mitochondria leads to the recruitment of autophagosomal membrane precursors to the surface of mitochondria and subsequent engulfment of the damaged mitochondria by the auto- phagosome6,19 (FIGS 1,4b). This recruitment is expected to occur through association of the LIR elements in OPTN or CALCOCO2 with ATG8 proteins within the growing autophagosome93,99. However, the canonical model for ATG8 receptor recognition does not explain recent studies reporting that human cells lacking all six ATG8 proteins are still capable of building autophagosomes around damaged mitochondria100. Thus, the canonical association of LIR sequences in autophagy receptors with ATG8 proteins does not seem to be required for this form of selective autophagy (FIG. 4b). Moreover, cells lacking the ATG8 conjugation system still support signifi cant (~30%) flux for starvation-induced bulk autophagy, with a decreased frequency of autophagosomal closure and decreased rates of autophagosomal inner membrane breakdown upon fusion with lysosomes101,102. Thus, capture of some types of autophagic cargo may occur independently of ATG8–LIR interactions. It is possible that such ATG8-conjugation-independent forms of what is otherwise considered to be selective autophagy involve interaction of distinct sequences in either the autophagy receptor or associated TBK1 with the autophagosomal machinery (FIG. 4b). Nevertheless, ATG8 proteins and the ATG8 lipidation machinery are required for mitophagic flux, and their absence correlates with defects in fusion of lysosomes with autophagosomes100,103.
Recent studies have proposed a role for prohibitin 2, a poorly understood mitochondrial inner membrane protein, as an autophagy receptor for mitochondria that functions by directly interacting with ATG8 pro- teins104. Given the proposed direct binding to ATG8, it is unclear whether prohibitin 2 plays a role in inducing mitophagy independently of the ubiquitin conjugation pathway. In addition, it is not clear why cells lacking OPTN, CALCOCO2 and TAX1BP1 fail to undergo mitophagy33–35 if prohibitin 2 is a direct autophagy receptor as proposed104. Further investigations are needed to understand when LIR–ATG8 interactions are required
and to understand any interplay between ubiquitin binding mitophagy receptors and prohibitin 2. In this regard, multiple prohibitin 2 ubiquitylation sites are detected in a parkin-dependent and PINK1-dependent manner and in a kinetically delayed manner relative to most primary parkin targets, consistent with ubiquitylation occurring after MOM rupture75.
Coupling parkin function to antigen presentation While much of the efforts to understand the role of parkin and PINK1 in disease have focused on the removal of mitochondria via mitophagy, parkin can also function to suppress the presentation of mitochondria-derived anti- gen105, suggesting a novel autoimmune mechanism for Parkinson disease in patients with mutations in parkin and PINK1.
In macrophages and dendritic cells as well as fibroblasts, parkin suppresses heat stress and lipopoly sac char ide (LPS)-dependent production of mito chondrial antigens by ubiquitin-dependent turnover of sorting nexin 9 (SNX9), a protein required for parkin- independent prod uction of mitochondrial-derived vesicles (MDVs)106. In the absence of parkin, MDVs transfer mitochondrial contents into endosomes, where peptides are ultimately presented on major histo compatibility complex class I molecules105. This results in targeting of T cells to antigen- presenting cells. These data suggest a non-cell autono- mous mechanism that could contribute to Parkinson disease, whereby cytotoxic T cell activity promotes the loss of dopaminergic neurons. Intriguingly, LPS induces selective loss of dopaminergic neurons in Prkn−/− mice107, raising the possibility that age-dependent neuroinflam- mation could underlie neuronal loss in humans105. Further explor ation of this system and the pathways by which parkin selectively degrades SNX9 to promote its proteasomal turnover to block production of MDVs might provide a new paradigm in parkin function.
Conclusions and future questions Parkin-dependent mitophagy has provided a paradigm for understanding the molecular mechanisms that couple ubiquitin chain synthesis to recruitment of autophagy receptors that are required to induce mitophagy and possibly other types of organellar autophagy.
Parkin is unique in that it is the only ubiquitin ligase known to be activated by binding to pSer65-Ub. It is intriguing to consider that parkin may have evolved to function only in the presence of pSer65-Ub. PINK1 is the only known Ser65-ubiquitin kinase, although pSer65-Ub is also seen in budding yeast108, which lacks an obvious PINK1 orthologue. Therefore, mitophagy is the only signalling system that has been discovered for parkin activation. A plethora of candidate parkin substrates were reported before the discovery that parkin is activated by PINK1 and pSer65-Ub109–115, but virtually none of these studies examined what we now understand to be the active form of parkin. It therefore remains unclear whether there are alternative protein kinases that can phosphorylate parkin and/or ubiquitin in the context of other signalling pathways that parkin has been implicated in. Interestingly, pSer65-Ub can be detected at very low
Starvation-induced bulk autophagy The process by which nutrient deprivation leads to engulfment of cytosolic contents in autophagosomes followed by delivery to lysosomes.
Dendritic cells Antigen-presenting immune cells that activate T cells.
T cells Lymphocytes that function in cell-mediated immunity and contain the T cell receptor on their cell surface.
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levels in PINK1-null cells47, suggesting the existence of additional Ser65-ubiquitin kinases. Moreover, parkin is required for xenophagic removal of M. tuberculosis from mammalian cells, but this function is apparently independent of PINK1, suggesting that parkin activation for this form of xenophagy may involve a distinct kinase signalling pathway26. Exploration of other signals that promote pSer65-Ub may yield new insights into signalling pathways regulated by parkin.
Our understanding of pSer65-Ub in animals is limited. In brain tissue of mice lacking parkin, pSer65-Ub is increased upon mutations in POLG, which encodes the mitochondrial DNA polymerase gamma, catalytic subunit, which leads to mitochondrial stress as a result of defects in respiratory chain assembly116. Moreover, pSer65-Ub is detectable in human brain using specific antibodies, and the signal increases with age and disease in limited patient samples but is absent when PINK1 is mutated117. Thus, much work still needs to be done to understand pSer65-Ub pathways in normal and patho- genic conditions. Signals that activate PINK1 to promote parkin function in both embryos and haematopoietic stem cells remain to be identified (BOX 1).
Our understanding of the activation mechanism for parkin and the role for pSer65-Ub has advanced rapidly since the discovery of pSer65-Ub in 2014, but several questions remain unresolved. We do not have a clear understanding of how pSer65-Ub and parkin phosphorylation modify parkin structure to fully activ ate its chain assembly function. The current structures48,64 with pSer65-Ub still have some elements of auto inhibition in place that are presumably removed upon full activation. An additional question concerns how parkin initially encounters PINK1. Parkin does not seem to form a stable complex with activated PINK1 on the TOM complex isolated from depolarized cells31, suggesting that if initial activ ation of parkin occurs in the context of a PINK1–TOM complex, this interaction is transient. Structural analysis of PINK1– parkin complexes may shed light on this question. More- over, it is unclear whether speci fic protein phosphatases act on either pSer65-parkin or pSer65-Ub, thereby potentially providing a second threshold to overcome in addition to that imposed by DUBs (BOX 3).
Because most studies in the pathway have employed cells with overexpressed parkin or PINK1, which can affect the amplitude and persistence of the feedforward system, we do not fully understand the temporal order of
individual steps in the pathway and the relative importance of PINK1-dependent phosphorylation of ubiquitin present on mitochondria before damage versus direct parkin activation, for example, in postmitotic neurons. It is possible that the levels of pre-existing ubiquitin on the MOM are cell-type dependent or are regulated by distinct mitochondrial E3s linked with mitochondrial dynamics, such as mitochondrial ubiquitin ligase activator of NFKB 1 (MUL1) or the E3 ubiquitin-protein ligase MARCH5 (REFS 89,118), thereby facilitating the feed forward initiation mechanism directly. Alternatively, USP30 or other DUBs may act to control the abundance of pre- existing ubiquitin on the MOM (BOX 3). Understanding the biochemical steps necessary for parkin activation may facilitate the identification of molecules that can promote parkin activation in the context of disease alleles in parkin itself or PINK1.
Finally, only recently has the link between parkin and mitochondrial antigen presentation been made. This link provides a completely novel regulatory function for parkin that may provide key insights into the fate of neurons in patients with Parkinson disease with mutations in PINK1 or parkin through a potential autoimmune mechanism. According to current models105,106, when under stress, antigen-presenting cells harbouring mutations in parkin produce MDVs that allow presentation of mitochondrial-derived antigens on the cell surface and subsequent T cell activation. Neurons that present mitochondrial- derived antigens on their surface could be subsequently recognized by mitochondrial antigen-specific T cells, thereby triggering a cytotoxic response ultimately leading to neuronal death through an autoimmune-type mechanism. Major questions concern how signals downstream of stressors linked with mitochondrial antigen presentation are coupled to parkin and PINK1 activation, the extent to which parkin activation in antigen-presenting cells is uncoupled from the canonical mitophagy system, and how parkin selectively recog- nizes SNX9 for ubiquitylation to suppress mitochondrial antigen presentation. Furthermore, it is crucial to know the cell types in vivo in which the pathway is active and thereby might be targeted by cytotoxic T cells. The availability of mice expressing reporters of mitophagic flux119,120 will greatly facilitate a physiological understanding of spatial and temporal control of mitophagy and the genetic requirements for the PINK1– parkin system across a broad range of tissues.
1. Haynes, C. M., Petrova, K., Benedetti, C., Yang, Y. & Ron, D. ClpP mediates activation of a mitochondrial unfolded protein response in C. elegans. Dev. Cell 13, 467–480 (2007).
2. Munch, C. & Harper, J. W. Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation. Nature 534, 710–713 (2016).
3. Lin, Y. F. & Haynes, C. M. Metabolism and the UPRmt. Mol. Cell 61, 677–682 (2016).
4. Shpilka, T. & Haynes, C. M. The mitochondrial UPR: mechanisms, physiological functions and implications in ageing. Nat. Rev. Mol. Cell Biol. http://dx.doi.org/ 10.1038/nrm.2017.110 (2017).
5. Lin, Y. F. et al. Maintenance and propagation of a deleterious mitochondrial genome by the mitochondrial unfolded protein response. Nature 533, 416–419 (2016).
6. Pickrell, A. M. & Youle, R. J. The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson’s disease. Neuron 85, 257–273 (2015).
7. Yamano, K., Matsuda, N. & Tanaka, K. The ubiquitin signal and autophagy: an orchestrated dance leading to mitochondrial degradation. EMBO Rep. 17, 300–316 (2016).
8. De Duve, C. & Wattiaux, R. Functions of lysosomes. Annu. Rev. Physiol. 28, 435–492 (1966).
9. Kitada, T. et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392, 605–608 (1998).
10. Narendra, D. P. et al. PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS Biol. 8, e1000298 (2010).
11. Valente, E. M. et al. Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science 304, 1158–1160 (2004).
12. Walden, H. & Muqit, M. M. Ubiquitin and Parkinson’s disease through the looking glass of genetics. Biochem. J. 474, 1439–1451 (2017).
13. Clark, I. E. et al. Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature 441, 1162–1166 (2006).
14. Park, J. et al. Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin. Nature 441, 1157–1161 (2006). References 13 and 14 provide the initial genetic evidence that PINK1 and parkin function in a common pathway to control mitochondrial function.
15. Narendra, D., Tanaka, A., Suen, D. F. & Youle, R. J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J. Cell Biol. 183, 795–803 (2008). This study provides a link between parkin recruitment to mitochondria and a role in mitophagy.
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16. Maruyama, H. et al. Mutations of optineurin in amyotrophic lateral sclerosis. Nature 465, 223–226 (2010).
17. Cirulli, E. T. et al. Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways. Science 347, 1436–1441 (2015).
18. Freischmidt, A. et al. Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia. Nat. Neurosci. 18, 631–636 (2015). References 17 and 18 report the identification of TBK1 mutations in patients with sporadic and familial ALS.
19. Weishaupt, J. H., Hyman, T. & Dikic, I. Common molecular pathways in amyotrophic lateral sclerosis and frontotemporal dementia. Trends Mol. Med. 22, 769–783 (2016).
20. Novak, I. et al. Nix is a selective autophagy receptor for mitochondrial clearance. EMBO Rep. 11, 45–51 (2010).
21. Chourasia, A. H. et al. Mitophagy defects arising from BNip3 loss promote mammary tumor progression to metastasis. EMBO Rep. 16, 1145–1163 (2015).
22. Li, W. et al. MicroRNA-137 is a novel hypoxia- responsive microRNA that inhibits mitophagy via regulation of two mitophagy receptors FUNDC1 and NIX. J. Biol. Chem. 289, 10691–10701 (2014).
23. Ito, K. et al. Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance. Science 354, 1156–1160 (2016).
24. Boland, M. L., Chourasia, A. H. & Macleod, K. F. Mitochondrial dysfunction in cancer. Front. Oncol. 3, 292 (2013).
25. Ney, P. A. Mitochondrial autophagy: origins, significance, and role of BNIP3 and NIX. Biochim. Biophys. Acta 1853, 2775–2783 (2015).
26. Manzanillo, P. S. et al. The ubiquitin ligase parkin mediates resistance to intracellular pathogens. Nature 501, 512–516 (2013).
27. Okatsu, K., Kimura, M., Oka, T., Tanaka, K. & Matsuda, N. Unconventional PINK1 localization to the outer membrane of depolarized mitochondria drives Parkin recruitment. J. Cell Sci. 128, 964–978 (2015).
28. Jin, S. M. et al. Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL. J. Cell Biol. 191, 933–942 (2010).
29. Greene, A. W. et al. Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment. EMBO Rep. 13, 378–385 (2012).
30. Yamano, K. & Youle, R. J. PINK1 is degraded through the N-end rule pathway. Autophagy 9, 1758–1769 (2013).
31. Lazarou, M., Jin, S. M., Kane, L. A. & Youle, R. J. Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin. Dev. Cell 22, 320–333 (2012).
32. Wong, Y. C. & Holzbaur, E. L. Optineurin is an autophagy receptor for damaged mitochondria in parkin-mediated mitophagy that is disrupted by an ALS-linked mutation. Proc. Natl Acad. Sci. USA 111, E4439–E4448 (2014).
33. Lazarou, M. et al. The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature 524, 309–314 (2015).
34. Heo, J. M., Ordureau, A., Paulo, J. A., Rinehart, J. & Harper, J. W. The PINK1-PARKIN mitochondrial ubiquitylation pathway drives a program of OPTN/ NDP52 recruitment and TBK1 activation to promote mitophagy. Mol. Cell 60, 7–20 (2015).
35. Richter, B. et al. Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria. Proc. Natl Acad. Sci. USA 113, 4039–4044 (2016). References 32–35 define the roles of ubiquitinbinding autophagy receptors in the capture of ubiquitylated mitochondria to promote mitophagy.
36. Bingol, B. & Sheng, M. Mechanisms of mitophagy: PINK1, Parkin, USP30 and beyond. Free Radic. Biol. Med. 100, 210–222 (2016).
37. Chaugule, V. K. et al. Autoregulation of Parkin activity through its ubiquitin-like domain. EMBO J. 30, 2853–2867 (2011).
38. Wauer, T. & Komander, D. Structure of the human Parkin ligase domain in an autoinhibited state. EMBO J. 32, 2099–2112 (2013).
39. Trempe, J. F. et al. Structure of parkin reveals mechanisms for ubiquitin ligase activation. Science 340, 1451–1455 (2013).
40. Riley, B. E. et al. Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases. Nat. Commun. 4, 1982 (2013). References 37–40 define the structure of autoinhibited parkin and define structural elements that impinge upon RING1 and RING2 functions.
41. Fajner, V., Maspero, E. & Polo, S. Targeting HECT-type E3 ligases — insights from catalysis, regulation and inhibitors. FEBS Lett. 591, 2636–2647 (2017).
42. Wenzel, D. M., Lissounov, A., Brzovic, P. S. & Klevit, R. E. UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids. Nature 474, 105–108 (2011).
43. Tang, M. Y. et al. Structure-guided mutagenesis reveals a hierarchical mechanism of Parkin activation. Nat. Commun. 8, 14697 (2017).
44. Sha, D., Chin, L. S. & Li, L. Phosphorylation of parkin by Parkinson disease-linked kinase PINK1 activates parkin E3 ligase function and NF-κB signaling. Hum. Mol. Genet. 19, 352–363 (2010).
45. Kondapalli, C. et al. PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65. Open Biol. 2, 120080 (2012).
46. Shiba-Fukushima, K. et al. PINK1-mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocation of Parkin and regulates mitophagy. Sci. Rep. 2, 1002 (2012). References 45 and 46 identify Ser65 in parkin as a target for PINK1.
47. Ordureau, A. et al. Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis. Mol. Cell 56, 360–375 (2014). This study describes a feedforward mechanism for parkin activation by PINK1 and pSer65Ub binding.
48. Wauer, T., Simicek, M., Schubert, A. & Komander, D. Mechanism of phospho-ubiquitin-induced PARKIN activation. Nature 524, 370–374 (2015). This study provides the first structural analysis of pSer65Ub bound to parkin and defines multiple mechanisms of parkin activation.
49. Aguirre, J. D., Dunkerley, K. M., Mercier, P. & Shaw, G. S. Structure of phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation. Proc. Natl Acad. Sci. USA 114, 298–303 (2017).
50. Caulfield, T. R. et al. Phosphorylation by PINK1 releases the UBL domain and initializes the conformational opening of the E3 ubiquitin ligase Parkin. PLoS Comput. Biol. 10, e1003935 (2014).
51. Kazlauskaite, A. et al. Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65. Biochem. J. 460, 127–139 (2014).
52. Koyano, F. et al. Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature 510, 162–166 (2014).
53. Kane, L. A. et al. PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity. J. Cell Biol. 205, 143–153 (2014).
54. Wauer, T. et al. Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis. EMBO J. 34, 307–325 (2015). References 47 and 51–54 identify Ser65 of ubiquitin as a substrate for PINK1 and demonstrate that pSer65Ub activates parkin.
55. Okatsu, K. et al. Phosphorylated ubiquitin chain is the genuine Parkin receptor. J. Cell Biol. 209, 111–128 (2015).
56. Sauve, V. et&nbs

building and decoding ubiquitin chains for mitophagy

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