ABSTRACT
Protein ubiquitination is a multi-functional post-translational modification that affects all cellular processes. Its versatility arises from architecturally complex polyubiquitin chains, in which individual ubiquitin moieties may be ubiquitinated on one or multiple residues, and/or modified by phosphorylation and acetylation1-3. Advances in mass spectrometry have enabled the mapping of individual ubiquitin modifications that generate the ubiquitin code; however, the architecture of polyubiquitin signals has remained largely inaccessible. Here we introduce Ub-clipping as a methodology by which to understand polyubiquitin signals and architectures. Ub-clipping uses an engineered viral protease, Lbpro∗, to incompletely remove ubiquitin from substrates and leave the signature C-terminal GlyGly dipeptide attached to the modified residue; this simplifies the direct assessment of protein ubiquitination on substrates and within polyubiquitin. Monoubiquitin generated by Lbpro∗ retains GlyGly-modified residues, enabling the quantification of multiply GlyGly-modified branch-point ubiquitin. Notably, we find that a large amount (10-20%) of ubiquitin in polymers seems to exist as branched chains. Moreover, Ub-clipping enables the assessment of co-existing ubiquitin modifications. The analysis of depolarized mitochondria reveals that PINK1/parkin-mediated mitophagy predominantly exploits mono- and short-chain polyubiquitin, in which phosphorylated ubiquitin moieties are not further modified. Ub-clipping can therefore provide insight into the combinatorial complexity and architecture of the ubiquitin code.
Subject(s)
Peptide Hydrolases/metabolism , Ubiquitin/chemistry , Ubiquitin/metabolism , Glycine/chemistry , Glycine/metabolism , HCT116 Cells , HeLa Cells , Humans , Mitophagy , Polyubiquitin/chemistry , Polyubiquitin/metabolism , Protein Kinases/metabolism , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
Mutations in the E3 ubiquitin ligase parkin (PARK2, also known as PRKN) and the protein kinase PINK1 (also known as PARK6) are linked to autosomal-recessive juvenile parkinsonism (AR-JP)1,2; at the cellular level, these mutations cause defects in mitophagy, the process that organizes the destruction of damaged mitochondria3,4. Parkin is autoinhibited, and requires activation by PINK1, which phosphorylates Ser65 in ubiquitin and in the parkin ubiquitin-like (Ubl) domain. Parkin binds phospho-ubiquitin, which enables efficient parkin phosphorylation; however, the enzyme remains autoinhibited with an inaccessible active site5,6. It is unclear how phosphorylation of parkin activates the molecule. Here we follow the activation of full-length human parkin by hydrogen-deuterium exchange mass spectrometry, and reveal large-scale domain rearrangement in the activation process, during which the phospho-Ubl rebinds to the parkin core and releases the catalytic RING2 domain. A 1.8 Å crystal structure of phosphorylated human parkin reveals the binding site of the phospho-Ubl on the unique parkin domain (UPD), involving a phosphate-binding pocket lined by AR-JP mutations. Notably, a conserved linker region between Ubl and the UPD acts as an activating element (ACT) that contributes to RING2 release by mimicking RING2 interactions on the UPD, explaining further AR-JP mutations. Our data show how autoinhibition in parkin is resolved, and suggest a mechanism for how parkin ubiquitinates its substrates via an untethered RING2 domain. These findings open new avenues for the design of parkin activators for clinical use.
Subject(s)
Protein Kinases/metabolism , Ubiquitin-Protein Ligases/metabolism , Binding Sites , Deuterium Exchange Measurement , Enzyme Activation , Humans , Mass Spectrometry , Models, Molecular , Phosphorylation , Protein Domains , Tumor Suppressor Proteins/metabolism , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Protein Ligases/chemistry , UbiquitinationABSTRACT
Autosomal-recessive juvenile Parkinsonism (AR-JP) is caused by mutations in a number of PARK genes, in particular the genes encoding the E3 ubiquitin ligase Parkin (PARK2, also known as PRKN) and its upstream protein kinase PINK1 (also known as PARK6). PINK1 phosphorylates both ubiquitin and the ubiquitin-like domain of Parkin on structurally protected Ser65 residues, triggering mitophagy. Here we report a crystal structure of a nanobody-stabilized complex containing Pediculus humanus corporis (Ph)PINK1 bound to ubiquitin in the 'C-terminally retracted' (Ub-CR) conformation. The structure reveals many peculiarities of PINK1, including the architecture of the C-terminal region, and reveals how the N lobe of PINK1 binds ubiquitin via a unique insertion. The flexible Ser65 loop in the Ub-CR conformation contacts the activation segment, facilitating placement of Ser65 in a phosphate-accepting position. The structure also explains how autophosphorylation in the N lobe stabilizes structurally and functionally important insertions, and reveals the molecular basis of AR-JP-causing mutations, some of which disrupt ubiquitin binding.
Subject(s)
Pediculus/enzymology , Protein Kinases/chemistry , Protein Kinases/metabolism , Ubiquitin/chemistry , Ubiquitin/metabolism , Animals , Binding Sites , Crystallography, X-Ray , Mitophagy , Models, Molecular , Mutation , Phosphorylation , Protein Kinases/genetics , Protein Kinases/immunology , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/immunologyABSTRACT
The Ser/Thr protein kinase PINK1 phosphorylates the well-folded, globular protein ubiquitin (Ub) at a relatively protected site, Ser65. We previously showed that Ser65 phosphorylation results in a conformational change in which Ub adopts a dynamic equilibrium between the known, common Ub conformation and a distinct, second conformation wherein the last ß-strand is retracted to extend the Ser65 loop and shorten the C-terminal tail. We show using chemical exchange saturation transfer (CEST) nuclear magnetic resonance experiments that a similar, C-terminally retracted (Ub-CR) conformation also exists at low population in wild-type Ub. Point mutations in the moving ß5 and neighbouring ß-strands shift the Ub/Ub-CR equilibrium. This enabled functional studies of the two states, and we show that while the Ub-CR conformation is defective for conjugation, it demonstrates improved binding to PINK1 through its extended Ser65 loop, and is a superior PINK1 substrate. Together our data suggest that PINK1 utilises a lowly populated yet more suitable Ub-CR conformation of Ub for efficient phosphorylation. Our findings could be relevant for many kinases that phosphorylate residues in folded protein domains.
Subject(s)
Protein Kinases/metabolism , Ubiquitin/metabolism , Crystallization , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Models, Structural , Molecular Conformation , Phosphorylation , Point Mutation , Protein Domains , Protein Kinases/genetics , Protein Stability , Substrate Specificity , Ubiquitin/chemistry , Ubiquitin/geneticsABSTRACT
The protein kinase PINK1 was recently shown to phosphorylate ubiquitin (Ub) on Ser65, and phosphoUb activates the E3 ligase Parkin allosterically. Here, we show that PINK1 can phosphorylate every Ub in Ub chains. Moreover, Ser65 phosphorylation alters Ub structure, generating two conformations in solution. A crystal structure of the major conformation resembles Ub but has altered surface properties. NMR reveals a second phosphoUb conformation in which ß5-strand slippage retracts the C-terminal tail by two residues into the Ub core. We further show that phosphoUb has no effect on E1-mediated E2 charging but can affect discharging of E2 enzymes to form polyUb chains. Notably, UBE2R1- (CDC34), UBE2N/UBE2V1- (UBC13/UEV1A), TRAF6- and HOIP-mediated chain assembly is inhibited by phosphoUb. While Lys63-linked poly-phosphoUb is recognized by the TAB2 NZF Ub binding domain (UBD), 10 out of 12 deubiquitinases (DUBs), including USP8, USP15 and USP30, are impaired in hydrolyzing phosphoUb chains. Hence, Ub phosphorylation has repercussions for ubiquitination and deubiquitination cascades beyond Parkin activation and may provide an independent layer of regulation in the Ub system.
Subject(s)
Phosphoproteins/metabolism , Polyubiquitin/metabolism , Protein Multimerization/physiology , Ubiquitination/physiology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Allosteric Regulation/physiology , Endopeptidases/genetics , Endopeptidases/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , Humans , Hydrolysis , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Phosphoproteins/genetics , Phosphorylation/physiology , Polyubiquitin/genetics , Protein Structure, Tertiary , Serine/genetics , Serine/metabolism , TNF Receptor-Associated Factor 6/genetics , TNF Receptor-Associated Factor 6/metabolism , Thiolester Hydrolases/genetics , Thiolester Hydrolases/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Specific Proteases/genetics , Ubiquitin-Specific Proteases/metabolismABSTRACT
Nonalcoholic fatty liver disease is associated with chronic oxidative stress. In our study, we explored the antioxidant effect of antidiabetic metformin on chronic [high-fat diet (HFD)-induced] and acute oxidative stress induced by short-term warm partial ischemia-reperfusion (I/R) or on a combination of both in the liver. Wistar rats were fed a standard diet (SD) or HFD for 10 wk, half of them being administered metformin (150 mg·kg body wt(-1)·day(-1)). Metformin treatment prevented acute stress-induced necroinflammatory reaction, reduced alanine aminotransferase and aspartate aminotransferase serum activity, and diminished lipoperoxidation. The effect was more pronounced in the HFD than in the SD group. The metformin-treated groups exhibited less severe mitochondrial damage (markers: cytochrome c release, citrate synthase activity, mtDNA copy number, mitochondrial respiration) and apoptosis (caspase 9 and caspase 3 activation). Metformin-treated HFD-fed rats subjected to I/R exhibited increased antioxidant enzyme activity as well as attenuated mitochondrial respiratory capacity and ATP resynthesis. The exposure to I/R significantly increased NADH- and succinate-related reactive oxygen species (ROS) mitochondrial production in vitro. The effect of I/R was significantly alleviated by previous metformin treatment. Metformin downregulated the I/R-induced expression of proinflammatory (TNF-α, TLR4, IL-1ß, Ccr2) and infiltrating monocyte (Ly6c) and macrophage (CD11b) markers. Our data indicate that metformin reduces mitochondrial performance but concomitantly protects the liver from I/R-induced injury. We propose that the beneficial effect of metformin action is based on a combination of three contributory mechanisms: increased antioxidant enzyme activity, lower mitochondrial ROS production, and reduction of postischemic inflammation.
Subject(s)
Antioxidants/pharmacology , Liver/drug effects , Metformin/pharmacology , Non-alcoholic Fatty Liver Disease/drug therapy , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolism , Reperfusion Injury/prevention & control , Adenosine Triphosphate/metabolism , Animals , Anti-Inflammatory Agents/pharmacology , Apoptosis/drug effects , Cytoprotection , Diet, High-Fat , Disease Models, Animal , Energy Metabolism/drug effects , Inflammation Mediators/metabolism , Lipid Peroxidation/drug effects , Liver/metabolism , Liver/pathology , Male , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism , Mitochondria, Liver/pathology , Non-alcoholic Fatty Liver Disease/etiology , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Rats, Wistar , Reperfusion Injury/etiology , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Time FactorsABSTRACT
Damaged mitochondria undergo mitophagy, a specialized form of autophagy that is initiated by the protein kinase PINK1 and the ubiquitin E3 ligase Parkin. Ubiquitin-specific protease USP30 antagonizes Parkin-mediated ubiquitination events on mitochondria and is a key negative regulator of mitophagy. Parkin and USP30 both show a preference for assembly or disassembly, respectively, of Lys6-linked polyubiquitin, a chain type that has not been well studied. Here we report crystal structures of human USP30 bound to monoubiquitin and Lys6-linked diubiquitin, which explain how USP30 achieves Lys6-linkage preference through unique ubiquitin binding interfaces. We assess the interplay between USP30, PINK1 and Parkin and show that distally phosphorylated ubiquitin chains impair USP30 activity. Lys6-linkage-specific affimers identify numerous mitochondrial substrates for this modification, and we show that USP30 regulates Lys6-polyubiquitinated TOM20. Our work provides insights into the architecture, activity and regulation of USP30, which will aid drug design against this and related enzymes.
Subject(s)
Deubiquitinating Enzymes/chemistry , Deubiquitinating Enzymes/metabolism , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Thiolester Hydrolases/chemistry , Thiolester Hydrolases/metabolism , Ubiquitin/chemistry , Ubiquitin/metabolism , Humans , Protein Binding , Protein Kinases/metabolism , Substrate Specificity , Ubiquitin-Protein Ligases/metabolismABSTRACT
The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.