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1.
Sci Rep ; 14(1): 7739, 2024 04 02.
Article in English | MEDLINE | ID: mdl-38565869

ABSTRACT

Mutations in PINK1 and Parkin cause early-onset Parkinson's Disease (PD). PINK1 is a kinase which functions as a mitochondrial damage sensor and initiates mitochondrial quality control by accumulating on the damaged organelle. There, it phosphorylates ubiquitin, which in turn recruits and activates Parkin, an E3 ubiquitin ligase. Ubiquitylation of mitochondrial proteins leads to the autophagic degradation of the damaged organelle. Pharmacological modulation of PINK1 constitutes an appealing avenue to study its physiological function and develop therapeutics. In this study, we used a thermal shift assay with insect PINK1 to identify small molecules that inhibit ATP hydrolysis and ubiquitin phosphorylation. PRT062607, an SYK inhibitor, is the most potent inhibitor in our screen and inhibits both insect and human PINK1, with an IC50 in the 0.5-3 µM range in HeLa cells and dopaminergic neurons. The crystal structures of insect PINK1 bound to PRT062607 or CYC116 reveal how the compounds interact with the ATP-binding pocket. PRT062607 notably engages with the catalytic aspartate and causes a destabilization of insert-2 at the autophosphorylation dimer interface. While PRT062607 is not selective for PINK1, it provides a scaffold for the development of more selective and potent inhibitors of PINK1 that could be used as chemical probes.


Subject(s)
Cyclohexylamines , Protein Kinases , Pyrimidines , Ubiquitin-Protein Ligases , Humans , Protein Kinases/metabolism , HeLa Cells , Ubiquitin-Protein Ligases/metabolism , Phosphorylation , Ubiquitin/metabolism , Adenosine Triphosphate/metabolism
2.
Parkinsonism Relat Disord ; 98: 62-69, 2022 05.
Article in English | MEDLINE | ID: mdl-35487127

ABSTRACT

INTRODUCTION: Spastic paraplegia type 4 (SPG4), resulting from heterozygous mutations in the SPAST gene, is the most common form among the heterogeneous group of hereditary spastic paraplegias (HSPs). We aimed to study genetic and clinical characteristics of SPG4 across Canada. METHODS: The SPAST gene was analyzed in a total of 696 HSP patients from 431 families by either HSP-gene panel sequencing or whole exome sequencing (WES). We used Multiplex ligation-dependent probe amplification to analyze copy number variations (CNVs), and performed in silico structural analysis of selected mutations. Clinical characteristics of patients were assessed, and long-term follow-up was done to study genotype-phenotype correlations. RESULTS: We identified 157 SPG4 patients from 65 families who carried 41 different SPAST mutations, six of which are novel and six are CNVs. We report novel aspects of mutations occurring in Arg499, a case with homozygous mutation, a family with probable compound heterozygous mutations, three patients with de novo mutations, three cases with pathogenic synonymous mutation, co-occurrence of SPG4 and clinically isolated syndrome, and novel or rarely reported signs and symptoms seen in SPG4 patients. CONCLUSION: Our study demonstrates that SPG4 is a heterogeneous type of HSP, with diverse genetic features and clinical manifestations. In rare cases, biallelic inheritance, de novo mutation, pathogenic synonymous mutations and CNVs should be considered.


Subject(s)
Spastic Paraplegia, Hereditary , Spastin , Adenosine Triphosphatases/genetics , DNA Copy Number Variations , Humans , Mutation , Paraplegia/genetics , Phenotype , Spastic Paraplegia, Hereditary/diagnosis , Spastic Paraplegia, Hereditary/genetics , Spastin/genetics
3.
Mol Cell ; 82(1): 44-59.e6, 2022 01 06.
Article in English | MEDLINE | ID: mdl-34875213

ABSTRACT

Mutations in PINK1 cause autosomal-recessive Parkinson's disease. Mitochondrial damage results in PINK1 import arrest on the translocase of the outer mitochondrial membrane (TOM) complex, resulting in the activation of its ubiquitin kinase activity by autophosphorylation and initiation of Parkin-dependent mitochondrial clearance. Herein, we report crystal structures of the entire cytosolic domain of insect PINK1. Our structures reveal a dimeric autophosphorylation complex targeting phosphorylation at the invariant Ser205 (human Ser228). The dimer interface requires insert 2, which is unique to PINK1. The structures also reveal how an N-terminal helix binds to the C-terminal extension and provide insights into stabilization of PINK1 on the core TOM complex.


Subject(s)
Insect Proteins/metabolism , Mitochondria/enzymology , Mitochondrial Precursor Protein Import Complex Proteins/metabolism , Protein Kinases/metabolism , Tribolium/enzymology , Animals , Cell Line, Tumor , Enzyme Activation , Enzyme Stability , Humans , Insect Proteins/genetics , Kinetics , Mitochondria/genetics , Mitochondrial Precursor Protein Import Complex Proteins/genetics , Molecular Docking Simulation , Mutation , Phosphorylation , Protein Interaction Domains and Motifs , Protein Kinases/genetics , Structure-Activity Relationship , Tribolium/genetics
4.
Clin Genet ; 100(1): 51-58, 2021 07.
Article in English | MEDLINE | ID: mdl-33713342

ABSTRACT

GCH1 mutations have been associated with dopa-responsive dystonia (DRD), Parkinson's disease (PD) and tetrahydrobiopterin (BH4 )-deficient hyperphenylalaninemia B. Recently, GCH1 mutations have been reported in five patients with hereditary spastic paraplegia (HSP). Here, we analyzed a total of 400 HSP patients (291 families) from different centers across Canada by whole exome sequencing (WES). Three patients with heterozygous GCH1 variants were identified: monozygotic twins with a p.(Ser77_Leu82del) variant, and a patient with a p.(Val205Glu) variant. The former variant is predicted to be likely pathogenic and the latter is pathogenic. The three patients presented with childhood-onset lower limb spasticity, hyperreflexia and abnormal plantar responses. One of the patients had diurnal fluctuations, and none had parkinsonism or dystonia. Phenotypic differences between the monozygotic twins were observed, who responded well to levodopa treatment. Pathway enrichment analysis suggested that GCH1 shares processes and pathways with other HSP-associated genes, and structural analysis of the variants indicated a disruptive effect. In conclusion, GCH1 mutations may cause HSP; therefore, we suggest a levodopa trial in HSP patients and including GCH1 in the screening panels of HSP genes. Clinical differences between monozygotic twins suggest that environmental factors, epigenetics, and stochasticity could play a role in the clinical presentation.


Subject(s)
GTP Cyclohydrolase/genetics , Mutation/genetics , Spastic Paraplegia, Hereditary/genetics , Adult , Canada , Child , Female , Humans , Levodopa/therapeutic use , Male , Middle Aged , Parkinsonian Disorders/genetics , Pedigree , Phenotype , Spastic Paraplegia, Hereditary/drug therapy , Twins, Monozygotic/genetics
5.
Structure ; 29(6): 572-586.e6, 2021 06 03.
Article in English | MEDLINE | ID: mdl-33529594

ABSTRACT

The Parkin co-regulated gene protein (PACRG) binds at the inner junction between doublet microtubules of the axoneme, a structure found in flagella and cilia. PACRG binds to the adaptor protein meiosis expressed gene 1 (MEIG1), but how they bind to microtubules is unknown. Here, we report the crystal structure of human PACRG in complex with MEIG1. PACRG adopts a helical repeat fold with a loop that interacts with MEIG1. Using the structure of the axonemal doublet microtubule from the protozoan Chlamydomonas reinhardtii and single-molecule fluorescence microscopy, we propose that PACRG binds to microtubules while simultaneously recruiting free tubulin to catalyze formation of the inner junction. We show that the homologous PACRG-like protein also mediates dual tubulin interactions but does not bind MEIG1. Our findings establish a framework to assess the function of the PACRG family of proteins and MEIG1 in regulating axoneme assembly.


Subject(s)
Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Chlamydomonas reinhardtii/metabolism , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Tubulin/metabolism , Axoneme/metabolism , Binding Sites , Crystallography, X-Ray , Humans , Microfilament Proteins/genetics , Microscopy, Fluorescence , Molecular Chaperones/genetics , Multiprotein Complexes/chemistry , Mutation , Protein Binding , Protein Conformation , Protein Domains , Single Molecule Imaging
6.
Elife ; 92020 01 17.
Article in English | MEDLINE | ID: mdl-31951202

ABSTRACT

Microtubules are cytoskeletal structures involved in stability, transport and organization in the cell. The building blocks, the α- and ß-tubulin heterodimers, form protofilaments that associate laterally into the hollow microtubule. Microtubule also exists as highly stable doublet microtubules in the cilia where stability is needed for ciliary beating and function. The doublet microtubule maintains its stability through interactions at its inner and outer junctions where its A- and B-tubules meet. Here, using cryo-electron microscopy, bioinformatics and mass spectrometry of the doublets of Chlamydomonas reinhardtii and Tetrahymena thermophila, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction-associated protein, FAP126, thus presenting the complete answer to the inner junction identity and localization. Our structural study of the doublets shows that the inner junction serves as an interaction hub that involves tubulin post-translational modifications. These interactions contribute to the stability of the doublet and hence, normal ciliary motility.


Subject(s)
Cilia/metabolism , Protein Processing, Post-Translational , Chlamydomonas reinhardtii/metabolism , Computational Biology , Cryoelectron Microscopy/methods , Mass Spectrometry , Microtubules/metabolism , Plant Proteins/metabolism , Protozoan Proteins/metabolism , Tetrahymena thermophila/metabolism
7.
Nat Commun ; 10(1): 1142, 2019 03 08.
Article in English | MEDLINE | ID: mdl-30850593

ABSTRACT

FIC proteins regulate molecular processes from bacteria to humans by catalyzing post-translational modifications (PTM), the most frequent being the addition of AMP or AMPylation. In many AMPylating FIC proteins, a structurally conserved glutamate represses AMPylation and, in mammalian FICD, also supports deAMPylation of BiP/GRP78, a key chaperone of the unfolded protein response. Currently, a direct signal regulating these FIC proteins has not been identified. Here, we use X-ray crystallography and in vitro PTM assays to address this question. We discover that Enterococcus faecalis FIC (EfFIC) catalyzes both AMPylation and deAMPylation and that the glutamate implements a multi-position metal switch whereby Mg2+ and Ca2+ control AMPylation and deAMPylation differentially without a conformational change. Remarkably, Ca2+ concentration also tunes deAMPylation of BiP by human FICD. Our results suggest that the conserved glutamate is a signature of AMPylation/deAMPylation FIC bifunctionality and identify metal ions as diffusible signals that regulate such FIC proteins directly.


Subject(s)
Adenosine Monophosphate/metabolism , Bacterial Proteins/chemistry , Calcium/metabolism , Chemokine CCL7/chemistry , Heat-Shock Proteins/chemistry , Protein Processing, Post-Translational , Adenosine Monophosphate/chemistry , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Biocatalysis , Calcium/chemistry , Cations, Divalent , Chemokine CCL7/genetics , Chemokine CCL7/metabolism , Cloning, Molecular , Crystallography, X-Ray , Endoplasmic Reticulum Chaperone BiP , Enterococcus faecalis/genetics , Enterococcus faecalis/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Magnesium/chemistry , Magnesium/metabolism , Mice , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity
8.
Pathog Dis ; 76(2)2018 03 01.
Article in English | MEDLINE | ID: mdl-29617857

ABSTRACT

During the last decade, FIC proteins have emerged as a large family comprised of a variety of bacterial enzymes and a single member in animals. The air de famille of FIC proteins stems from a domain of conserved structure, which catalyzes the post-translational modification of proteins (PTM) by a phosphate-containing compound. In bacteria, examples of FIC proteins include the toxin component of toxin/antitoxin modules, such as Doc-Phd and VbhT-VbhA, toxins secreted by pathogenic bacteria to divert host cell processes, such as VopS, IbpA and AnkX, and a vast majority of proteins of unknown functions. FIC proteins catalyze primarily the transfer of AMP (AMPylation), but they are not restricted to this PTM and also carry out other modifications, for example by phosphocholine or phosphate. In a recent twist, animal FICD/HYPE was shown to catalyze both AMPylation and de-AMPylation of the endoplasmic reticulum BIP chaperone to regulate the unfolded protein response. FICD shares structural features with some bacterial FIC proteins, raising the possibility that bacteria also encode such dual activities. In this review, we discuss how structural, biochemical and cellular approaches have fertilized each other to understand the mechanism, regulation and function of FIC proteins from bacterial pathogens to humans.


Subject(s)
Adenosine Monophosphate/metabolism , Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Gene Expression Regulation , Membrane Proteins/metabolism , Protein Processing, Post-Translational , Bacteria , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Carrier Proteins/chemistry , Carrier Proteins/genetics , Evolution, Molecular , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Nucleotidyltransferases , Protein Conformation
9.
Biochem J ; 474(7): 1259-1272, 2017 03 23.
Article in English | MEDLINE | ID: mdl-28196833

ABSTRACT

Active, GTP-bound small GTPases need to be attached to membranes by post-translational lipid modifications in order to process and propagate information in cells. However, generating and manipulating lipidated GTPases has remained difficult, which has limited our quantitative understanding of their activation by guanine nucleotide exchange factors (GEFs) and their termination by GTPase-activating proteins. Here, we replaced the lipid modification by a histidine tag in 11 full-length, human small GTPases belonging to the Arf, Rho and Rab families, which allowed to tether them to nickel-lipid-containing membranes and characterize the kinetics of their activation by GEFs. Remarkably, this strategy uncovered large effects of membranes on the efficiency and/or specificity in all systems studied. Notably, it recapitulated the release of autoinhibition of Arf1, Arf3, Arf4, Arf5 and Arf6 GTPases by membranes and revealed that all isoforms are efficiently activated by two GEFs with different regulatory regimes, ARNO and Brag2. It demonstrated that membranes stimulate the GEF activity of Trio toward RhoG by ∼30 fold and Rac1 by ∼10 fold, and uncovered a previously unknown broader specificity toward RhoA and Cdc42 that was undetectable in solution. Finally, it demonstrated that the exceptional affinity of the bacterial RabGEF DrrA for the phosphoinositide PI(4)P delimits the activation of Rab1 to the immediate vicinity of the membrane-bound GEF. Our study thus validates the histidine-tag strategy as a potent and simple means to mimic small GTPase lipidation, which opens a variety of applications to uncover regulations brought about by membranes.


Subject(s)
ADP-Ribosylation Factor 1/metabolism , GTPase-Activating Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Histidine/metabolism , Oligopeptides/metabolism , Phosphatidylinositols/metabolism , ADP-Ribosylation Factor 1/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enzyme Activation , GTPase-Activating Proteins/genetics , Gene Expression , Guanine Nucleotide Exchange Factors/genetics , Histidine/genetics , Humans , Legionella pneumophila/chemistry , Membranes, Artificial , Oligopeptides/genetics , Phosphatidylinositols/genetics , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , cdc42 GTP-Binding Protein/genetics , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolism , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolism , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolism
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