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1.
Sci Rep ; 14(1): 7739, 2024 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-38565869

RESUMEN

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.


Asunto(s)
Ciclohexilaminas , Proteínas Quinasas , Pirimidinas , Ubiquitina-Proteína Ligasas , Humanos , Proteínas Quinasas/metabolismo , Células HeLa , Ubiquitina-Proteína Ligasas/metabolismo , Fosforilación , Ubiquitina/metabolismo , Adenosina Trifosfato/metabolismo
2.
Parkinsonism Relat Disord ; 98: 62-69, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35487127

RESUMEN

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.


Asunto(s)
Paraplejía Espástica Hereditaria , Espastina , Adenosina Trifosfatasas/genética , Variaciones en el Número de Copia de ADN , Humanos , Mutación , Paraplejía/genética , Fenotipo , Paraplejía Espástica Hereditaria/diagnóstico , Paraplejía Espástica Hereditaria/genética , Espastina/genética
3.
Mol Cell ; 82(1): 44-59.e6, 2022 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-34875213

RESUMEN

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.


Asunto(s)
Proteínas de Insectos/metabolismo , Mitocondrias/enzimología , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/metabolismo , Proteínas Quinasas/metabolismo , Tribolium/enzimología , Animales , Línea Celular Tumoral , Activación Enzimática , Estabilidad de Enzimas , Humanos , Proteínas de Insectos/genética , Cinética , Mitocondrias/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/genética , Simulación del Acoplamiento Molecular , Mutación , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Proteínas Quinasas/genética , Relación Estructura-Actividad , Tribolium/genética
4.
Clin Genet ; 100(1): 51-58, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33713342

RESUMEN

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.


Asunto(s)
GTP Ciclohidrolasa/genética , Mutación/genética , Paraplejía Espástica Hereditaria/genética , Adulto , Canadá , Niño , Femenino , Humanos , Levodopa/uso terapéutico , Masculino , Persona de Mediana Edad , Trastornos Parkinsonianos/genética , Linaje , Fenotipo , Paraplejía Espástica Hereditaria/tratamiento farmacológico , Gemelos Monocigóticos/genética
5.
Structure ; 29(6): 572-586.e6, 2021 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-33529594

RESUMEN

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.


Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Chlamydomonas reinhardtii/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Tubulina (Proteína)/metabolismo , Axonema/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Humanos , Proteínas de Microfilamentos/genética , Microscopía Fluorescente , Chaperonas Moleculares/genética , Complejos Multiproteicos/química , Mutación , Unión Proteica , Conformación Proteica , Dominios Proteicos , Imagen Individual de Molécula
6.
Elife ; 92020 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-31951202

RESUMEN

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.


Asunto(s)
Cilios/metabolismo , Procesamiento Proteico-Postraduccional , Chlamydomonas reinhardtii/metabolismo , Biología Computacional , Microscopía por Crioelectrón/métodos , Espectrometría de Masas , Microtúbulos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Protozoarias/metabolismo , Tetrahymena thermophila/metabolismo
7.
Nat Commun ; 10(1): 1142, 2019 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-30850593

RESUMEN

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.


Asunto(s)
Adenosina Monofosfato/metabolismo , Proteínas Bacterianas/química , Calcio/metabolismo , Quimiocina CCL7/química , Proteínas de Choque Térmico/química , Procesamiento Proteico-Postraduccional , Adenosina Monofosfato/química , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biocatálisis , Calcio/química , Cationes Bivalentes , Quimiocina CCL7/genética , Quimiocina CCL7/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Chaperón BiP del Retículo Endoplásmico , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Magnesio/química , Magnesio/metabolismo , Ratones , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato
8.
Pathog Dis ; 76(2)2018 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-29617857

RESUMEN

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.


Asunto(s)
Adenosina Monofosfato/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Bacterias , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Evolución Molecular , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Nucleotidiltransferasas , Conformación Proteica
9.
Biochem J ; 474(7): 1259-1272, 2017 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-28196833

RESUMEN

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.


Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Histidina/metabolismo , Oligopéptidos/metabolismo , Fosfatidilinositoles/metabolismo , Factor 1 de Ribosilacion-ADP/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Proteínas Activadoras de GTPasa/genética , Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Histidina/genética , Humanos , Legionella pneumophila/química , Membranas Artificiales , Oligopéptidos/genética , Fosfatidilinositoles/genética , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo
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