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BACKGROUND: Multiple sclerosis (MS) paramagnetic rim lesions (PRLs) are markers of chronic active biology and exhibit complex iron and myelin changes that may complicate quantification when using conventional MRI approaches. PURPOSE: To conduct a multiparametric MRI analysis of PRLs. STUDY TYPE: Retrospective/longitudinal. SUBJECTS: Ninety-five progressive MS subjects with at least one persistent PRL who were enrolled in the CONSONANCE trial. FIELD STRENGTH/SEQUENCE: 3-T/Susceptibility-weighted, T1-weighted, T2-weighted, and fluid-attenuated inversion recovery. ASSESSMENT: Acute/chronic PRLs and non-PRLs were measured at screening, 24, 48, and 96 weeks using quantitative magnetic susceptibility (QS), R2*, and standardized T1w/T2w ratio (sT1w/T2w). PRL analyses were performed for whole lesion, core, and rim. The correlations between PRL core and rim sT1w/T2w, QS, and R2* were assessed. STATISTICAL TESTS: Linear mixed models. A P-value <0.05 was considered significant. RESULTS: There was a significant decrease in sT1w/T2w (-0.24 ± -5.3 × 10-3) and R2* (-3.6 ± 2.2 Hz) but a significant increase in QS (+21 ± 1.3 ppb) using whole-lesion analysis of chronic PRLs compared to non-PRLs at screening. Tissue damage accumulated at the 96-week time point was more evident in acute/chronic PRLs compared to acute/chronic non-PRLs (ΔsT1w/T2w = -0.21/-0.24 ± 0.033/0.0053; ΔR2* = -4.4/-3.6 ± 1.4/2.2 Hz). New, acute PRL sT1w/T2w significantly increased in lesion core (+4.3 × 10-3 ± 1.2 × 10-4) and rim (+5.6 × 10-3 ± 1.2 × 10-4) 24 weeks post lesion inception, suggestive of partial recovery. Chronic PRLs, contrastingly, showed significant decreases in sT1w/T2w over the initial 24 weeks for both core (-2.1 × 10-4 ± 2.0 × 10-5) and rim (-2.4 × 10-4 ± 2.0 × 10-5), indicative of irreversible tissue damage. Significant positive correlations between PRL core and rim sT1w/T2w (R2 = 0.53), R2* (R2 = 0.69) and QS (R2 = 0.52) were observed. DATA CONCLUSION: Multiparametric assessment of PRLs has the potential to be a valuable tool for assessing complex iron and myelin changes in chronic active PRLs of progressive MS patients. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: Stage 3.
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Serine phosphorylation is a key post-translational modification that regulates diverse biological processes. Powerful analytical methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair that directs the efficient incorporation of phosphoserine (pSer (1)) into recombinant proteins in Escherichia coli. Moreover, combining the orthogonal pair with a metabolically engineered E. coli enables the site-specific incorporation of a nonhydrolyzable analog of pSer. Our approach enables quantitative decoding of the amber stop codon as pSer, and we purify, with yields of several milligrams per liter of culture, proteins bearing biologically relevant phosphorylations that were previously challenging or impossible to access--including phosphorylated ubiquitin and the kinase Nek7, which is synthetically activated by a genetically encoded phosphorylation in its activation loop.
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Aminoacil-ARNt Sintetasas/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Fosfoserina/metabolismo , Procesamiento Proteico-Postraduccional , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/metabolismo , Secuencia de Bases , Codón de Terminación/química , Codón de Terminación/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Código Genético , Modelos Moleculares , Datos de Secuencia Molecular , Quinasas Relacionadas con NIMA , Conformación de Ácido Nucleico , Fosforilación , Fosfoserina/química , Ingeniería de Proteínas , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina/química , Ubiquitina/genética , Ubiquitina/metabolismoRESUMEN
Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser(65))--which lies within its ubiquitin-like domain (Ubl)--and indirectly through phosphorylation of ubiquitin at Ser(65). How Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin(Phospho-Ser65) binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser(65) by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin(Phospho-Ser65), thereby promoting Parkin Ser(65) phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser(65) phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin(Phospho-Ser65) to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser(65). Finally, purified Parkin maximally phosphorylated at Ser(65) in vitro cannot be further activated by the addition of ubiquitin(Phospho-Ser65). Our results thus suggest that a major role of ubiquitin(Phospho-Ser65) is to promote PINK1-mediated phosphorylation of Parkin at Ser(65), leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser(65)-binding pocket on the surface of Parkin that is critical for the ubiquitin(Phospho-Ser65) interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin(Phospho-Ser65), which could aid in the development of Parkin activators that mimic the effect of ubiquitin(Phospho-Ser65).
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Proteínas Quinasas/metabolismo , Serina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Células HEK293 , Humanos , Espectrometría de Masas , Mutagénesis Sitio-Dirigida , Mutación , Fosforilación , Unión Proteica , Proteínas Quinasas/genética , Estructura Terciaria de Proteína , Serina/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Ubiquitin phosphorylation is emerging as an important regulatory layer in the ubiquitin system. This is exemplified by the phosphorylation of ubiquitin on Ser65 by the Parkinson's disease-associated kinase PINK1, which mediates the activation of the E3 ligase Parkin. Additional phosphorylation sites on ubiquitin might also have important cellular roles. Here we report a versatile strategy for preparing phosphorylated ubiquitin. We biochemically and structurally characterise semisynthetic phospho-Ser65-ubiquitin. Unexpectedly, we observed disulfide bond formation between ubiquitin molecules, and hence a novel crystal form. The method outlined provides a direct approach to study the combinatorial effects of phosphorylation on ubiquitin function. Our analysis also suggests that disulfide engineering of ubiquitin could be a useful strategy for obtaining alternative crystal forms of ubiquitin species thereby facilitating structural validation.
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Serina/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Disulfuros/química , Activación Enzimática , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilación , Conformación Proteica , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser65 within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser65 that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser65 in ubiquitin lies in a similar motif to Ser65 in the Ubl domain of Parkin. Remarkably, PINK1 directly phosphorylates Ser65 of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser65 (ΔUbl-Parkin) is robustly activated by ubiquitinPhospho-Ser65, but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser65 (UblPhospho-Ser65) can also activate ΔUbl-Parkin similarly to ubiquitinPhospho-Ser65. Thirdly, we establish that ubiquitinPhospho-Ser65, but not non-phosphorylated ubiquitin or UblPhospho-Ser65, activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser65 and ubiquitin at Ser65, since only mutation of both proteins at Ser65 completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser65, but also by phosphorylating ubiquitin at Ser65. We propose that phosphorylation of Parkin at Ser65 serves to prime the E3 ligase enzyme for activation by ubiquitinPhospho-Ser65, suggesting that small molecules that mimic ubiquitinPhospho-Ser65 could hold promise as novel therapies for Parkinson's disease.
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Proteínas Quinasas/genética , Serina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Células HEK293 , Humanos , Datos de Secuencia Molecular , Fosforilación/genética , Proteínas Quinasas/fisiología , Serina/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The discovery of mutations in genes encoding protein kinase PTEN-induced kinase 1 (PINK1) and E3 ubiquitin ligase Parkin in familial Parkinson's disease and their association with mitochondria provides compelling evidence that mitochondrial dysfunction is a major contributor to neurodegeneration in Parkinson's disease. In recent years, tremendous progress has been made in the understanding of how PINK1 and Parkin enzymes are regulated and how they influence downstream mitochondrial signalling processes. We provide a critical overview of the key advances in the field and also discuss the outstanding questions, including novel ways in which this knowledge could be exploited to develop therapies against Parkinson's disease.
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Degeneración Nerviosa/patología , Enfermedad de Parkinson/genética , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Humanos , Mitocondrias/enzimología , Mitocondrias/patología , Mutación , Degeneración Nerviosa/etiología , Degeneración Nerviosa/genética , Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/patología , Fosforilación , Proteínas Quinasas/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Mutations in PINK1 and Parkin are associated with early-onset Parkinson's disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser(65)) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser(65) phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Miro1. We observe that Parkin efficiently ubiquitylates Miro1 at highly conserved lysine residues, 153, 230, 235, 330 and 572, upon phosphorylation by PINK1. We have further established an E2-ubiquitin discharge assay to assess Parkin activity and observe robust discharge of ubiquitin-loaded UbcH7 E2 ligase upon phosphorylation of Parkin at Ser(65) by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser(65) phosphorylation may activate Parkin E3 ligase activity. For the first time, to the best of our knowledge, we report the effect of Parkin disease-associated mutations in substrate-based assays using full-length untagged recombinant Parkin. Our mutation analysis indicates an essential role for the catalytic cysteine Cys431 and reveals fundamental new knowledge on how mutations may confer pathogenicity via disruption of Miro1 ubiquitylation, free ubiquitin chain formation or by impacting Parkin's ability to discharge ubiquitin from a loaded E2. This study provides further evidence that phosphorylation of Parkin at Ser(65) is critical for its activation. It also provides evidence that Miro1 is a direct Parkin substrate. The assays and reagents developed in this study will be important to uncover new insights into Parkin biology as well as aid in the development of screens to identify small molecule Parkin activators for the treatment of Parkinson's disease.
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Proteínas Mitocondriales/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alineación de Secuencia , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
The pyridoxal 5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) catalyses the first step of de novo sphingolipid biosynthesis. The core human enzyme is a membrane-bound heterodimer composed of two subunits (hLCB1 and hLCB2a/b), and mutations in both hLCB1 (e.g., C133W and C133Y) and hLCB2a (e.g., V359M, G382V, and I504F) have been identified in patients with hereditary sensory and autonomic neuropathy type I (HSAN1), an inherited disorder that affects sensory and autonomic neurons. These mutations result in substrate promiscuity, leading to formation of neurotoxic deoxysphingolipids found in affected individuals. Here we measure the activities of the hLCB2a mutants in the presence of ssSPTa and ssSPTb and find that all decrease enzyme activity. High resolution structural data of the homodimeric SPT enzyme from the bacterium Sphingomonas paucimobilis (Sp SPT) provides a model to understand the impact of the hLCB2a mutations on the mechanism of SPT. The three human hLCB2a HSAN1 mutations map onto Sp SPT (V246M, G268V, and G385F), and these mutant mimics reveal that the amino acid changes have varying impacts; they perturb the PLP cofactor binding, reduce the affinity for both substrates, decrease the enzyme activity, and, in the most severe case, cause the protein to be expressed in an insoluble form.
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Proteínas Bacterianas/metabolismo , Neuropatías Hereditarias Sensoriales y Autónomas/enzimología , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Subunidades de Proteína/metabolismo , Fosfato de Piridoxal/metabolismo , Serina C-Palmitoiltransferasa/genética , Serina C-Palmitoiltransferasa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Humanos , Cinética , Modelos Moleculares , Imitación Molecular , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Mutación , Multimerización de Proteína , Quinonas/metabolismo , Serina C-Palmitoiltransferasa/química , Espectrofotometría Ultravioleta , Sphingomonas/enzimología , Especificidad por SustratoRESUMEN
Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser(65). We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser(65). We further show that phosphorylation of Parkin at Ser(65) leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser(65) or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr(257), which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser(65) and/or PINK1 at Thr(257) represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.