RESUMEN
Understanding the mechanism and fidelity of transcription by the RNA polymerase (RNAP) requires measurement of the dissociation constant (K(d)) of correct and incorrect NTPs and their incorporation rate constants (k(pol)). Currently, such parameters are obtained from radiometric-based assays that are both tedious and discontinuous. Here, we report a fluorescence-based assay for measuring the real-time kinetics of single-nucleotide incorporation during transcription elongation. The fluorescent adenine analogue 2-aminopurine was incorporated at various single positions in the template or the nontemplate strand of the promoter-free elongation substrate. On addition of the correct NTP to the T7 RNAP-DNA, 2-aminopurine fluorescence increased rapidly and exponentially with a rate constant similar to the RNA extension rate obtained from the radiometric assay. The fluorescence stopped-flow assay, therefore, provides a high-throughput way to measure the kinetic parameters of RNA synthesis. Using this assay, we report the k(pol) and K(d) of all four correct NTP additions by T7 RNAP, which showed a range of values of 145-190 s(-1) and 28-124 µM, respectively. The fluorescent elongation substrates were used to determine the misincorporation kinetics as well, which showed that T7 RNAP discriminates against incorrect NTP both at the nucleotide binding and incorporation steps. The fluorescence-based assay should be generally applicable to all DNA-dependent RNAPs, as they use similar elongation substrates. It can be used to elucidate the mechanism, fidelity, and sequence dependency of transcription and is a rapid means to screen for inhibitors of RNAPs for therapeutic purposes.
Asunto(s)
2-Aminopurina/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Colorantes Fluorescentes/química , Mediciones Luminiscentes/métodos , ARN/biosíntesis , Transcripción Genética , ARN Polimerasas Dirigidas por ADN/química , Fluorescencia , Cinética , Nucleótidos/metabolismoRESUMEN
Mutations in LRRK2 (leucine-rich repeat kinase 2) have been identified as major genetic determinants of Parkinson's disease (PD). The most prevalent mutation, G2019S, increases LRRK2's kinase activity, therefore understanding the sites and substrates that LRRK2 phosphorylates is critical to understanding its role in disease aetiology. Since the physiological substrates of this kinase are unknown, we set out to reveal potential targets of LRRK2 G2019S by identifying its favored phosphorylation motif. A non-biased screen of an oriented peptide library elucidated F/Y-x-T-x-R/K as the core dependent substrate sequence. Bioinformatic analysis of the consensus phosphorylation motif identified several novel candidate substrates that potentially function in neuronal pathophysiology. Peptides corresponding to the most PD relevant proteins were efficiently phosphorylated by LRRK2 in vitro. Interestingly, the phosphomotif was also identified within LRRK2 itself. Autophosphorylation was detected by mass spectrometry and biochemical means at the only F-x-T-x-R site (Thr 1410) within LRRK2. The relevance of this site was assessed by measuring effects of mutations on autophosphorylation, kinase activity, GTP binding, GTP hydrolysis, and LRRK2 multimerization. These studies indicate that modification of Thr1410 subtly regulates GTP hydrolysis by LRRK2, but with minimal effects on other parameters measured. Together the identification of LRRK2's phosphorylation consensus motif, and the functional consequences of its phosphorylation, provide insights into downstream LRRK2-signaling pathways.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Línea Celular , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Guanosina Trifosfato/metabolismo , Humanos , Hidrólisis , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
Leucine-rich repeat kinase 2 (LRRK2) is a large, complex, multidomain protein containing kinase and GTPase enzymatic activities and multiple protein-protein interaction domains. Mutations linked to autosomal dominant forms of Parkinson's disease result in amino acid changes throughout the protein and alterations in both its enzymatic properties and interactions. The best characterized mutation to date, G2019S, leads to increased kinase activity, and mutations in the GTPase domain, such as R1441C and R1441G, have also been reported to influence kinase activity. Therefore, an examination of LRRK2's properties as a kinase is important for understanding the mechanisms underlying the disorder and has the potential to lead to therapeutics. These findings also suggest that there may be complex interplay between the functional domains of LRRK2. Here, we review LRRK2's biochemical functions based on structural and kinetic studies of the enzymatic domains, its potential substrates and the role of its interactions. Despite the field's embryonic understanding of the true relevance of these substrates and interactions, initial studies are providing clues with respect to its pathophysiological functions. Together, these findings should increase our understanding of mechanisms underlying Parkinson's disease and place LRRK2 as a unique molecular target for effective therapeutic development.
Asunto(s)
Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/fisiología , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Modelos Biológicos , Mutación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiologíaRESUMEN
Mutations in leucine-rich repeat kinase 2 (LRRK2) comprise the leading cause of autosomal dominant Parkinson's disease, with age of onset and symptoms identical to those of idiopathic forms of the disorder. Several of these pathogenic mutations are thought to affect its kinase activity, so understanding the roles of LRRK2, and modulation of its kinase activity,may lead to novel therapeutic strategies for treating Parkinson's disease. In this study, highly purified, baculovirus-expressed proteins have been used,for the first time providing large amounts of protein that enable a thorough enzymatic characterization of the kinase activity of LRRK2.Although LRRK2 undergoes weak autophosphorylation, it exhibits high activity towards the peptidic substrate LRRKtide, suggesting that it is a catalytically efficient kinase. We have also utilized a time-resolved fluorescence resonance energy transfer (TR-FRET) assay format (Lantha-ScreenTM) to characterize LRRK2 and test the effects of nonselective kinase inhibitors. Finally, we have used both radiometric and TR-FRETassays to assess the role of clinical mutations affecting LRRK2's kinase activity. Our results suggest that only the most prevalent clinical mutation,G2019S, results in a robust enhancement of kinase activity with LRRKtideas the substrate. This mutation also affects binding of ATP to LRRK2,with wild-type binding being tighter (Km,app of 57 lm) than with theG2019S mutant (Km,app of 134 lm). Overall, these studies delineate the catalytic efficiency of LRRK2 as a kinase and provide strategies by which a therapeutic agent for Parkinson's disease may be identified.