ABSTRACT
Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD). A disease-causing point mutation R1441H/G/C in the GTPase domain of LRRK2 leads to overactivation of its kinase domain. However, the mechanism by which this mutation alters the normal function of its GTPase domain [Ras of complex proteins (Roc)] remains unclear. Here, we report the effects of R1441H mutation (RocR1441H) on the structure and activity of Roc. We show that Roc forms a stable monomeric conformation in solution that is catalytically active, thus demonstrating that LRRK2 is a bona fide self-contained GTPase. We further show that the R1441H mutation causes a twofold reduction in GTPase activity without affecting the structure, thermal stability, and GDP-binding affinity of Roc. However, the mutation causes a twofold increase in GTP-binding affinity of Roc, thus suggesting that the PD-causing mutation R1441H traps Roc in a more persistently activated state by increasing its affinity for GTP and, at the same time, compromising its GTP hydrolysis.
Subject(s)
GTP Phosphohydrolases/metabolism , Models, Molecular , Mutation, Missense/genetics , Parkinson Disease/genetics , Protein Conformation , Protein Serine-Threonine Kinases/genetics , Blotting, Western , Chromatography, Gel , Circular Dichroism , Dimerization , Electrophoresis, Polyacrylamide Gel , GTP Phosphohydrolases/genetics , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Mass Spectrometry , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolismABSTRACT
Cerebrospinal fluid (CSF) has been used for biomarker discovery of neurodegenerative diseases in humans since biological changes in the brain can be seen in this biofluid. Inactivation of A-T-mutated protein (ATM), a multifunctional protein kinase, is responsible for A-T, yet biochemical studies have not succeeded in conclusively identifying the molecular mechanism(s) underlying the neurodegeneration seen in A-T patients or the proteins that can be used as biomarkers for neurologic assessment of A-T or as potential therapeutic targets. In this study, we applied a high-throughput LC/MS-based label-free protein quantification technology to quantitatively characterize the proteins in CSF samples in order to identify differentially expressed proteins that can serve as potential biomarker candidates for A-T. Among 204 identified CSF proteins with high peptide-identification confidence, thirteen showed significant protein expression changes. Bioinformatic analysis revealed that these 13 proteins are either involved in neurodegenerative disorders or cancer. Future molecular and functional characterization of these proteins would provide more insights into the potential therapeutic targets for the treatment of A-T and the biomarkers that can be used to monitor or predict A-T disease progression. Clinical validation studies are required before any of these proteins can be developed into clinically useful biomarkers.
ABSTRACT
BACKGROUND: Overexpression of superoxide dismutase 1 (SOD1) has been shown to be one of the factors involved in causing cisplatin resistance in ovarian cancer. Reduction of SOD1 expression is expected to restore, at least partially, cisplatin sensitivity in ovarian cancer chemotherapy. Here, we explored the potential of RNAi as a therapy for reversal of cisplatin resistance. MATERIALS AND METHODS: SOD1-specific small-interfering RNA (siRNA) was synthesized and transfected into cisplatin-resistant cell line A2780/CP prior to treatment with 15 muM cisplatin. Cell survival was assessed by clonogenic assay. RESULTS: An enhanced cisplatin sensitivity was observed in the A2780/CP cells treated with SOD1-specific siRNA, compared to non-siRNA-treated or scrambled-siRNA-treated control cells. CONCLUSION: Specifically targeting SOD1 could lead to sensitization of cisplatin-resistant ovarian cancer cells, and SOD1 may be used as a potential target for chemosensitizers.
Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Gene Knockdown Techniques/methods , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/enzymology , Superoxide Dismutase/deficiency , Cell Line, Tumor , Drug Resistance, Neoplasm , Female , Humans , Ovarian Neoplasms/genetics , RNA, Small Interfering/genetics , Superoxide Dismutase/biosynthesis , Superoxide Dismutase/genetics , Superoxide Dismutase-1 , TransfectionABSTRACT
Protein quantification in a complex protein mixture presents a daunting task in biochemical analysis. Antibody-based immunoassays are traditional methods for protein quantification. However, there are issues associated with accuracy and specificity in these assays, especially when the changes are small (e.g., <2-fold). With recent developments in mass spectrometry, monitoring a selected peptide, thus protein, in a complex biological sample has become possible. In this study, we demonstrate a simple mass spectrometry-based method for selective measurement of a moderately low abundant protein, superoxide dismutase 1 (SOD1), in cisplatin-sensitive and cisplatin-resistant human ovarian cancer cells. Selected-reaction-monitoring (SRM) technology was employed to specifically analyze the target peptides in a pair of human ovarian cancer cell lines: 2008/2008-C13*5.25 (cisplatin-sensitive/cisplatin-resistant, respectively). The observed 1.47-fold higher expression in the resistant cell line is consistent with findings by other approaches. This robust liquid chromatography/mass spectrometry (LC/MS) method provides a powerful tool for targeted proteomic verification and/or validation studies.
