Biochemical Characterization of Highly Purified Leucine-Rich Repeat Kinases 1 and 2 Demonstrates Formation of Homodimers
| العنوان: | Biochemical Characterization of Highly Purified Leucine-Rich Repeat Kinases 1 and 2 Demonstrates Formation of Homodimers |
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| المؤلفون: | Elisa Greggio, Alexandra Beilina, Fangye Gao, Luigi Bubacco, Laura Civiero, Mark R. Cookson, Ivan Mičetić, Marc De Maeyer, Elisa Belluzzi, Renée Vancraenenbroeck, Jean-Marc Taymans, Veerle Baekelandt, Evy Lobbestael, Lauran Reyniers |
| المساهمون: | Yue, Zhenyu |
| المصدر: | PLoS ONE PLoS ONE, Vol 7, Iss 8, p e43472 (2012) |
| سنة النشر: | 2012 |
| مصطلحات موضوعية: | Proteomics, Protein Folding, Protein domain, Biophysics, lcsh:Medicine, Plasma protein binding, Protein Serine-Threonine Kinases, Leucine-rich repeat, Leucine-Rich Repeat Proteins, Leucine-Rich Repeat Serine-Threonine Protein Kinase-2, Biochemistry, Signaling Pathways, Adenosine Triphosphate, Protein structure, Microscopy, Electron, Transmission, Neurobiology of Disease and Regeneration, Humans, Point Mutation, Phosphorylation, lcsh:Science, Protein Interactions, Biology, Enzyme Kinetics, Chromatography, Multidisciplinary, Chemistry, Kinase, Physics, Circular Dichroism, lcsh:R, Lentivirus, Autophosphorylation, Proteins, Immunogold labelling, Immunohistochemistry, Enzymes, nervous system diseases, HEK293 Cells, Microscopy, Fluorescence, Membrane protein, Mutation, lcsh:Q, Molecular Neuroscience, Dimerization, Research Article, Neuroscience, Protein Binding |
| الوصف: | Leucine-rich repeat kinase 1 and 2 (LRRK1 and LRRK2) are large multidomain proteins containing kinase, GTPase and multiple protein-protein interaction domains, but only mutations in LRRK2 are linked to familial Parkinson's disease (PD). Independent studies suggest that LRRK2 exists in the cell as a complex compatible with the size of a dimer. However, whether this complex is truly a homodimer or a heterologous complex formed by monomeric LRRK2 with other proteins has not been definitively proven due to the limitations in obtaining highly pure proteins suitable for structural characterization. Here, we used stable expression of LRRK1 and LRRK2 in HEK293T cell lines to produce recombinant LRRK1 and LRRK2 proteins of greater than 90% purity. Both purified LRRKs are folded, with a predominantly alpha-helical secondary structure and are capable of binding GTP with similar affinity. Furthermore, recombinant LRRK2 exhibits robust autophosphorylation activity, phosphorylation of model peptides in vitro and ATP binding. In contrast, LRRK1 does not display significant autophosphorylation activity and fails to phosphorylate LRRK2 model substrates, although it does bind ATP. Using these biochemically validated proteins, we show that LRRK1 and LRRK2 are capable of forming homodimers as shown by single-particle transmission electron microscopy and immunogold labeling. These LRRK dimers display an elongated conformation with a mean particle size of 145 Å and 175 Å respectively, which is disrupted by addition of 6M guanidinium chloride. Immunogold staining revealed double-labeled particles also in the pathological LRRK2 mutant G2019S and artificial mutants disrupting GTPase and kinase activities, suggesting that point mutations do not hinder the dimeric conformation. Overall, our findings indicate for the first time that purified and active LRRK1 and LRRK2 can form dimers in their full-length conformation. ispartof: PLoS One vol:7 issue:8 ispartof: location:United States status: published |
| وصف الملف: | Print-Electronic; application/pdf |
| اللغة: | English |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::2c01f9fa14593a494640c30fef31f9ca http://hdl.handle.net/11577/2528892 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....2c01f9fa14593a494640c30fef31f9ca |
| قاعدة البيانات: | OpenAIRE |
| الوصف غير متاح. |