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Biochemical characterization of highly purified leucine-rich repeat kinases 1 and 2 demonstrates formation of homodimers.

التفاصيل البيبلوغرافية
العنوان: Biochemical characterization of highly purified leucine-rich repeat kinases 1 and 2 demonstrates formation of homodimers.
المؤلفون: Laura Civiero, Renée Vancraenenbroeck, Elisa Belluzzi, Alexandra Beilina, Evy Lobbestael, Lauran Reyniers, Fangye Gao, Ivan Micetic, Marc De Maeyer, Luigi Bubacco, Veerle Baekelandt, Mark R Cookson, Elisa Greggio, Jean-Marc Taymans
المصدر: PLoS ONE, Vol 7, Iss 8, p e43472 (2012)
بيانات النشر: Public Library of Science (PLoS), 2012.
سنة النشر: 2012
المجموعة: LCC:Medicine
LCC:Science
مصطلحات موضوعية: Medicine, Science
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.
نوع الوثيقة: article
وصف الملف: electronic resource
اللغة: English
تدمد: 1932-6203
Relation: http://europepmc.org/articles/PMC3430690?pdf=render; https://doaj.org/toc/1932-6203
DOI: 10.1371/journal.pone.0043472
URL الوصول: https://doaj.org/article/fe841f27320647b584d617a48e129e58
رقم الأكسشن: edsdoj.fe841f27320647b584d617a48e129e58
قاعدة البيانات: Directory of Open Access Journals
الوصف
تدمد:19326203
DOI:10.1371/journal.pone.0043472