Leucine rich repeat kinase 2 and dominantly inherited Parkinson disease

富含亮氨酸重复激酶 2 与显性遗传性帕金森病

• 批准号: 7732369
• 负责人: Mark Cookson
• 金额: $ 76.71万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732369
• 关键词: Accounting; Biochemistry; Biological Models; Complex; Enzymes; Frequencies; Genes; Genetic; Guanosine Triphosphate Phosphohydrolases; Immunoprecipitation; In Vitro; Inherited; Introns; Length; Link; Mammalian Cell; Maps; Mediating; Mutation; Neurons; Parkinson Disease; Parkinsonian Disorders; Phosphorylation; Phosphotransferases; Population; Protein Region; Proteins; Recombinant Proteins; Regulation; Research; Structure; Techniques; Tertiary Protein Structure; Testing; Toxic effect; Yeasts; base; design; dimer; disease-causing mutation; leucine-rich repeat kinase 2; mutant; novel therapeutics; protein function; research study; size; small molecule; therapeutic target; tool; yeast two hybrid system

The most common known cause of inherited parkinsonism is the gene Leucine rich repeat kinase 2 (LRRK2). Depending on the population, LRRK2 accounts for between 1 and 30% of all cases of Parkinson disease, including some apparently sporadic cases. The LRRK2 protein is large and complex, containing kinase and GTPase domains linked by an intervening sequence called the COR region. Most of the confirmed pathogenic mutations are in this tridomain region, suggesting that enzyme regulation and activity are likely important for understanding how LRRK2 mutations cause disease. Furthermore, given the frequency of mutations and the presence of two domains that could be targeted by small molecules, LRRK2 is an attractive target for novel therapeutics for Parkinson disease. In the past year, we have concentrated on understanding some of the basic biochemistry of LRRK2. Specifically, we have identified that the large protein forms dimers in a variety of situations and have begun to map the critical regions responsible for dimer formation. We have shown that both transfected and endogenous LRRK2 are present in native protein complexes (identified using non-denaturing techniques) slightly larger than the predicted size of a LRRK2 homodimer. Using complementary techniques, including yeast two hybrid immunoprecipitation and in vitro pulldowns, we confirmed that the central portion of LRRK2 mediates much of the dimer formation. Furthermore, we showed that using full length LRRK2 protein, phosphorylation of LRRK2 by itself (autophosphorylation) occurs within each dimer. This has implications for the design of experiments to test the idea that LRRK2 might regulate itself and we are currently attempting to map the specific regions of the protein that act as phosphorylation acceptors. We have also begun to approach this problem using structural tools. Our collaborators have solved the structure of the GTPase or ROC (for Ras of Complex proteins) domain. We have shown that this is an active enzyme using protein prepared from mammalian cells or as a single domain produced as a recombinant protein. Importantly, we found that a mutation within the ROC domain (R1441C) will weaken the formation of a dimer within LRRK2, thus potentially changing regulation of the protein. We suggest that this altered intramolecular regulation results in an overstimulated kinase activity and, eventually, to toxic effects in neurons. We plan to further explore these phenomena by developing a more detailed map of the various interactions of LRRK2 and identifying how this impacts protein function.

遗传性帕金森氏症的最常见原因是基因亮氨酸富集重复激酶2（LRRK2）。 根据人口的不同，LRRK2占所有帕金森病病例的1-30％，其中包括一些零星的病例。 LRRK2蛋白是大而复杂的，其中包含由称为COR区域的中间序列连接的激酶和GTPase结构域。 大多数已确认的致病突变都在该三域区域，这表明酶调节和活性对于理解LRRK2突变如何引起疾病很重要。 此外，鉴于突变的频率和两个可以由小分子靶向的域的存在，LRRK2是帕金森氏病新型治疗剂的有吸引力的靶标。 在过去的一年中，我们专注于了解LRRK2的一些基本生物化学。 具体而言，我们已经确定在各种情况下大蛋白质形成二聚体，并开始绘制负责二聚体形成的关键区域。 我们已经表明，转染和内源性LRRK2都存在于天然蛋白质复合物（使用非污染技术鉴定）中，比LRRK2同二聚体的预测尺寸略大。 使用互补技术，包括酵母两杂交免疫沉淀和体外下拉，我们证实了LRRK2的中央部分介导了许多二聚体形成。 此外，我们表明，使用全长LRRK2蛋白，LRRK2本身（自磷酸化）的磷酸化发生在每个二聚体内。 这对实验的设计具有影响，以测试LRRK2可能调节自身的想法，而我们目前正在尝试映射充当磷酸化受体的蛋白质的特定区域。 我们还开始使用结构工具来解决此问题。 我们的合作者解决了GTPase或ROC（用于复杂蛋白质的RAS）域的结构。 我们已经证明，这是一种使用由哺乳动物细胞制备的蛋白或作为重组蛋白产生的单个结构蛋白的活性酶。 重要的是，我们发现ROC结构域（R1441C）内的突变会削弱LRRK2内二聚体的形成，从而可能改变蛋白质的调节。 我们建议这种改变的分子内调节导致激酶活性过多，并最终导致神经元的毒性作用。 我们计划通过开发LRRK2各种相互作用的更详细的图并确定这如何影响蛋白质功能来进一步探索这些现象。

项目成果

Differential protein-protein interactions of LRRK1 and LRRK2 indicate roles in distinct cellular signaling pathways.

LRRK1 和 LRRK2 的差异蛋白-蛋白相互作用表明它们在不同的细胞信号传导途径中的作用。

• DOI: 10.1111/jnc.12798
• 发表时间: 2014
• 期刊: Journal of neurochemistry
• 影响因子: 4.7
• 作者: Reyniers,Lauran;DelGiudice,MariaGrazia;Civiero,Laura;Belluzzi,Elisa;Lobbestael,Evy;Beilina,Alexandra;Arrigoni,Giorgio;Derua,Rita;Waelkens,Etienne;Li,Yan;Crosio,Claudia;Iaccarino,Ciro;Cookson,MarkR;Baekelandt,Veerle;Greggio,
• 通讯作者: Greggio,

LRRK2: dropping (kinase) inhibitions and seeking an (immune) response.

• DOI: 10.1111/jnc.12691
• 发表时间: 2014-06
• 期刊: Journal of neurochemistry
• 影响因子: 4.7
• 作者: Mamais A;Cookson MR
• 通讯作者: Cookson MR