Using C. elegans to study titin and obscurin (UNC-89)

使用线虫研究 titin 和 obscurin (UNC-89)

• 批准号: 7847206
• 负责人: GUY Martin BENIAN
• 金额: $ 1.01万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-10 至 2009-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7847206
• 关键词: ATP phosphohydrolase; Alleles; Binding; Biochemical; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cardiomyopathies; Complex; DH Domain; Data; Development; Elements; Family; Genes; Goals; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; Homology Modeling; Human; Hybrids; Insecta; Libraries; Mammals; Mechanics; Molecular Genetics; Muscle; Mutation; Myofibrils; Myosin Light Chain Kinase; Myosin Regulatory Light Chains; Nematoda; Organism; PH Domain; Paralysed; Phosphotransferases; Physiological; Property; Protein Isoforms; Protein Kinase; Proteins; Reporting; Sarcomeres; Screening procedure; Signal Transduction; Stretching; Structure; Testing; Udd-Markesbery Muscular Dystrophy; connectin; fascinate; insight; loss of function; member; mutant; novel; obscurin; polypeptide; projectin; research study; response; rho; single molecule; stretchin; titin kinase; twitchin

DESCRIPTION (provided by applicant): Myofibrils contain giant polypeptides (700,000 Da--4 MDa) that have either one or two protein kinase domains. We propose to gain insights into two of these giants, titin and obscurin, by studying their C. elegans homologs, exploiting the molecular genetics available in this organism. Although the kinase domains of the giant proteins show greatest homology to myosin light chain kinases (MLCKs), the true substrates for these giant kinases are not clearly known. We recently discovered that a single gene in the worm encodes 3 titin-related polypeptides of 2.2, 1.2 and 0.3 MDa. The Ce titin protein kinase domain has protein kinase activity and is regulated by a novel mechanism. Obscurin is closely related to a C. elegans protein, UNC-89, that we described previously. Our recent data shows that some isoforms of UNC-89 have 2 MLCK-like protein kinase domains, similar to what has recently been reported for some isoforms of obscurin. In addition to signaling via protein kinase domains, obscurin and UNC-89 are likely to signal via Rho-like GTPases, since each protein has a dbl (DH) homology domain. Single molecules of the largest isoforms of mammalian titin span half a sarcomere, from M-line to Z-disc. In the I-band, various types of experiments have shown that titin is elastic. In the I-band, the "small" approximately 700 kDa "novex-3" titin interacts with obscurin. It has been suggested that a novex-3 titin / obscurin complex in the I-band is elastic, and might detect strain imposed on the sarcomere during stretch and in response, signals through the DH domain to GTPases. Our preliminary data indicates that loss-of-function for Ce titins results in paralysis and developmental arrest. We plan to characterize mutations in Ce titin, determine Ce titin's kinase substrate and other binding partners, and study structural and mechanical properties of its likely elastic elements. For UNC-89, we will explore the structure and activity of its kinase domains, identify the substrates or binding partners for its kinase domains, and identify and study binding partners for its SH3, DH and PH domains.

描述（由申请人提供）：肌原纤维包含具有一或两个蛋白激酶结构域的巨型多肽（700,000 DA-4 MDA）。我们建议通过研究其秀丽隐杆线虫同源物，利用这种生物体可用的分子遗传学来了解这两个巨人钛和遮盖蛋白。尽管巨型蛋白的激酶结构域与肌球蛋白轻链激酶（MLCKS）表现出最大的同源性，但这些巨型激酶的真实底物并不清楚。我们最近发现，蠕虫中的单个基因编码3个与TITIN相关的多肽为2.2、1.2和0.3 MDA。 CE Titin蛋白激酶结构域具有蛋白激酶活性，并受一种新机制调节。我们先前描述的黑素蛋白与秀丽隐杆线虫蛋白UNC-89密切相关。我们最近的数据表明，某些UNC-89的同工型具有2个MLCK样蛋白激酶结构域，类似于最近报道的某些obsobulin的同工型。除了通过蛋白激酶结构域通过信号传导外，近距离蛋白和UNC-89可能通过类似Rho样GTPase发出信号，因为每种蛋白质都有DBL（DH）同源性域。哺乳动物滴定的最大同工型的单分子跨越了半肌肉，从M线到Z-DISC。在I波段中，各种类型的实验表明Titin是弹性的。在i波段中，“小”大约700 kDa“ novex-3”山雀与遮盖蛋白相互作用。有人提出，I波段中的NoVex-3滴定 /掩盖蛋白复合物是弹性的，并且可能在拉伸过程中检测到肌膜上施加的菌株，并在响应中通过DH结构域向GTPase发出信号。我们的初步数据表明，CE Titins的功能丧失会导致瘫痪和发育停滞。我们计划表征Ce Titin中的突变，确定CE Titin的激酶底物和其他结合伙伴，并研究其可能的弹性元素的结构和机械性能。对于UNC-89，我们将探索其激酶结构域的结构和活性，确定其激酶域的底物或结合伴侣，并确定和研究其SH3，DH和PH域的结合伙伴。

项目成果

The DH-PH region of the giant protein UNC-89 activates RHO-1 GTPase in Caenorhabditis elegans body wall muscle.

• DOI: 10.1016/j.jmb.2008.08.083
• 发表时间: 2008-11-21
• 期刊: JOURNAL OF MOLECULAR BIOLOGY
• 影响因子: 5.6
• 作者: Qadota, Hiroshi;Blangy, Anne;Xiong, Ge;Benian, Guy M.
• 通讯作者: Benian, Guy M.

A LIM-9 (FHL)/SCPL-1 (SCP) complex interacts with the C-terminal protein kinase regions of UNC-89 (obscurin) in Caenorhabditis elegans muscle.

LIM-9 (FHL)/SCPL-1 (SCP) 复合物与秀丽隐杆线虫肌肉中 UNC-89（暗色蛋白）的 C 末端蛋白激酶区域相互作用。

• DOI: 10.1016/j.jmb.2009.01.016
• 发表时间: 2009
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Xiong,Ge;Qadota,Hiroshi;Mercer,KristinaB;McGaha,LeeAnne;Oberhauser,AndresF;Benian,GuyM
• 通讯作者: Benian,GuyM

A C. elegans homolog of huntingtin-associated protein 1 is expressed in chemosensory neurons and in a number of other somatic cell types.

• DOI: 10.1007/s12031-008-9109-z
• 发表时间: 2009-01
• 期刊: JOURNAL OF MOLECULAR NEUROSCIENCE
• 影响因子: 3.1
• 作者: Mercer, Kristina B.;Szlam, Sarah M.;Manning, Erin;Gernert, Kim M.;Walthall, Walter W.;Benian, Guy M.;Gutekunst, Claire-Anne
• 通讯作者: Gutekunst, Claire-Anne