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

描述（申请人提供）：肌原纤维含有巨大的多肽（700，000 Da--4 MDa），具有一个或两个蛋白激酶结构域。我们建议通过研究它们的C. elegans同源物，利用这种生物体中可用的分子遗传学。虽然巨蛋白的激酶结构域与肌球蛋白轻链激酶（MLCK）具有最大的同源性，但这些巨蛋白激酶的真正底物尚不清楚。我们最近发现，在蠕虫的一个单一的基因编码3个肌联蛋白相关的多肽的2.2，1.2和0.3 MDa。Cetitin蛋白激酶结构域具有蛋白激酶活性，并由一种新的机制调节。Obscurin与C. elegans蛋白质，我们之前描述过的p1189。我们最近的数据表明，一些亚型的β-89有2 MLCK样蛋白激酶结构域，类似于最近报道的一些亚型的obscurin。除了通过蛋白激酶结构域进行信号传导外，obscurin和β-89可能通过Rho样GTP酶进行信号传导，因为每种蛋白质都具有dbl（DH）同源结构域。哺乳动物肌联蛋白最大亚型的单分子跨越半个肌节，从M线到Z盘。在I波段，各种类型的实验表明肌联蛋白是弹性的。在I-带中，“小的”约700 kDa的“novex-3”肌联蛋白与obscurin相互作用。已经提出，I带中的novex-3肌联蛋白/暗蛋白复合物是弹性的，并且可以检测在拉伸期间施加在肌节上的应变，并且作为响应，通过DH结构域向GTP酶发出信号。我们的初步数据表明，Ce titins的功能丧失导致瘫痪和发育停滞。我们计划表征Cetitin的突变，确定Cetitin的激酶底物和其他结合伙伴，并研究其可能的弹性元件的结构和机械特性。对于β-89，我们将探索其激酶结构域的结构和活性，鉴定其激酶结构域的底物或结合伴侣，并鉴定和研究其SH 3，DH和PH结构域的结合伴侣。

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.