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

描述（由申请人提供）：肌原纤维含有巨大的多肽（700,000 Da- 4mda），具有一个或两个蛋白激酶结构域。我们建议通过研究它们在秀丽隐杆线虫中的同源物，利用这种生物体中可用的分子遗传学，来深入了解其中的两个巨人，titin和obscurin。尽管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，蠕虫中的一个基因编码3个与titin相关的2.2、1.2和0.3 MDa的多肽。cetitin蛋白激酶结构域具有蛋白激酶活性，并受一种新的机制调控。Obscurin与我们之前描述的秀丽隐杆线虫蛋白UNC-89密切相关。我们最近的数据显示，UNC-89的一些异构体具有2个mlck样蛋白激酶结构域，与最近报道的一些obscurin异构体相似。除了通过蛋白激酶结构域发出信号外，obscurin和UNC-89可能通过rho样GTPases发出信号，因为每种蛋白都有一个dbl （DH）同源结构域。哺乳动物titin的最大同种异构体的单分子横跨半个肌节，从m -线到z -盘。在i波段，各种类型的实验表明titin是有弹性的。在i波段，“小的”约700 kDa的“novx -3”titin与obscurin相互作用。有研究表明，i波段的novex-3 titin / obscurin复合物具有弹性，可以检测拉伸过程中施加在肌节上的应变，并通过DH结构域向GTPases发出响应信号。我们的初步数据表明，Ce - titin的功能丧失会导致瘫痪和发育停止。我们计划表征Ce titin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件的结构和力学特性。对于UNC-89，我们将探索其激酶结构域的结构和活性，确定其激酶结构域的底物或结合伙伴，并确定和研究其SH3， DH和PH结构域的结合伙伴。

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.