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

描述(由申请人提供)：肌原纤维含有含有一个或两个蛋白激酶结构域的巨型多肽(700,000 Da-4MDA)。我们建议通过研究线虫的同源物，利用这种有机体中可用的分子遗传学，来深入了解其中的两个巨人，titin和Darcurin。虽然这些巨型蛋白的激酶域与肌球蛋白轻链激酶(MLCKs)有最大的同源性，但这些巨型蛋白的真正底物尚不清楚。我们最近发现，蠕虫中的单个基因编码2.2、1.2和0.3个丙二醛的3个肌动蛋白相关多肽。Ce-titin蛋白激酶结构域具有蛋白激酶活性，并受一种新机制的调节。Obscurin与我们之前描述的线虫蛋白UNC-89密切相关。我们最近的数据显示，UNC-89的某些亚型具有2个MLCK样蛋白激活域，与最近报道的某些Oblcurin亚型相似。除了通过蛋白激酶结构域发出信号外，Oblcurin和UNC-89还可能通过Rho样GTP酶发出信号，因为每个蛋白质都有一个DBL(DH)同源结构域。哺乳动物肌动蛋白最大亚型的单分子横跨半个肌节，从M线到Z盘。在I波段，各种类型的实验都表明Titin是有弹性的。在I-带中，约700 kDa的“小”novex-3“titin与obcurin相互作用。有人认为，I-带中的Novex-3Titin/Oblcurin复合体是有弹性的，可能会检测到在拉伸过程中施加在肌节上的应变，并作为响应，通过DH域向GTP酶发出信号。我们的初步数据表明，CeTiins的功能丧失会导致瘫痪和发育停滞。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性质。对于UNC-89，我们将探索其激活域的结构和活性，确定其激活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.