Jurewicz Ewelina, Ostrowska Zofia, Jozwiak Jolanta, Redowicz Maria Jolanta, Lesniak Wieslawa, Moraczewska Joanna, Filipek Anna
Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland.
Biochim Biophys Acta. 2013 Mar;1833(3):761-6. doi: 10.1016/j.bbamcr.2012.12.010. Epub 2012 Dec 22.
The CacyBP/SIP protein interacts with several targets, including actin. Since the majority of actin filaments are associated with tropomyosin, in this work we characterized binding of CacyBP/SIP to the actin-tropomyosin complex and examined the effects of CacyBP/SIP on actin filament functions. By using reconstituted filaments composed of actin and AEDANS-labeled tropomyosin, we observed that binding of CacyBP/SIP caused an increase in tropomyosin fluorescence intensity indicating the occurrence of conformational changes within the filament. We also found that CacyBP/SIP bound directly to tropomyosin and that these proteins did not compete with each other for binding to actin. Electron microscopy showed that in the absence of tropomyosin CacyBP/SIP destabilized actin filaments, but tropomyosin reversed this effect. Actin-activated myosin S1 ATPase activity assays, performed using a colorimetric method, indicated that CacyBP/SIP reduced ATPase activity and that the presence of tropomyosin enhanced this inhibitory effect. Thus, our results suggest that CacyBP/SIP, through its interaction with both actin and tropomyosin, regulates the organization and functional properties of the thin filament.
CacyBP/SIP蛋白可与包括肌动蛋白在内的多种靶标相互作用。由于大多数肌动蛋白丝都与原肌球蛋白相关联，因此在本研究中，我们对CacyBP/SIP与肌动蛋白 - 原肌球蛋白复合物的结合进行了表征，并研究了CacyBP/SIP对肌动蛋白丝功能的影响。通过使用由肌动蛋白和AEDANS标记的原肌球蛋白组成的重组丝，我们观察到CacyBP/SIP的结合导致原肌球蛋白荧光强度增加，这表明丝内发生了构象变化。我们还发现CacyBP/SIP直接与原肌球蛋白结合，并且这些蛋白质在与肌动蛋白结合时不会相互竞争。电子显微镜显示，在没有原肌球蛋白的情况下，CacyBP/SIP会使肌动蛋白丝不稳定，但原肌球蛋白可逆转这种效应。使用比色法进行的肌动蛋白激活的肌球蛋白S1 ATP酶活性测定表明，CacyBP/SIP会降低ATP酶活性，并且原肌球蛋白的存在会增强这种抑制作用。因此，我们的结果表明，CacyBP/SIP通过其与肌动蛋白和原肌球蛋白的相互作用，调节细肌丝的组织和功能特性。
