Department of Biochemistry and Cell Biology, Faculty of Natural Sciences, Kazimierz Wielki University in Bydgoszcz, Poland; Department of Chemistry and Biochemistry, University of California, Los Angeles, USA .
Department of Biochemistry and Cell Biology, Faculty of Natural Sciences, Kazimierz Wielki University in Bydgoszcz, Poland.
Arch Biochem Biophys. 2020 Mar 30;682:108280. doi: 10.1016/j.abb.2020.108280. Epub 2020 Jan 26.
Tropomyosin and cofilin are involved in the regulation of actin filament dynamic polymerization and depolymerization. Binding of cofilin changes actin filaments structure, leading to their severing and depolymerization. Non-muscle tropomyosin isoforms were shown before to differentially regulate the activity of cofilin 1; products of TPM1 gene stabilized actin filaments, but products of TPM3 gene promoted cofilin-dependent severing and depolymerization. Here, conformational changes at the longitudinal and lateral interface between actin subunits resulting from tropomyosin and cofilin 1 binding were studied using skeletal actin and yeast wild type and mutant Q41C and S265C actins. Cross-linking of F-actin and fluorescence changes in F-actin labeled with acrylodan at Cys41 (in D-loop) or Cys265 (in H-loop) showed that tropomyosin isoforms differentially regulated cofilin-induced conformational rearrangements at longitudinal and lateral filament interfaces. Tryptic digestion of F-Mg-actin confirmed the differences between tropomyosin isoforms in their regulation of cofilin-dependent changes at actin-actin interfaces. Changes in the fluorescence of AEDANS attached to C-terminal Cys of actin, as well as FRET between Trp residues in actin subdomain 1 and AEDANS, did not show differences in the conformation of the C-terminal segment of F-actin in the presence of different tropomyosins ± cofilin 1. Therefore, actin's D- and H-loop are the sites involved in regulation of cofilin activity by tropomyosin isoforms.
原肌球蛋白和副肌球蛋白参与调节肌动蛋白丝的动态聚合和解聚。副肌球蛋白的结合改变了肌动蛋白丝的结构，导致其断裂和解聚。以前已经表明，非肌肉原肌球蛋白同工型可以不同程度地调节肌球蛋白 1 的活性；TPM1 基因的产物稳定肌动蛋白丝，但 TPM3 基因的产物促进副肌球蛋白依赖性的断裂和解聚。在这里，使用骨骼肌肌动蛋白和酵母野生型及突变体 Q41C 和 S265C 肌动蛋白研究了原肌球蛋白和肌球蛋白 1 结合导致的肌动蛋白亚基纵向和横向界面的构象变化。F-肌动蛋白的交联和用丙烯酰丹标记的 Cys41（在 D 环）或 Cys265（在 H 环）的 F-肌动蛋白的荧光变化表明，原肌球蛋白同工型不同程度地调节了肌球蛋白诱导的纵向和横向丝界面构象重排。F-Mg-肌动蛋白的胰蛋白酶消化证实了原肌球蛋白同工型在调节肌球蛋白依赖性肌动蛋白-肌动蛋白界面变化方面的差异。连接到肌动蛋白 C 末端半胱氨酸的 AEDANS 的荧光变化以及肌动蛋白亚基 1 中的色氨酸残基与 AEDANS 之间的 FRET 均未显示不同原肌球蛋白±肌球蛋白 1 存在时 F-肌动蛋白 C 末端片段构象的差异。因此，肌动蛋白的 D 环和 H 环是原肌球蛋白同工型调节肌球蛋白活性的位点。
