Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA.
Departments of Neuroscience and Pharmacology, The University of Texas of Southwestern Medical Center, Dallas, TX, USA.
J Biomol Struct Dyn. 2023 May;41(8):3295-3304. doi: 10.1080/07391102.2022.2050947. Epub 2022 Mar 28.
Polymerization and depolymerization of actin play an essential role in eukaryotic cells. Actin exists in cells in both monomeric (G-actin) and filamentous (polymer, F-actin) forms. Actin binding proteins (ABPs) facilitate the transition between these two states, and their interactions with these two states of actin are critical for actin-based cellular processes. Rapid depolymerization of actin is assisted in the brain and/or other cells by its oxidation by the enzyme Mical (yielding Mox-actin), and/or by the binding of Inverted Formin 2 (INF2) - which can also accelerate filaments formation. At their stoichiometric molar ratio INF2 and actin yield the 8S complex (consisting of 4 actin monomers: 2 INF2 dimer molecules). Using biochemical and biophysical methods, we investigate the structural arrangement of actin in the 8S particles and the interaction of INF2 with actin and Mox-actin. To that end, we show 2 D class averages of 8S particles obtained by negative staining electron microscopy. We also show that: (i) 8S particles can seed rapid actin assembly; (ii) Mox-actin and INF2 form 8S particles at proteins ratios similar to those of unoxidized actin; (iii) chemical crosslinkings suggest that actin monomers are in a parallel orientation in the 8S particles of both actin and Mox-actin; and (iv) INF2 accelerates the disassembly of Mox-F-actin. Our results provide better understanding of actin's arrangement in the 8S particles formed during actin depolymerization and in the early polymerization stages of both actin and Mox-actin.Communicated by Ramaswamy H. Sarma.
肌动蛋白的聚合和解聚在真核细胞中起着至关重要的作用。肌动蛋白以单体（G-肌动蛋白）和丝状（聚合，F-肌动蛋白）两种形式存在于细胞中。肌动蛋白结合蛋白（ABP）促进这两种状态之间的转变，它们与肌动蛋白这两种状态的相互作用对于基于肌动蛋白的细胞过程至关重要。在大脑和/或其他细胞中，通过酶 Mical 对肌动蛋白的氧化（产生 Mox-肌动蛋白）以及/或者通过倒位形成蛋白 2（INF2）的结合来辅助肌动蛋白的快速解聚，INF2 也可以加速纤维丝的形成。在其化学计量摩尔比下，INF2 和肌动蛋白产生 8S 复合物（由 4 个肌动蛋白单体组成：2 个 INF2 二聚体分子）。使用生化和生物物理方法，我们研究了肌动蛋白在 8S 颗粒中的结构排列以及 INF2 与肌动蛋白和 Mox-肌动蛋白的相互作用。为此，我们展示了通过负染色电子显微镜获得的 8S 颗粒的 2D 类平均图像。我们还表明：（i）8S 颗粒可以促进肌动蛋白的快速组装；（ii）Mox-肌动蛋白和 INF2 在与未氧化肌动蛋白相似的蛋白比例下形成 8S 颗粒；（iii）化学交联表明，在肌动蛋白和 Mox-肌动蛋白的 8S 颗粒中，肌动蛋白单体呈平行取向；（iv）INF2 加速 Mox-F-肌动蛋白的解聚。我们的结果提供了对肌动蛋白在肌动蛋白解聚过程中形成的 8S 颗粒以及肌动蛋白和 Mox-肌动蛋白早期聚合阶段中的排列的更好理解。由 Ramaswamy H. Sarma 传达。
