The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirm this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin, and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics.

中文翻译：

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PFN2 和 NAA80 协同作用，有效乙酰化肌动蛋白的 N 末端。


肌动蛋白细胞骨架对于细胞形状、分裂和细胞内力的产生具有极其重要的意义。 Profilin 与球状 (G-) 肌动蛋白结合并调节肌动蛋白丝的形成。尽管 profilin 是公认的肌动蛋白调节因子，但占主导地位的 profilin 1 (PFN1) 与含量较低的 profilin 2 (PFN2) 的不同作用仍然是个谜。在本研究中，我们利用相互作用蛋白质组学发现PFN2是肌动蛋白N末端乙酰转移酶NAA80的相互作用伴侣，并通过分析超速离心进一步证实了这一点。使用 NAA80 和不同的 Profilin 进行的酶测定表明，PFN2 结合特异性地增加了 NAA80 的内在催化活性。 NAA80 通过富含脯氨酸的环结合 PFN2，删除该环会消除 PFN2 结合。小角度 X 射线散射显示 NAA80、肌动蛋白和 PFN2 形成三元复合物，并且 NAA80 在 N 末端和富含脯氨酸的环中具有部分无序区域，后者在 PFN2 结合时部分有序。此外，PFN2 通过富含脯氨酸的环与 NAA80 结合，促进肌动蛋白球状结构域和 NAA80 之间的结合，从而促进肌动蛋白的乙酰化。然而，大多数细胞 NAA80 稳定地与 PFN2 结合，而不是与肌动蛋白结合，我们认为该复合物在 G-肌动蛋白并入肌丝之前将其乙酰化。总之，我们揭示了 PFN2 作为肌动蛋白 N 端乙酰转移酶 NAA80 的稳定相互作用子和调节剂的功能特异性作用，并建立了 NAA80 介导的肌动蛋白 N 端乙酰化的操作方式，这是一种对细胞骨架动力学产生重大影响的修饰。