CAREER: Isoform-Dependent Redox Regulation of Actin

本奖项全部或部分由《2021年美国救援计划法案》（公法117- 2）资助。肌动蛋白是真核细胞中一种必需的细胞骨架蛋白，它经历了可逆的单体到聚合物的转变。这导致了各种各样的功能配置，从肌动蛋白参与肌肉收缩到突触可塑性。为了支持各种各样的功能，肌动蛋白聚合物被不断地分解成单体，并以严格调节的方式重新组装成聚合物。本研究的重点是通过一种新的还原/氧化（氧化还原）机制调节肌动蛋白动力学。这一机制是由micical酶（分子与Cas L相互作用）精心安排的，micical酶通过靶向氧化其序列中的两个保守氨基酸来促进肌动蛋白聚合物的分解，而这一过程反过来又可以被蛋氨酸亚氧化物还原酶（MsrB/SelR）逆转。在其聚合物形式下，肌动蛋白不仅作为micical的底物，而且作为这些酶产生过氧化氢的活化剂，这可能导致健康和疾病中更广泛的氧化还原信号。细胞分裂、肌肉、心脏和神经元发育等基本细胞过程需要micical的活性，然而，对不同细胞类型中肌动蛋白结构如何受到micical /MsrB氧化还原机制影响的分子水平理解是有限的，本研究将解决这一问题。获得的知识将有助于更全面地了解基本的依赖于动作蛋白的细胞过程。这项工作的广泛影响包括研究和教育的紧密结合。该项目将对具有竞争力的STEM劳动力的发展产生积极影响，使妇女、第一代学生和代表性不足的少数民族充分参与STEM，通过开发免费的网络资源提高当地STEM教育工作者的发展，提高公众的科学素养。美国救援计划的资金为这位调查员在职业生涯的关键阶段提供了支持。肌动蛋白细胞骨架是真核细胞生存所必需的。在哺乳动物中，多种基于肌动蛋白的结构是由六种不同的同种异构体构建的，这些异构体以细胞类型特异性的方式表达。这些细胞骨架结构是如何根据它们的肌动蛋白异构体组成进行差异调节的，这是一个悬而未决的基本问题。该项目的目标是了解肌动蛋白同工异构体的动力学是如何由micical和MsrB酶协调的新机制进行差异调节的，该机制涉及肌动蛋白序列中两个蛋氨酸的位点特异性氧化/还原。要验证的中心假设是，在mical诱导的氧化、肌动蛋白动力学、稳定性、相互作用和mical介导的过氧化氢生产的激活是肌动蛋白异构体依赖的。这将通过结合合成生物学、生物化学、生物物理学和TIRF成像来解决，并将允许定义肌动蛋白的关键特征，这些特征决定了它对mical诱导的拆卸和mrsb驱动的重组的易感性。这些知识将广泛适用，并允许基于局部肌动蛋白异构体组成的化学激活的生物学后果的预测。所获得的知识将有助于深入了解micical /MsrB氧化还原机制在不同细胞、组织和生物体中的作用。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117- 2).Actin is an essential cytoskeletal protein in eukaryotic cells that undergoes reversible monomer-to-polymer transitions. This results in a diverse array of functional configurations, ranging from actin involvement in muscle contraction to synaptic plasticity. To support such variety of functions, actin polymers are continuously disassembled to monomers and reassembled to polymers in a tightly regulated manner. This research is focused on regulation of actin dynamics by a novel reduction/oxidation (redox) mechanism. This mechanism is orchestrated by MICAL enzymes (Molecule Interacting with Cas L) that promote disassembly of actin polymers via targeted oxidation of two conserved amino acids in its sequence which, in turn, can be reversed by Methionine sulfoxide reductases (MsrB/SelR). In its polymer form, actin serves not only as a substrate of Mical but also an activator of a hydrogen peroxide production by these enzymes which potentially can lead to a broader redox signaling in health and disease. Essential cellular processes such as cell division, muscle, heart, and neuronal development require activity of MICAL, however, molecular level understanding of how actin structures in different cell types are impacted by Mical/MsrB redox mechanism is limited and will be addressed by this research. Acquired knowledge will contribute to a more complete understanding of fundamental actin-dependent cellular processes. The Broader Impacts of this work comprise a tight integration of research and education. This project will have a positive impact on development of a competitive STEM workforce, full participation of women, first generation students, and underrepresented minorities in STEM, improved STEM educator development at the level of local increased public scientific literacy via development of a free web resource. American Rescue Plan funding provides support for this investigator at a critical stage in her career.The actin cytoskeleton is indispensable for viability of eukaryotic cell. In mammals, a variety of actin-based structures are built from six different isoforms that are expressed in a cell type-specific manner. How these cytoskeletal structures are differentially regulated based on their actin isoform composition is an open fundamental question. The goal of this project is to understand how dynamics of actin isoforms are differentially regulated by a novel mechanism orchestrated by Mical and MsrB enzymes which involves site-specific oxidation/reduction of two methionines in actin’s sequence. The central hypothesis to be tested is that upon Mical-induced oxidation, actin dynamics, stability, interactions, and activation of Mical-mediated hydrogen peroxide production are actin isoform-dependent. This will be addressed by combining synthetic biology, biochemistry, biophysics, and TIRF imaging, and will allow defining the key features of actin that determine it susceptibility to Mical-induced disassembly and MrsB-driven reassembly. This knowledge will be broadly applicable and allow for prediction of the biological consequences of Mical activation based on the local actin isoform composition. The acquired knowledge will contribute to in-depth understanding of how the Mical/MsrB redox mechanism functions in different cells, tissues, and organisms.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.