Mechanism and effects of communication between actin and gene regulatory complexes

肌动蛋白与基因调控复合物之间通讯的机制和影响

• 批准号: 10432121
• 负责人: Ryan David Mohan
• 金额: $ 38.03万
• 依托单位: UNIVERSITY OF MISSOURI KANSAS CITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10432121
• 关键词: Actins; Affinity Chromatography; Antibodies; Binding; Biological Process; Blindness; Cell Nucleus; Cells; Characteristics; Column Chromatography; Communication; Complex; Coupled; Cytoplasm; Cytoskeletal Modeling; DNA Repair Pathway; Defect; Disease; Dominant-Negative Mutation; Ensure; Equilibrium; Eye; F-Actin; Functional disorder; Gene Expression; Genetic Transcription; Genome; Homologous Gene; Image; Immunoprecipitation; In Situ Hybridization; Knowledge; Lamins; Lead; Link; Location; Longevity; Mass Spectrum Analysis; Measures; Membrane; Messenger RNA; Molecular; Multienzyme Complexes; Mutate; Mutation; Nerve Degeneration; Nervous system structure; Neuro-Ocular System; Nuclear; Nuclear Inner Membrane; Nuclear Pore; Nuclear Pore Complex; Nuclear Protein; Pathway interactions; Phenotype; Polymers; Protein Family; Proteins; Publishing; Regulation; Regulator Genes; Regulatory Pathway; Reporter; Retina; Role; SAGA; SCA7 protein; Shapes; Site; Symptoms; System; Tail; Testing; Tissues; Transcript; Transcription Coactivator; WAVE protein; Wiskott-Aldrich Syndrome; base; experimental study; fly; in vivo; insight; mRNA Export; mutant; novel; novel therapeutics; polyglutamine; polymerization; prevent; promoter; protein complex; recruit; repaired; spatiotemporal

Abstract Although many studies have demonstrated correlations between cytoskeletal dynamics, genome organization, and gene expression, the underlying mechanisms linking them remain unclear. Recently, we discovered crosstalk between the Wiskott-Aldrich syndrome protein family verprolin homolog (WAVE) regulatory complex (WRC), which promotes actin polymerization, and the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, a transcriptional coactivator. Their relationship is established through the sharing of subunits comprising the SAGA deubiquitinase module, including the deubiquitinase Non-stop. Deubiquitinase module mutations—for example, polyglutamine expansion in ataxin 7 (ATXN7)—lead to a spectrum of phenotypes not explained by SAGA’s transcriptional coactivator function, including nervous system degeneration and blindness. In both the nervous system and the eye, the WRC is an essential promoter of actin polymerization, which is facilitated by the constitutively active enzymatic subunit WAVE. WAVE activity and localization are regulated by the remaining WRC subunits, ensuring spatial and temporal control of actin polymerization. Misregulation of actin polymerizing complexes results in a similar spectrum of phenotypes as seen in SAGA mutants, including nervous system degeneration and blindness. This suggests that SAGA is important in the nervous system and eye because it is required to control the WRC in these tissues. We found that the SAGA deubiquitinase module leaves SAGA to bind the WRC. There, Non-stop deubiquitinates WAVE, increasing its level in both the cytoplasm and the nucleus. Therefore, we hypothesize that SAGA controls WRC complex composition, amount, and location; and it is through these activities that SAGA accomplishes functions we had previously attributed to SAGA alone. This hypotheses will be investigated in three aims. First, we will identify and characterize nuclear WAVE-containing complexes through affinity purification and column chromatography coupled to mass spectrometry. Because WAVE activity is regulated in part by its localization, the locations of these complexes will be determined, and the effects of Atxn7 polyglutamine expansion on complex composition and localization will be tested. Second, interactions between the SAGA deubiquitinase module and WRC will be disrupted in cells and flies to determine which SAGA/WRC functions require them. Lastly, the effects of SAGA deubiquitinase-WRC interactions on blindness and neurodegeneration will be investigated in flies, by disrupting them and measuring phenotypes characteristic of Atxn7 polyglutamine expansion. These studies will provide novel insight on the causes of neurodegeneration and blindness, in addition to the links between transcriptional and cytoskeletal regulatory complexes.

摘要 尽管许多研究已经证明了细胞骨架动力学、基因组组织、 与基因表达之间的联系，其潜在机制尚不清楚。最近，我们发现 Wiskott-Aldrich综合征蛋白家族Verprolin同系物（WAVE）调节复合物之间的串扰 (WRC)和Spt-Ada-Gcn 5-乙酰转移酶（佐贺）复合物， 转录辅激活因子它们之间的关系是通过共享组成 佐贺去泛素化酶模块，包括去泛素化酶不停。去泛素化酶模块突变 例如，共济失调蛋白7（ATXN 7）中的多聚谷氨酰胺扩增-导致一系列表型， 佐贺的转录辅激活因子功能包括神经系统变性和失明。两者中 在神经系统和眼睛中，WRC是肌动蛋白聚合的重要促进剂， 组成型活性酶亚基WAVE。WAVE的活性和定位由 剩余的WRC亚基，确保肌动蛋白聚合的空间和时间控制。肌动蛋白失调 聚合复合物导致与佐贺突变体中所见相似的表型谱，包括 神经系统退化和失明这表明佐贺在神经系统中很重要， 因为需要控制这些组织中的WRC。我们发现佐贺去泛素化酶模块 让佐贺来绑定WRC。在那里，不停地去泛素化WAVE，增加其在两个组织中的水平。 细胞质和细胞核。因此，我们假设佐贺控制WRC复合物的组成， 数量和位置;正是通过这些活动，佐贺完成了我们以前的功能， 只属于佐贺。这些假设将在三个目标进行研究。首先，我们将确定和 通过亲和纯化和柱色谱法表征含核WAVE的复合物 与质谱联用由于WAVE活动部分受其定位的调节， 这些复合物将被确定，Atxn 7多聚谷氨酰胺扩增对复合物的影响 将测试组成和定位。第二，佐贺去泛素化酶模块之间的相互作用 和WRC将在细胞和果蝇中被破坏，以确定哪些佐贺/WRC功能需要它们。最后 佐贺去泛素化酶-WRC相互作用对失明和神经变性的影响将在 苍蝇，通过破坏它们和测量Atxn 7多聚谷氨酰胺扩增的表型特征。这些 研究将提供新的见解神经变性和失明的原因，除了链接 在转录和细胞骨架调节复合物之间。