通過誘變表徵新型微管分支因子 SSNA1;Characterization of the novel microtubule-branching factor SSNA1 by mutagenesis

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Title:	通過誘變表徵新型微管分支因子 SSNA1;Characterization of the novel microtubule-branching factor SSNA1 by mutagenesis
Authors:	普秧卡;Vinothkumar, Priyanka
Contributors:	生命科學系
Keywords:	SSNA1;誘變;SSNA1;Mutagenesis
Date:	2021-08-11
Issue Date:	2021-12-07 11:27:28 (UTC+8)
Publisher:	國立中央大學
Abstract:	Sj?gren 綜合徵核自身抗原 1 (SSNA1) 最初被確定為 Sj?gren 綜合徵患者自身抗體的主要靶點，它影響了西方世界估計 0.5% 的人口，但其生物學功能和醫學相關性在很大程度上是未知的。除了其核定位外，SSNA1 還定位於幾個基於微管的結構，包括中心體和中體，因此可能調節細胞分裂。最近，據報導，除了中心體之外，SSNA1 還存在於神經元中，並促進軸突生長、伸長和分支。此外，最近的一份報告表明，SSNA1 重組蛋白自組裝成小簇和纖維，促進體外微管成核和分支。為了闡明 SSNA1 在微管重塑中的作用，我們開始在體外和細胞中系統地研究哺乳動物 SSNA1。首先，我們觀察到當在細胞中表達時，SSNA1 會自我相互作用並形成液滴狀結構，這讓人想起液液相分離 (LLPS) 現象。由於 LLPS 是由蛋白質內在無序區域賦予的多價相互作用介導的，我們使用 IUPred2A 和 PONDR 等生物信息學軟件分析了 SSNA1 的一級氨基酸序列，以預測疾病傾向。我們發現 SSNA1 帶有本質上無序的 N 和 C 末端。為了進一步確定液滴形成所需的基本殘基或基序，我們構建了一個 SSNA1 突變體文庫，其中包括一系列丙氨酸掃描和截短突變體以及幾種可能受有絲分裂和/或中心體定位激?調節的抗磷酸化和模擬突變體.通過在體外和體內表徵這些突變體，我們現在已經確定了控制 SSNA1 自組裝的關鍵基序和磷酸化依賴性殘基。;Sj?gren syndrome nuclear autoantigen 1 (SSNA1) was originally identified as a major target of autoantibodies in patients with Sj?gren syndrome, which affects an estimated 0.5% of the population of the western world, but its biological function and medical relevance are largely unknown. In addition to its nuclear localization, SSNA1 has been localized to several microtubule-based structures including centrosomes and the mid-body and may thus regulate cell division. Recently SSNA1 was also reported to be present in neurons apart from centrosomes and promotes axon growth, elongation and branching. Moreover, a recent report demonstrated that SSNA1 recombinant proteins self-assemble into small clusters and fibrils that promote microtubule nucleation and branching in vitro. To elucidate the role of SSNA1 in microtubule remodeling, we set out to systematically investigate mammalian SSNA1 in vitro and in cells. First, we observed that when expressed in cells, SSNA1 self-interacts and forms droplet-like structures, which is reminiscent of the liquid-liquid phase separation (LLPS) phenomenon. Since LLPS is mediated by multivalent interactions conferred by the intrinsically disordered regions of the proteins, we analyzed the primary amino acid sequence of SSNA1 using bioinformatics softwares such as IUPred2A and PONDR to predict disorder propensity. We found that SSNA1 carries intrinsically disordered N and C-termini. To further identify essential residues or motifs required for droplet formation, we constructed a SSNA1 mutant library which includes a series of alanine-scan and truncated mutants as well as several phosphorylation-resistant and mimicking mutants potentially regulated by mitotic and/or centrosome-localized kinases. By characterizing these mutants in vitro and in vivo, we have identified key motifs and phosphorylation-dependent residues that control SSNA1 self-assembly.
Appears in Collections:	[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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