阿拉伯芥 HSA32 之生化性質分析

Abstract

Heat-Stress-Associated 32 kDa Protein (HSA32)透過抑制 Heat Shock Protein 101 (HSP101)的降解，參與了阿拉伯芥和水稻之熱順應記憶(HAM，heat acclimation memory)，同時，HSP101 防止 HSA32 被快速降解。然而，HSP101 是如何調控 HSA32 的蛋白質穩定性與兩者之間的交互作用機制尚不清楚。為了暸解 HSA32 的 穩定性和其蛋白質功能之分子機制，我嘗試用生物化學實驗方法來探討阿拉伯芥 HSA32 的蛋白質特性。首先，透過大腸桿菌蛋白質表達系統表現阿拉伯芥 HSA32， 發現 HSA32 在大腸桿菌中形成不可溶的沈澱，且與 HSP101 共同表現時，只略微 增加了 HSA32 的溶解度，因此推論單獨 HSP101 並無法在大腸桿菌中顯著影響 HSA32 的溶解性，且透過大腸桿菌蛋白質表達系統純化 HSA32 目前面臨困難。接 著，我嘗試用酵母菌表現系統，但在純化 HSA32 上依然面臨困難。因此，我透過 使用膠體過濾層析(SEC)和蔗糖梯度離心法直接分離阿拉伯芥蛋白粗抽取，發現 HSA32 會形成高密度的大分子聚集體，無論有無 HSP101 存在，HSA32 的分佈均 未有顯著不同。並且，透過穿隧式電子顯微鏡觀察免疫金染色的阿拉伯芥切片發現， 熱處理後 HSA32-GFP 會在細胞質中聚集。研究結果雖然無法推斷 HSA32 的蛋白 結構與功能，且無法直接證明 HSA32 與 HSP101 間的交互機制，但提出了 HSA32 是一個易聚集的蛋白，此特性可能在植物生理功能中扮演重要角色。

Heat-Stress-Associated 32 kDa Protein (HSA32) is involved in plant heat acclimation memory (HAM) in Arabidopsis and rice by suppressing the degradation of Heat Shock Protein 101 (HSP101). Conversely, HSP101 prevents the rapid degradation of HSA32. To elucidate the molecular mechanisms underlying the stability and function of HSA32, I utilized biochemical approaches to characterize the protein properties of HSA32. Here, I demonstrate that Arabidopsis HSA32 forms insoluble aggregates with a slight increase in solubility when co-expressed with HSP101 in Escherichia coli. Overexpression of His- tagged HSA32 in Saccharomyces cerevisiae does not result in visible His signal when analyzed by immunoblotting, highlighting the challenges encountered in using these heterologous expression systems to characterize the protein properties of HSA32. Fractionation of Arabidopsis crude extract using size-exclusion chromatography (SEC) and sucrose gradient centrifugation, followed by immunoblot analysis, demonstrates that HSA32 forms macromolecular assemblies. However, whether in the presence or absence of HSP101, the distribution of HSA32 shows no significant difference, possibly due to the disruption of native interactions following cell breakage. Immunogold-labeling TEM revealed that HSA32 forms protein condensates in response to HS in transgenic plants. Together, my study shows that HSA32 is an aggregation-prone protein that forms aggregates upon cell breakage, and forms protein condensates following HS in vivo.