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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 常怡雍 | zh_TW |
| dc.contributor.advisor | Yee-yung Charng | en |
| dc.contributor.author | 李泓毅 | zh_TW |
| dc.contributor.author | Hong-Yi Li | en |
| dc.date.accessioned | 2024-08-15T16:09:40Z | - |
| dc.date.available | 2024-08-16 | - |
| dc.date.copyright | 2024-08-15 | - |
| dc.date.issued | 2024 | - |
| dc.date.submitted | 2024-08-09 | - |
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Plant Cell Environ 32, 917-927 (2009). https://doi.org:10.1111/j.1365-3040.2009.01972.x | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/94195 | - |
| dc.description.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。 | zh_TW |
| dc.description.abstract | 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. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2024-08-15T16:09:40Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2024-08-15T16:09:40Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 誌謝 i
摘要 ii Abstract iii Contents v Chapter 1. Introduction 1 1.1 Heat tolerance in plants 1 1.2 Heat acclimation memory 2 1.3 Heat-Stress-Associated 32-kD Protein 3 1.4 Role of Heat Shock Protein 101 in thermotolerance 4 1.5 Molecular mechanism of HAM mediated by HSA32 and HSP101 6 1.6 Aims of the studies 7 Chapter 2. Materials and Methods 8 2.1 In silico predictions 8 2.2 Plant materials and heat treatment 9 2.3 Purification of HSA32 in denaturing condition 9 2.4 High-throughput mini dialysis 10 2.5 Co-expression of HSA32 with HSP101 in E. coli 11 2.6 Fractionation of soluble and insoluble protein from E. coli 11 2.7 Cloning, protein expression and purification using Saccharomyces cerevisiae 12 2.8 Protein extraction from Arabidopsis 13 2.9 Size-exclusion chromatography 14 2.10 Sucrose density gradient sedimentation 15 2.11 SDS-PAGE and immunoblotting 15 2.12 RNA extraction and quantitative reverse transcription PCR 16 2.13 Transmission electron microscopy and immunogold labeling 17 Chapter 3. Results 18 3.1 HSA32 is predicted to be a TIM barrel protein containing several short aggregation-prone regions 18 3.2 HSA32 is predicted to hold trimeric structure 19 3.3 HSA32 mainly forms amorphous, insoluble aggregates after purification from E. coli… 20 3.4 The solubility of HSA32 slightly increases when co-expressed with HSP101 in E. coli 21 3.5 HSA32 post-transcriptional stabilization of HSP101 in E. coli 22 3.6 Challenges in purifying HSA32 from S. cerevisiae due to undetectable His tag and ineffective nickel affinity column 23 3.7 HSA32 consistently appears in high molecular weight fractions regardless of heat stress or the presence of HSP101 in Arabidopsis crude extract 23 3.8 HSA32 forms protein condensates in vivo 25 Chapter 4. Discussion 27 4.1 Aggregation properties of HSA32 27 4.1.1 HSA32 is a TIM-barrel fold protein 27 4.1.2 HSA32 is an aggregation-prone protein with several APRs 28 4.1.3 HSA32 failed to refold to native structures in vitro 29 4.1.4 Currently, purifying HSA32 from yeast poses challenges 31 4.2 Interaction between HSA32 and HSP101 may be weak or indirect 32 4.2.1 No significant resolubilization of HSA32 was observed despite co-expression with HSP101 in E. coli 32 4.2.2 Co-expression of HSA32 leads to an accumulation of HSP101 protein levels…. 33 4.2.3 HSP101 may exhibit a disaggregase activity that effectively disassociates protein condensates of HSA32 33 4.3 The aggregation of HSA32 observed in plant crude extract may be attributed to non-specific interactions 34 4.4 HSA32 forms protein condensates in response to HS in vivo 35 4.5 Conclusion 36 References 37 Figures 45 Figure 1. Schematic model of HSA32 involvement in heat acclimation memory 45 Figure 2. HSA32 is a TIM-barrel protein 46 Figure 3. HSA32 is predicted to have several aggregation prone regions at unstructured regions 48 Figure 4. Trimeric HSA32 have highest structure similarity score in homomer prediction 49 Figure 5. HSA32 forms insoluble aggregates and failed to resolubilize after dialysis in vitro.. 50 Figure 7. HSP101 slightly increases the solubility of HSA32. In turn, HSA32 post-transcriptionally promotes the accumulation of HSP101 53 Figure 8. Loss-of-function mutations of HSA32 fail to promote the accumulation of HSP101 54 Figure 9. His tag at C terminus of HSA32 were cleaved when overexpress in S. cerevisiae 55 Figure 10. SEC fractionation of Arabidopsis crude extracts shows that the distribution of HSA32 is not significantly different with or without the presence of HSP101 56 Figure 11. Western blot analysis of sucrose gradient fractions of HSA32 demonstrates no significant difference in the presence or absence of HSP101 58 Figure 12. Membrane-less protein condensates were identified by TEM of immunogold labeled transgenic Arabidopsis seedlings 60 Appendix 62 Supplementary Table 1. List of primer 62 Supplementary Table 2. List of antibodies 63 Supplementary figure 1. Aggregation prone regions of HSA32 65 Supplementary figure 2. Identification of self-assembly hotspots of HSA32 66 Supplementary figure 3. Chromatogram of size-exclusion chromatography 67 | - |
| dc.language.iso | en | - |
| dc.title | 阿拉伯芥 HSA32 之生化性質分析 | zh_TW |
| dc.title | Biochemical Characterization of Arabidopsis HSA32 | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 112-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 蕭傳鐙;楊健志;葉靖輝 | zh_TW |
| dc.contributor.oralexamcommittee | Chwan-Deng Hsiao;Chien-Chih Yang;Ching-Hui Yeh | en |
| dc.subject.keyword | 阿拉伯芥,HSA32,HSP101,熱逆境,熱順應記憶,易聚集蛋白, | zh_TW |
| dc.subject.keyword | Arabidopsis,HSA32,HSP101,heat stress,heat acclimation memory,aggregation-prone protein, | en |
| dc.relation.page | 68 | - |
| dc.identifier.doi | 10.6342/NTU202403618 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2024-08-12 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科技學系 | - |
| 顯示於系所單位： | 生化科技學系 | |
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