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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳昆峯 | zh_TW |
| dc.contributor.advisor | Kuen-Phon Wu | en |
| dc.contributor.author | 陳沛慈 | zh_TW |
| dc.contributor.author | Pei-Tzu Chen | en |
| dc.date.accessioned | 2023-10-03T16:09:52Z | - |
| dc.date.available | 2023-11-10 | - |
| dc.date.copyright | 2023-10-03 | - |
| dc.date.issued | 2023 | - |
| dc.date.submitted | 2023-06-26 | - |
| dc.identifier.citation | 1. Ivashkiv, L.B. and L.T. Donlin, Regulation of type I interferon responses. Nat Rev Immunol, 2014. 14(1): p. 36-49.
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Mol Cancer Ther, 2008. 7(12): p. 3780-8. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90457 | - |
| dc.description.abstract | 泛素化(Ubiquitination)作為其中一項蛋白質後轉譯修飾途徑，對於細胞功能調節上影響甚鉅。ISG15是一種類泛素蛋白質(Ubiquitin-like protein)，與其特定的相關修飾酵素E1 (UBA7)、 E2 (UBE2L6)、E3 (HERC5)皆受到第一類干擾素(Type I Interferon)、病毒和細菌感染的強烈誘導，而ISG15既可以做為獨立蛋白通過與其他蛋白交互作用以影響其功能，也可以作為標記直接與目標蛋白產生鍵結(ISGylation)，並且通常與抗病毒等免疫反應相關。然而，不同於ubiquitin修飾途徑上各個相關酵素的作用與機制上的豐富研究，ISG15與其專屬修飾系統尚未被完整理解。在此，除了設法得知UBA7的蛋白結構資訊外，我們還進一步發現 UBA7 與UBE2L6兩者之間的催化中心半胱氨酸，非常容易產生雙硫鍵鍵結，且該鍵結負向面影響二者的酵素活性，因而阻礙了 ISGylation。但是相比於其他不同修飾途徑中同功能的蛋白酵素，這並不是一個常見的情況。通常需要在一定氧化劑添加，才能產生E1及E2之間的雙硫鍵鍵結，而這被認為是細胞面臨氧化壓力下的調控手段之一。目前還尚未有與之相關的文獻闡述，而本篇研究目的目的是去了解UBA7、UBE2L6與不同種類E1,E2之間的結構、物性、及化性等差異，找出導致兩者極容易產生雙硫鍵鍵結的機制與原因，以便更全面的認識該修飾途徑的調控方式。在此，我們發現E1與E2之間的結合親和力與酵素自身催化中心的微環境特性為非常關鍵的決定因素，影響了E1及E2之間雙硫鍵的產生的速率以及對於所需環境、蛋白濃度的要求。有鑑於先前的研究報導指出，目標蛋白質受到ISG15修飾可以造成功能上的獲得或喪失，因此只需一小部分的蛋白質被進行ISGylation，即可發揮強大的作用。故我們推測雙硫鍵的產生可能是一種對於ISG15修飾途徑中負回饋調節型式，以避免造成過量非預期的ISG15修飾。 | zh_TW |
| dc.description.abstract | The expression of the ubiquitin-like molecule ISG15 and its associated E1, E2, and E3 enzymes is induced by type I interferons, highlighting their role in antiviral defense. However, the precise mechanism of ISG15 activation, conjugation, and ISGylation remains unknown. In this study, we identified a unique disulfide-covalent form between the catalytic cysteines of UBA7 and UBE2L6. This disulfide bond inhibits UBA7 and downstream catalytic activity, rendering the ISGylation process inactive. Interestingly, we found that the oxidation of the catalytic cysteines of E1 and E2 in response to environmental ROS factors is crucial for stress response. This novel finding suggests an additional regulatory mechanism for ISGylation. Through enzymatic kinetics and biophysical analysis, we unraveled the intricate interactions between UBA7 and UBE2L6, proposing a mechanism for the formation of the UBA7-UBE2L6 disulfide bond. Firstly, UBA1 possesses a longer cysteine capping loop that shields the catalytic cysteine, facilitating thioester bond formation with ubiquitin. In contrast, UBA7's shorter capping loop exposes its active site, making it more prone to solvent exposure and higher deprotonation potential. Secondly, the presence of acidic residues in UBA1's loop increases the active site's pKa value, reducing its reactivity. Thirdly, UBA7 exhibits a significantly stronger binding affinity for UBE2L6 compared to UBA1 and its respective E2s. The findings shed light on the structural factors governing UBA7 activity through E1-E2 disulfide formation and reveal an autoinhibitory regulatory mechanism in the ISG15-mediated pathway. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-10-03T16:09:52Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2023-10-03T16:09:52Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 摘要 i
Abstract ii Abbreviations iv 1. Introduction 1 1.1. ISG15 signaling and IFN response 1 1.1.1. The innate antiviral immunity 1 1.1.2. ISG15 and its conjugation 4 1.2. Modification pathway for ISG15 8 1.3. Ubiquitin-like modifier-activating enzyme and thioester transfer reaction 11 1.3.1. Ubiquitin-like modifier-activating enzymes (E1) 11 1.3.2. UBA7, UBA1, and UBA6 13 1.4. What is unknown? 17 2. Materials and Methods 20 2.1. DNA Constructs 20 2.2. Protein expression and purification 26 2.3. Fluorescent probe labeling ISG15 29 2.4. Biochemical assays 31 2.4.1. Transthioesterification kinetic 31 2.4.2. E1-E2 disulfide formation rate 32 2.4.3. bUBA7~bISG15 thioester formation rate 32 2.4.4. Data analysis with ImageJ and pro Fit 7 32 2.5. Biolayer Interferometry (BLI) assay for measuring protein binding affinity 34 2.6. Determination of the pKa value of E1 or E2 catalytic cysteine 36 2.6.1. pH Dependence inactivation of the active site of E1s[43] 36 2.6.2. Determination of thiol pKa by isothermal titration microcalorimetry[45] 39 2.7. Hydrogen exchange mass spectrometry (HDX-MS) 41 2.7.1. Brief introduction of HDX-MS[46] 41 2.7.2. Procedure 42 2.8. AlphaFold structure prediction 45 2.9. Size exclusion chromatography with multi-angle light scatter (SEC-MALS) 46 2.10. Cryo-EM sample preparation and data collection 47 3. Results 49 3.1. Disulfide formation between the catalytic cysteines of UBA7 and UBE2L6 49 3.1.1. A cryptic and atypical disulfide-bonded form 49 3.2. UBA7 structure and E1-E2 complex prediction with AlphaFold 54 3.2.1. The overall structure of UBA7 54 3.2.2. E1-E2 complex models 55 3.3. Insight into ubiquitin-like E1 and E2 interaction using HDX-MS 62 3.3.1. Deuterium uptake for bUBA7, bUBA7-UbcB8, hUBA1, and hUBA1-UbcH7 62 3.3.2. UBA7 Catalytic mechanism and active site remodeling 66 3.4. Chimeric proteins for the E1 functional study 74 3.5. The UFD and SCCH domains of E1s 78 3.5.1. UFD, while necessary, is not the most critical determinant for bUBA7 78 3.6. E2 specificity 81 3.7. High binding affinity and E1- E2 disulfide formation 87 3.7.1. High binding affinity between bUBA7 and UbcB8 87 3.7.2. The correlation between binding affinity and disulfide formation 89 3.7.3. E2~ISG15 production 91 3.8. The deprotonation potential of E1 & E2 catalytic cysteine 98 3.8.1. From deprotonation to oxidation state (S-S) 98 3.8.2. The cysteine capping loop and the pKa of UBA7’s catalytic cysteine 99 3.8.3. UbcB8 / UbcH8 / UbcH7 have lower pKa compared to other E2s 101 4. Discussion and conclusion 110 4.1. ROS and PTM 110 4.1.1. SUMO E1~E2 disulfide 111 4.1.2. Ubiquitin E1~E2 disulfide 114 4.1.3. Atg8 E1-E2 disulfide 117 4.1.4. What about the ISGylation pathway 118 4.2. UBA7~UBE2L6 formation and UBA7 evolution 120 4.3. Different viewpoints for ISGylation 124 4.4. Conclusion 128 5. References 130 | - |
| dc.language.iso | en | - |
| dc.title | 深入解析ISG15調控途徑中UBA7與UBE2L6之間雙硫鍵形成機制 | zh_TW |
| dc.title | Insight into the switch-off mechanism of disulfide-bonded UBA7- UBE2L6 in the ISG15-regulated pathway | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 陳瑞華;徐尚德;張語曲 | zh_TW |
| dc.contributor.oralexamcommittee | Ruey-Hwa Chen;Shang-Te Hsu;Yu-Chu Chang | en |
| dc.subject.keyword | 泛素,類泛素蛋白質,E1-E2雙硫鍵結,ISG15, | zh_TW |
| dc.subject.keyword | Ubiquitin,ubiquitin-like protein,ISG15,ISGylation,E1-E2 disulfide-bonded, | en |
| dc.relation.page | 137 | - |
| dc.identifier.doi | 10.6342/NTU202301154 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2023-06-28 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科學研究所 | - |
| dc.date.embargo-lift | 2028-06-25 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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