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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 許家維 | zh_TW |
| dc.contributor.advisor | Jia-Wei Hsu | en |
| dc.contributor.author | 張乃心 | zh_TW |
| dc.contributor.author | Nai-Hsin Chang | en |
| dc.date.accessioned | 2025-09-24T16:51:15Z | - |
| dc.date.available | 2025-09-25 | - |
| dc.date.copyright | 2025-09-24 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-08-05 | - |
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PLoS Biol 19(7): e3001361. 42. Zhu, H., H. Yu, H. Zhou, W. Zhu and X. Wang (2023). "Elevated Nuclear PHGDH Synergistically Functions with cMyc to Reshape the Immune Microenvironment of Liver Cancer." Adv Sci (Weinh) 10(17): e2205818. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/100207 | - |
| dc.description.abstract | 磷酸甘油酸脫氫酶（PHGDH）在傳統上被視為一種參與絲胺酸（serine）新生合成的代謝酵素，且已有研究指出其透過典型與非典型功能參與癌症調控。在本研究中，我們發現PHGDH具有一項先前未被揭示的非典型功能，即在不依賴其代謝活性的情況下，參與溶酶體（lysosome）生合成的調控。當PHGDH被剔除時，轉錄因子EB（TFEB）與轉錄因子E3（TFE3）發生核移位（nuclear translocation），進而促使溶酶體與自噬相關基因表現上升、蛋白質表現量增加，並導致溶酶體體積膨大。這些變化無法透過補充絲胺酸逆轉，卻可由失去酵素活性的PHGDH突變體所挽救，進一步支持此作用為一種與代謝功能無關的非典型機制。此外，PHGDH的剔除亦會降低mTOR活性，顯示在正常情況下，PHGDH可能透過維持mTOR訊號傳導，使TFEB與TFE3滯留於細胞質中。為進一步釐清其功能機制，我們進行蛋白質結構域的鑑定，並透過結構域剔除實驗發現，SUB2與REG結構域對於PHGDH調控溶酶體生合成至關重要。缺失任一結構域皆會損害PHGDH的溶酶體定位能力，並導致TFEB與TFE3進入細胞核，以及溶酶體膨大等表徵。本研究揭示了PHGDH在溶酶體生合成中的一項新穎且非典型的功能，該功能獨立於其代謝活性，拓展了我們對PHGDH生理角色的理解。 | zh_TW |
| dc.description.abstract | Phosphoglycerate dehydrogenase (PHGDH) is traditionally known for its metabolic role in de novo serine biosynthesis and has been implicated in cancer regulation through both canonical and non-canonical functions. In this study, we identify a novel role of PHGDH in regulating lysosome biogenesis independently of its metabolic activity. PHGDH depletion induces nuclear translocation of the transcription factors TFEB and TFE3, resulting in elevated expression of lysosome- and autophagy-related genes, increased protein levels, and lysosomal enlargement. These changes are not reversed by serine supplementation and are rescued by catalytically inactive PHGDH mutants, supporting a non-metabolic mechanism. Additionally, PHGDH depletion reduces mTOR activity, suggesting that PHGDH may sustain mTOR signaling to retain TFEB and TEF3 in the cytoplasm under normal conditions. Further analysis using domain-deletion mutants reveals that the SUB2 and REG domains are critical for PHGDH’s function in lysosome biogenesis. Specifically, loss of either domain impairs PHDGH’s lysosomal localization and its ability to prevent TFEB and TFE3 nuclear translocation and lysosomal enlargement. These findings uncover a previously unrecognized role for PHGDH in the regulation of lysosome biogenesis independent of its canonical metabolic function. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-09-24T16:51:15Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-09-24T16:51:15Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員會審定書 I
致謝 II 摘要 III Abstract IV Table of contents V Chapter 1 Introduction 1 1.1 Canonical and non-canonical functions of phosphoglycerate dehydrogenase (PHGDH) 1 1.2 Amino acid deprivation induces lysosomal acidification and enlargement 4 1.3 TFEB and TFE3-mediated transcriptional regulation of lysosome biogenesis 5 1.4 Study background 6 Chapter 2 Materials and Methods 9 2.1 Cell line and cell culture 9 2.2 siRNA transfection (knockdown) 9 2.3 Plasmid transfection 9 2.4 Plasmid construct 10 2.5 Immunofluorescence 10 2.6 Western Blotting 11 2.6.1 Sample preparation 11 2.6.2 SDS-PAGE and immunoblotting 12 2.7 Nuclear fractionation 12 2.8 qPCR 13 2.9 Membrane fractionation 13 2.10 Confocal microscopy 14 2.11 Analysis 14 2.12 Chemicals 15 2.12.1 Reagents 15 2.12.2 siRNA sequence 16 2.12.3 Antibodies 16 2.12.4 Primers 18 Chapter 3 Results 22 3.1 TFEB and TFE3 translocate to the nucleus and regulate gene expression upon PHGDH depletion 22 3.2 PHGDH depletion enlarges lysosomes and enhances lysosomal function 27 3.3 The PHGDH-caused lysosome enlargement is independent of PHGDH's canonical function 28 3.4 The PHGDH SUB2 domain is dominantly involved in the lysosome biogenesis pathway 30 Chapter 4 Discussion 32 4.1 The effects of PHGDH depletion 32 4.2 PHGDH deficiency leads to a decrease in mTOR activity 33 4.3 The lysosomal surface localization of PHGDH depends on its REG domain 34 4.4 The possible mechanism of PHGDH in the regulation of lysosome biogenesis 35 4.5 PHGDH depletion impairs cell viability in a way independent of serine-biosynthesis function 36 Chapter 5 Figures 38 Figure 1. Knocking down PHGDH leads to lysosome enlargement 38 Figure 2. PHGDH deprivation causes TFEB nuclear translocation 39 Figure 3. TFE3-GFP is enriched in the nuclear fraction when PHGDH is knocked down. 41 Figure 4. The TFEB-related genes are upregulated when PHGDH is knocked out 42 Figure 5. The protein levels of TFEB-target genes are increased 43 Figure 6. The upregulations of lysosome-related genes expression and LysoTracker content are mediated by TFEB and TFE3 44 Figure 7. The LAMP1-and LAMP2-positive compartments are enlarged when PHGDH is knocked out 46 Figure 8. The enlarged lysosomes also have a function when PHGDH is knocked out 47 Figure 9. Knocking down PHGDH decreases HeLa cell viability 48 Figure 10. Extracellular serine- and glycine-deprivation does not affect lysosome sizes in control cells 49 Figure 11. The increase in lysosome sizes is independent of serine deprivation when PHGDH is knocked out 50 Figure 12. The increase in LysoTracker content in PHGDH knockout cells is independent of serine deprivation 51 Figure 13. The decrease in cell viability caused by PHGDH knockdown is independent of serine deprivation 52 Figure 14. The TFEB nuclear translocation is independent of PHGDH enzyme activity. 53 Figure 15. The SUB2 and REG domains of PHGDH play a critical role in retaining TFEB in the cytoplasm. 54 Figure 16. The REG domain of PHGDH plays a more important role in regulating lysosome size 56 Figure 17. The SUB2 and REG domains of PHGDH are required for PHGDH lysosomal localization. 58 Figure 18. PHGDH is involved in the regulation of lysosome biogenesis 59 Figure 19. Loss of the SUB2 domain affects the conformation of the REG domain 60 Reference 62 | - |
| dc.language.iso | en | - |
| dc.subject | 磷酸甘油酸脫氫酶 | zh_TW |
| dc.subject | 溶酶體生合成 | zh_TW |
| dc.subject | 轉錄因子E3 | zh_TW |
| dc.subject | 轉錄因子EB | zh_TW |
| dc.subject | 非典型功能 | zh_TW |
| dc.subject | Non-canonical function | en |
| dc.subject | TFEB | en |
| dc.subject | TFE3 | en |
| dc.subject | PHGDH | en |
| dc.subject | Lysosome biogenesis | en |
| dc.title | 非典型磷酸甘油酸脫氫酶調控溶酶體的生合成 | zh_TW |
| dc.title | Non-canonical Phosphoglycerate Dehydrogenase Regulates Lysosome Biogenesis | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 劉雅雯;詹智強;李家瑋 | zh_TW |
| dc.contributor.oralexamcommittee | Ya-Wen Liu;Chih-Chiang Chan;Chia-Wei Lee | en |
| dc.subject.keyword | 溶酶體生合成,磷酸甘油酸脫氫酶,非典型功能,轉錄因子EB,轉錄因子E3, | zh_TW |
| dc.subject.keyword | Lysosome biogenesis,PHGDH,Non-canonical function,TFEB,TFE3, | en |
| dc.relation.page | 69 | - |
| dc.identifier.doi | 10.6342/NTU202503977 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2025-08-11 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科學研究所 | - |
| dc.date.embargo-lift | 2030-08-05 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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