去泛素酶是一類調節泛素活性的酵素，其中泛素特異性肽酶（DUB）與多種疾病相關。研究顯示，USP24在耐藥癌細胞中表達增加，透過促進轉運蛋白表現和誘導基因組不穩定性，增強抗藥性。因此，靶向USP24可能抑制或延緩治療引發的抗藥性。然而，USP24的結構尚未明確，我們基於USP7的結構模板，建立USP24催化中心模型，並透過分子對接篩選特異性抑制劑。近期開發的USP24特異性抑制劑NCI677397及其類似物USP24-i-101，能有效抑制癌症治療中的抗藥性。深入探究USP24催化結構域與抑制劑USP24-i-101之間的結構互作，是我們的首要目標，有助於未來對USP24-i-101進行優化。在本研究中，我們使用大腸桿菌表達系統構建USP24 catalytic domain的建構，但發現重組蛋白的溶解度並不理想。為了解決此問題，我們將蛋白表達系統轉換為哺乳動物表達系統。首先，我們成功將帶有His標籤的USP24 catalytic domain candidate no.8建構至pEGFP-C3載體中，並於HEK293細胞中進行表達。結果顯示蛋白的溶解度顯著改善。然而，蛋白純化結果顯示His-Nickel chromatography會因為結合非目標蛋白，進而影響蛋白的純度，這可能會導致之後的蛋白結晶的步驟受到干擾。為解決此問題，我們使用GFP-trap來純化目標蛋白，Coomassie Blue染色結果顯示，純度比使用His-Nickel chromatography來的更高。在成功解決蛋白溶解度及純度問題後，我們進一步構建含有TEV切割點的新序列，用來分離GFP與USP24 catalytic domain，結果顯示TEV蛋白酶能有效作用於GFP-TEV-USP24 catalytic domain重組蛋白，並成功分離出USP24 catalytic domain蛋白。在建立穩定表現的重組蛋白系統後，未來我們計畫提升蛋白的表達量，考慮利用腺病毒表現系統，或基於現有序列構建新的大腸桿菌系統載體。此外，我們也會運用生物資訊學及人工智慧技術，模擬USP24與USP24-i-101之間的交互作用，並嘗試進一步優化USP24-i-101的衍生物。綜上，探討USP24與USP24-i-101的結構關係，對開發針對USP24的抗藥性治療藥物至關重要。未來可將USP24抑制劑與化療或靶向藥物結合，形成雞尾酒療法，抑制抗藥性，並具有潛力成為臨床新藥。

Deubiquitinases are special enzymes which can regulating the activity of ubiquitin. Ubiquitin-specific peptidase is part of DUB superfamily which have been proved associated to many kinds of disease. Our previous studies revealed that ubiquitin-specific peptidase 24 (USP24) up-expression in drug resistant cancer cells through increase in transporters expression and inducing genomic instability, implying that targeting USP24 might inhibit, or delay drug resistance acquired from therapy. Because USP24 structure is yet determined now, so, we used USP7 structure as a parent template to model the catalytic motif of USP24, thereby screen the specific inhibitors by docking assay. Recently, a specific USP24 inhibitor, NCI677397, and its analog, USP24-i-101, have been developed to inhibit drug resistance in cancer therapy in vitro and in vivo. studying the structure interaction between catalytic domain of USP24 and USP24-i-101 in-depth is our priority, which will be beneficial for the optimization of USP24-i-101 in the future. In this study, we construct the USP24 catalytic domain in E. coli system, however, the recombinant protein solubility is not ideal. To solve this problem, we change the protein expression system into mammalian system. First, we successfully construct the USP24 catalytic domain candidate no.8 with His-tag into pEGFP-C3 vector and express in HEK293 cell. Result shows that the protein solubility has been well resolved. But the protein purification result indicated that His-Nickel chromatography will bind with no interest protein and further influence the protein purity, which may interrupt the protein crystallization step. For resolved this concern, we use GFP-trap to purify the target protein, Coomassie Blue result indicating the purity is better than using His-Nickel chromatography. After resolving solubility and purity issue, we construct a new sequence with TEV cutting site to separate GFP and USP24 catalytic domain, result indicating TEV protease can function well on GFP-TEV-USP24-catalytic domain protein and USP24-catalytic domain protein can be isolated. After building a stable express recombinant protein, we try to increase the expression quantity in the future, using adenovirus or constructing new plasmid with existing sequence for E. coli system. In addition, we will also use bioinformatics and AI technologies to study the interaction between modeling USP24 and USP24-i-101 and then try to optimize the USP24-i-101 derivatives. In summary, studying the relationship between the structure of USP24 and USP24-i-101 is important for the drug development of targeting USP24, which is highly potential to be as clinical therapeutics to inhibit drug resistance by cocktail treatment such as chemotherapy or target therapy drugs in the future.

Ambudkar SV, Kimchi-Sarfaty C, Sauna ZE, Gottesman MM. P-glycoprotein: from genomics to mechanism. Oncogene. 2003;22(47):7468-7485. doi:10.1038/sj.onc.1206948
Avvakumov GV, Walker JR, Xue S, et al. Amino-terminal dimerization, NRDP1/RNF41 binding, and inhibition of the deubiquitinase USP8. J Biol Chem. 2012;287(28):22417-22429. doi:10.1074/jbc.M112.362673
Bartoszewski R, Brewer JW, Rab A, Crossman DK, Bartoszewska S, Kapoor N, Fuller C, Collawn JF, Bebok Z. The unfolded protein response (UPR)-activated transcription factor X-box-binding protein 1 (XBP1) induces microRNA-346 expression that targets the human antigen peptide transporter 1 (TAP1) mRNA and governs immune regulatory genes. J Biol Chem. 2011;286(48):41862-41870. doi:10.1074/jbc.M111.252635
Bedford L, Lowe J, Dick LR, Mayer RJ, Brownell JE. Ubiquitin-like protein conjugation and the ubiquitin–proteasome system as drug targets. Nat Rev Drug Discov. 2011;10(1):29-46. doi:10.1038/nrd3321
Chen Z, Shi T, Zhang L, Zhu P, Deng M, Huang C, Hu T, Jiang L, Li J. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family in multidrug resistance: A review of the past decade. Cancer Lett. 2016;370(1):153-164. doi:10.1016/j.canlet.2015.10.010
Clague MJ, Coulson JM, Urbé S. Cellular functions of the DUBs. J Cell Sci. 2012;125(Pt 2):277-286. doi:10.1242/jcs.090985
Clague MJ, Urbé S, Komander D. Breaking the chains: deubiquitylating enzyme specificity begets function. Nat Rev Mol Cell Biol. 2019;20(6):338-352. doi:10.1038/s41580-019-0099-1
D’Arcy P, Wang X, Linder S. Deubiquitinase inhibition as a cancer therapeutic strategy. Pharmacol Ther. 2015;147:32-54. doi:10.1016/j.pharmthera.2014.11.002
Eichhorn PJ, Rodón L, Gonzalez-Juncà A, et al. USP15 stabilizes TGF-β receptor I and promotes oncogenesis through the activation of TGF-β signaling in glioblastoma. Nat Med. 2012;18(3):429-435. doi:10.1038/nm.2619
Eletr ZM, Wilkinson KD. Regulation of proteolysis by human deubiquitinating enzymes. Biochim Biophys Acta. 2014;1843(1):114-128. doi:10.1016/j.bbamcr.2013.06.027
Fraile JM, Quesada V, Rodríguez D, Freije JM, López-Otín C. Deubiquitinases in cancer: new functions and therapeutic options. Oncogene. 2012;31(19):2373-2388. doi:10.1038/onc.2011.443
Fulda S. Tumor resistance to apoptosis. Int J Cancer. 2009;124(3):511-515. doi:10.1002/ijc.24009
Fulda S. Evasion of apoptosis as a cellular stress response in cancer. Int J Cell Biol. 2010;2010:370835. doi:10.1155/2010/370835
Fulda S, Vucic D. Targeting IAP proteins for therapeutic intervention in cancer. Nat Rev Drug Discov. 2012;11(2):109-124. doi:10.1038/nrd3627
Glickman MH, Ciechanover A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev. 2002;82(2):373-428. doi:10.1152/physrev.00027.2001
Grabbe C, Husnjak K, Dikic I. The spatial and temporal organization of ubiquitin networks. Nat Rev Mol Cell Biol. 2011;12(5):295-307. doi:10.1038/nrm3099
Gu Y, Kaufman JL, Bernal L, et al. Upregulation of the USP24 deubiquitinase promotes drug resistance in multiple myeloma. J Clin Invest. 2020;130(9):4668-4679. doi:10.1172/JCI134716
Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646-674. doi:10.1016/j.cell.2011.02.013
Harrigan JA, Jacq X, Martin NM, Jackson SP. Deubiquitylating enzymes and drug discovery: emerging opportunities. Nat Rev Drug Discov. 2018;17(1):57-78. doi:10.1038/nrd.2017.152
Hayashi T, Motohashi H, Nakai A, et al. Human multidrug resistance-associated protein (MRP) 8/ABCC11 is a novel ATP-dependent efflux transporter for amphipathic anions. J Biol Chem. 2003;278(49):47052-47059. doi:10.1074/jbc.M308467200
Hoeller D, Hecker CM, Dikic I. Ubiquitin and ubiquitin-like proteins in cancer pathogenesis. Nat Rev Cancer. 2006;6(10):776-788. doi:10.1038/nrc1994
Huang TT, D’Andrea AD. Regulation of DNA repair by ubiquitylation. Nat Rev Mol Cell Biol. 2006;7(5):323-334. doi:10.1038/nrm1908
Hegde AN. The ubiquitin-proteasome pathway and synaptic plasticity. Learn Mem. 2010;17(7):314-327. doi:10.1101/lm.1506810
Issaenko OA, Amerik AY. Chalcone-based small-molecule inhibitors attenuate malignant phenotype via targeting deubiquitinating enzymes. Cell Cycle. 2012;11(9):1804-1817. doi:10.4161/cc.20134
Komander D, Clague MJ, Urbé S. Breaking the chains: structure and function of the deubiquitinases. Nat Rev Mol Cell Biol. 2009;10(8):550-563. doi:10.1038/nrm2731
Komander D, Rape M. The ubiquitin code. Annu Rev Biochem. 2012;81:203-229. doi:10.1146/annurev-biochem-060310-170328
Li M, Brooks CL, Kon N, Gu W. A dynamic role of HAUSP in the p53–Mdm2 pathway. Mol Cell. 2004;13(6):879-886. doi:10.1016/S1097-2765(04)00157-1
Li W, Bengtson MH, Ulbrich A, Matsuda A, Reddy VA, Orth A. Genome-wide and functional annotation of human E3 ubiquitin ligases identifies novel regulators of caspase-8 stability and apoptosis. Nat Biotechnol. 2008;26(12):1337-1346. doi:10.1038/nbt.1500
Liu J, Xia H, Kim M, et al. Beclin1 controls the levels of p53 by regulating the deubiquitination activity of USP10 and USP13. Cell. 2011;147(1):223-234. doi:10.1016/j.cell.2011.08.037
Liu Y, Lashuel HA, Choi S, Xing X, Case A, Ni J, Yeh LA, Cuny GD, Stein RL, Lansbury PT Jr. Discovery of inhibitors that elucidate the role of UCH-L1 activity in the H1299 lung cancer cell line. Chem Biol. 2003;10(9):837-846. doi:10.1016/j.chembiol.2003.08.009
Livnat-Levanon N, Glickman MH. Ubiquitin–proteasome system and mitochondria—reciprocity. Biochim Biophys Acta. 2011;1809(2):80-87. doi:10.1016/j.bbagrm.2010.07.004
Love KR, Catic A, Schlieker C, Ploegh HL. Mechanisms, biology and inhibitors of deubiquitinating enzymes. Nat Chem Biol. 2007;3(11):697-705. doi:10.1038/nchembio.2007.48
Luo J, Solimini NL, Elledge SJ. Principles of cancer therapy: oncogene and non-oncogene addiction. Cell. 2009;136(5):823-837. doi:10.1016/j.cell.2009.02.024
Luo K. Signaling cross talk between TGF-β/Smad and other signaling pathways. Cold Spring Harb Perspect Biol. 2017;9(1):a022137. doi:10.1101/cshperspect.a022137
Luo Z, Yu G, Lee HW, et al. The deubiquitinase USP24 is a regulator of hematopoietic stem cell maintenance. Nat Med. 2016;22(1):118-127. doi:10.1038/nm.4017
Malhotra J, Jabbour SK. Targeting the PI3K/Akt/mTOR pathway in non-small cell lung cancer. Transl Lung Cancer Res. 2011;1(1):19-26. doi:10.3978/j.issn.2218-6751.2011.06.01
Malhotra JD, Kaufman RJ. The endoplasmic reticulum and the unfolded protein response. Semin Cell Dev Biol. 2007;18(6):716-731. doi:10.1016/j.semcdb.2007.09.003
Mevissen TET, Komander D. Mechanisms of deubiquitinase specificity and regulation. Annu Rev Biochem. 2017;86:159-192. doi:10.1146/annurev-biochem-061516-044916
Nijman SM, Luna-Vargas MP, Velds A, et al. A genomic and functional inventory of deubiquitinating enzymes. Cell. 2005;123(5):773-786. doi:10.1016/j.cell.2005.11.007
Pal A, Young MA, Donato NJ. Emerging potential of therapeutic targeting of ubiquitin-specific proteases in the treatment of cancer. Cancer Res. 2014;74(18):4955-4966. doi:10.1158/0008-5472.CAN-13-3535
Qian M, Cai Q, Zhai B, et al. USP24 stabilizes the microtubule-associated protein Tau and promotes neurodegeneration. Nat Commun. 2015;6:7560. doi:10.1038/ncomms8560
Repnik U, Stoka V, Turk V, Turk B. Lysosomal membrane permeabilization in cell death: Concepts and challenges. Mitochondrion. 2014;19(Pt A):49-57. doi:10.1016/j.mito.2014.06.007
Rock KL, Lai JJ, Kono H. Innate and adaptive immune responses to cell death. Immunol Rev. 2011;243(1):191-205. doi:10.1111/j.1600-065X.2011.01040.x
Roscic A, Schlieker C. Mechanisms of action and physiological roles of deubiquitinases. Nat Rev Mol Cell Biol. 2010;11(12):885-895. doi:10.1038/nrm3017
Skaar JR, Pagan JK, Pagano M. Mechanisms and function of substrate recruitment by F-box proteins. Nat Rev Mol Cell Biol. 2013;14(6):369-381. doi:10.1038/nrm3582
Song MS, Salmena L, Pandolfi PP. The functions and regulation of the PTEN tumour suppressor. Nat Rev Mol Cell Biol. 2012;13(5):283-296. doi:10.1038/nrm3330
Sun Y. E3 ubiquitin ligases as cancer targets and biomarkers. Neoplasia. 2006;8(8):645-654. doi:10.1593/neo.06552
Sun XX, Qian Y, He H, et al. The deubiquitinase USP24 promotes DNA repair and chemoresistance. Cancer Res. 2018;78(12):3146-3157. doi:10.1158/0008-5472.CAN-17-3423
Todi SV, Paulson HL. Balancing act: deubiquitinating enzymes in the nervous system. Trends Neurosci. 2011;34(7):486-496. doi:10.1016/j.tins.2011.05.002
Torres J, Pulido R. The tumor suppressor PTEN is phosphorylated by CK2 at its C terminus: implications for the regulation of PTEN stability and function. Oncogene. 2001;20(53):6499-6506. doi:10.1038/sj.onc.1204781
van der Horst A, de Vries-Smits LM, Brenkman AB, van Triest MH, van den Broek NJ, Colland F, Maurice MM, Burgering BM. FOXO4 transcriptional activity is regulated by monoubiquitination and USP7/HAUSP. Nat Cell Biol. 2006;8(10):1064-1073. doi:10.1038/ncb1466
Wang H, Li Z, Xu J, et al. USP24 promotes drug resistance during cancer therapy. Cell Death Dis. 2021;12:123. doi:10.1038/s41419-021-03474-x
Wang Z, Ma Z, Guo L, et al. USP24 deubiquitinates and stabilizes p53 in response to DNA damage. Cell Mol Life Sci. 2019;76(18):3603-3617. doi:10.1007/s00018-019-03095-8
Wilkinson KD. Regulation of ubiquitin-dependent processes by deubiquitinating enzymes. FASEB J. 1997;11(14):1245-1256. doi:10.1096/fasebj.11.14.9392713
Wu Y, Chien CT, Zhou B, et al. USP24 regulates EGFR turnover and is critical for lung cancer cell growth. Oncogene. 2018;37(41):5480-5492. doi:10.1038/s41388-018-0331-3
Xu D, Shan B, Lee TH, et al. Phosphorylation of the deubiquitinase USP7 regulates p53 and MDM2 stability. Nat Cell Biol. 2016;18(2):205-214. doi:10.1038/ncb3286
Yang H, Lee H, Song D, et al. Deubiquitinases as cancer therapeutic targets: molecular mechanisms and translational opportunities. Cancer Res. 2018;78(16):4353-4364. doi:10.1158/0008-5472.CAN-17-3501
Zhang Y, Gao J, Chung KK, Huang H, Dawson VL, Dawson TM. Parkin functions as an E2-dependent ubiquitin–protein ligase and promotes the degradation of the synaptic vesicle-associated protein CDCrel-1. Proc Natl Acad Sci U S A. 2000;97(10):5433-5438. doi:10.1073/pnas.97.10.5433
Zhou P, Bogacki R, McReynolds L, Howley PM. Harnessing the ubiquitination machinery to target the degradation of specific proteins. Mol Cell. 2000;6(3):751-756. doi:10.1016/S1097-2765(00)00075-2
Zinngrebe J, Montinaro A, Peltzer N, Walczak H. Ubiquitin in the immune system. EMBO Rep. 2014;15(1):28-45. doi:10.1002/embr.201338025