泛素(Ubiquitin)是一種廣泛存在於真核細胞中的蛋白質。它藉由泛素化作用(Ubiquitination)的路徑所催化，接在特定蛋白質上，調控多種生理功能，例如DNA修復、激酶的活化，以及抗病毒作用。許多文獻指出，細胞中泛素化程度的改變會引發病理反應，尤其是癌細胞的惡化。歸納目前研究，在人類中僅有兩種E1酵素，相對於具有多種類型的E2、E3酵素，選擇其一當作標的，更能廣泛地探討泛素化路徑對於癌細胞惡化的影響。本研究藉由RNA干擾(RNA interference, RNAi)的方式，在多種癌細胞中(HeLa、H1299、MDA-MB-231、PC3)穩定抑制泛素活化酵素1(Ubiquitin-activating enzyme E1, UBE 1)的表現。實驗結果顯示，缺乏泛素活化酵素1表現，不論增生或爬行的能力皆顯著下降。透過西方墨點法(Western blot)分析，該細胞株中多種細胞週期相關蛋白，例如Cyclin B1、Cdc 2，以及Cyclin D1表現量下降；而對於細胞生長極為重要的NF-κB 訊息傳遞路徑，原本經由泛素化-蛋白酶體系統降解的IκB表現量提升，位於下游的活化態NF-κB則顯著下降。此外，細胞週期分析則可觀察該細胞株在相同時間點下，位於G1期的細胞數量較對照組低。為了更進一步了解缺乏此酵素對於癌細胞的影響，我利用不同化療藥物：2-去氧葡萄糖(2-Deoxyglucose, 2-DG)、每福敏(Metformin)、依托泊苷(Etoposide)分別處理細胞株。結果顯示處理2-DG對於UBE1穩定靜默株的毒殺能力最強。在使用4 mM濃度下，該細胞株即大量死亡。經由流式細胞儀分析，不論處理何種濃度的2-DG，UBE1穩定靜默株進入細胞凋亡的數量，都會顯著上升。綜合以上結果，在多種癌細胞中，抑制泛素活化酵素1的表現，會降低其惡性程度，並且可能透過NF-κB 訊息傳遞路徑的變化，使增生速度減緩。此外，該細胞株對2-DG的敏感度增加，提供合併藥物治療的方向。

Ubiquitin is a protein that ubiquitously exists in tissues of eukaryotic organisms. The conjugation of proteins with ubiquitin can regulate many kinds of cellular functions. In many cases, abnormal ubiquitination level in cells causes pathologic reactions, especially the development of cancer. Therefore, the members of ubiquitin pathway could be a great target as novel anticancer drugs. First, I choose the dominant type of E1 enzyme UBE1 as the target of this study. In this research, I establish the stable knockdown UBE1 cancer cell lines by RNAi (RNA interference), including HeLa, H1299, MDA-MB-231, and PC3. The results show that the loss of UBE1 expression decreases cell proliferation rate and cell migration ability. Western blot analysis indicates that many cell cycle proteins like cyclin B1, cdc 2, and cyclin D1 are downregulated in UBE1 knockdown cell. Flow cytometry assay demonstrates that lack of UBE1 causes the change of cell cycle distribution. Furthermore, loss of UBE1 expression causes accumulation of NF-κB inhibitor IκB and inactivation of NF-κB. In pharmacology experiment, I choose three anticancer drugs to analyze the effect on loss of UBE1 expression. In contrast to the mock cells, the sensitivity of these drugs are increased in UBE1 knockdown cells, especially 2-deoxyglucose. The knockdown cells which are treated with low dose 2-deoxyglucose (4 mM) are almost killed. I use the flow cytometry assay to observe cell into apoptosis. Taken together, this study reveals UBE1 can be a potential target for cancer therapy.
Key words: Ubiquitin Activating Enzyme E1; 2-Deoxyglucose; Cancer Therapy; Apoptosis

[1] Goldstein, G., et al. Isolation of a polypeptide that has lymphocyte- differentiating properties and is probably represented universally in living cells. Proc. Natl Acad. Sci. USA 72, 11-15 (1975).
[2] Glickman, M.H., Ciechanover, A. The ubiquitinproteasome proteolytic pathway: destruction for the sake of construction. Physiol. Rev. 82, 373-428 (2002).
[3] Rock, K.L. et al. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class. Mol. Cell 78, 761-771 (1994).
[4] Hershko, A., Cienchanover, A. The ubiquitin system for protein degradation. Annu. Rev. Biochem. 61, 761–807 (1992).
[5] Jin, J., Li, X., Gygi, S.P., Harper, J.W. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging. Nature 447, 1135–1138 (2007).
[6] Weissman, A.M., Shabek, N., Ciechanover, A. The predator becomes the prey: regulating the ubiquitin system by ubiquitylation and degradation. Nature Rev. Mol. Cell Biol. 12, 605–620 (2011).
[7] Lake, M.W., Wuebbens, M.M., Rajagopalan, K.V., Schindelin, H. Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB–MoaD complex. Nature 414, 325–329 (2001).
[8] Walden, H., Podgorski, M.S., Schulman, B.A. Insights into the ubiquitin transfer cascade from the structure of the activating enzyme for NEDD8. Nature 422, 330–334 (2003).
[9] Huang, D.T. et al. Structural basis for recruitment of Ubc12 by an E2 binding domain in NEDD8's E1. Mol. Cell 17, 341–350 (2005).
[10] Huang, D.T. et al. Basis for a ubiquitin-like protein thioester switch toggling E1–E2 affinity. Nature 445, 394–398 (2007).
[11] Bialas J., Groettrup M., Aichem A. Conjugation of the Ubiquitin Activating Enzyme UBE1 with the Ubiquitin-Like Modifier FAT10 Targets It for Proteasomal Degradation. PLoS ONE 10, e0120329 (2015).
[12] Ungermannova, D. et al. Largazole and its derivatives selectively inhibit ubiquitin activating enzyme (e1). PLoS ONE 7, e29208 (2012).
[13] Moudry P. et al. Ubiquitin-activating enzyme UBA1 is required for cellular response to DNA damage. Cell Cycle 11, 1573–1582(2012).
[14] Hershko, A., Ciechanover, A. Mechanisms of intracellular protein breakdown. Annu. Rev. Biochem. 51, 335−364 (1982).
[15] Shih, S.C. et al. A ubiquitin-binding motif required for intramolecular ubiquitylation, the CUE domain. EMBO J. 22, 1273–1281 (2003).
[16] Haglund, K. et al. Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation. Nature Cell Biol. 5, 461–466 (2003).
[17] Ikeda, F., Dikic, I. Atypical ubiquitin chains: new molecular signals. 'Protein Modifications: Beyond the Usual Suspects' review series. EMBO Rep. 9, 536–542 (2008).
[18] Iwai, K., Tokunaga, F. Linear polyubiquitination: a new regulator of NF-κB activation. EMBO Rep. 10, 706–713 (2009).
[19] Xu, P. et al. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation.Cell 137, 133–145 (2009).
[20] Ziv, I. et al. A perturbed ubiquitin landscape distinguishes between ubiquitin in trafficking and in proteolysis. Mol. Cell Proteomics 10, M111.009753 (2011).
[21] Dammer, E. B. et al. Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of Alzheimer disease. J. Biol. Chem. 286, 10457–10465 (2011).
[22] Gerlach, B. et al. Linear ubiquitination prevents inflammation and regulates immune signalling. Nature 471, 591–596 (2011).
[23] Ikeda, F. et al. SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis. Nature 471, 637–641 (2011).
[24] Mallery, D. L., Vandenberg, C. J., Hiom, K. Activation of the E3 ligase function of the BRCA1/BARD1 complex by polyubiquitin chains. EMBO J. 21, 6755–6762 (2002).
[25] Hashizume, R. et al. The RING heterodimer BRCA1–BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation. J. Biol. Chem. 276, 14537–14540 (2001).
[26] Matsumoto, M. L. et al. K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody.Mol. Cell 39, 477–484 (2010).
[27] Dynek, J. N. et al. c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling. EMBO J. 29, 4198–4209 (2010).
[28] Birsa, N. et al. Lysine 27 ubiquitination of the mitochondrial transport protein miro is dependent on serine 65 of the parkin ubiquitin ligase. J. Biol. Chem. 289, 14569–14582 (2014).
[29] Chastagner, P., Israël, A., Brou, C. Itch/AIP4 mediates deltex degradation through the formation of K29-linked polyubiquitin chains. EMBO Rep. 7, 1147–1153 (2006).
[30] Al-Hakim, A.K. et al. Control of AMPK-related kinases by USP9X and atypical Lys/Lys-linked polyubiquitin chains.Biochem. J. 411, 249–260 (2008).
[31] Hershko, A., Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479(1998).
[32] Chen, Z.J., Sun, L.J. Nonproteolytic functions of ubiquitin in cell signaling. Mol. Cell 33, 275–286 (2009).
[33] Fan, Y. et al. Lysine 63-linked polyubiquitination of TAK1 at lysine 158 is required for tumor necrosis factor α- and interleukin-1β-induced IKK/NF-κB and JNK/AP-1 activation. J. Biol. Chem. 285, 5347–5360 (2010).
[34] Skaug, B., Jiang, X., Chen, Z.J. The role of ubiquitin in NF-κB regulatory pathways. Annu. Rev. Biochem. 78, 769–796 (2009).
[35] Yang, W. L. et al. The E3 ligase TRAF6 regulates Akt ubiquitination and activation. Science 325, 1134–1138 (2009).
[36] Chan, C.H. et al. The Skp2–SCF E3 ligase regulates Akt ubiquitination, glycolysis, herceptin sensitivity, and tumorigenesis. Cell 149, 1098–1111 (2012).
[37] Ikeda, F. et al. SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis. Nature 471, 637–641 (2011).
[38] Virdee, S., Ye, Y., Nguyen, D.P., Komander, D., Chin, J.W. Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. Nature Chem. Biol. 6, 750–757 (2010).
[39] Yang, R., Pasunooti, K.K., Li, F., Liu, X.-W., Liu, C.-F. Synthesis of K48-linked diubiquitin using dual native chemical ligation at lysine. Chem. Commun. 46, 7199–7201 (2010).
[40] Kumar, K.S.A., Spasser, L., Erlich, L.A., Bavikar, S.N., Brik, A. Total chemical synthesis of di-ubiquitin chains. Angew. Chem. Int. Ed. 49, 9126– 9131 (2010).
[41] Xu, G. W. et al. The ubiquitin-activating enzyme E1 as a therapeutic target for the treatment of leukemia and multiple myeloma. Blood 115, 2251–2259 (2010).
[42] Osada T., et al. Increased ubiquitin immunoreactivity in hepatocellular carcinomas and precancerous lesions of the liver. J Hepatol. 26, 1266–1273 (1997).
[43] Morelva Tde, M., Antonio, L.B. Immunohistochemical expression of ubiquitin and telomerase in cervical cancer. Virchows Arch. 455, 235–243 (2009).
[44] Ishibashi, Y. et al. Quantitative analysis of free ubiquitin and multi-ubiquitin chain in colorectal cancer. Cancer Lett. 211, 111–117 (2004).
[45] Rogers S., Wells R., Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science 234, 364–368(1986).
[46] Ciechanover, A., Finley, D., Varshavsky, A. Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85. Cell 37, 57–66 (1984)
[47] Pugh, C.W. et al. Activation of hypoxia-inducible factor-1; definition of regulatory domains within the subunit. J Biol. Chem. 272, 11205–11214 (1997).
[48] Cockman, M. E. et al. Hypoxia inducible factor-α binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein. J. Biol. Chem. 275, 25733–25741 (2000).
[49] Ohh, M. et al. Ubiquitination of hypoxia-inducible factor requires direct binding to the β-domain of the von Hippel-Lindau protein. Nature Cell Biol. 2, 423–427 (2000).
[50] Yang, J. C. et al. A randomized trial of bevacizumab, an anti-VEGF antibody, for metastatic renal cancer. N. Engl. J. Med. 349, 427–434 (2003).
[51] Siemeister, G. et al. Reversion of deregulated expression of vascular endothelial growth factor in human renal carcinoma cells by von Hippel-Lindau tumor suppressor protein. Cancer Res. 56, 2299-2301 (1996).
[52] Werness, B.A., Levine, A.J., Howley, P.M. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248, 76–79 (1990).
[53] Levine, A.J. p53, the cellular gatekeeper for growth and division. Cell 88, 323–331 (1997).
[54] Haupt Y., Maya R., Kazaz A., Oren M. Mdm2 promotes the rapid degradation of p53. Nature 387: 296-299, (1997).
[55] Momand, J., Jung, D., Wilczynski, S., Niland, J. The MDM2 gene amplification database. Nucleic Acids Res. 26, 3453–3459 (1998).
[56] Pagano, M. et al. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 269, 682–685 (1995).
[57] Moller M. p27 in cell cycle control and cancer. Leuk Lymph 39, 19–27(2000)
[58] Slingerland, J., Pagano, M. Regulation of the cdk inhibitor p27 and its deregulation in cancer. J. Cell Physiol. 183, 10–17 (2000)
[59] Loda, M. et al. Increased proteasome-dependent degradation of the cyclin -dependent kinase inhibitor p27 in aggressive colorectal carcinomas. Nature Med. 3, 231–234 (1997).
[60] Kudo, Y. et al. High expression of S-phase kinase-interacting protein 2, human F-box protein, correlates with poor prognosis in oral squamous cell carcinomas. Cancer Res. 61, 7044–7047 (2001).
[61] Latres, E. et al. Role of the F-box protein Skp2 in lymphomagenesis. Proc. Natl Acad. Sci. USA 98, 2515–2520 (2001).
[62] Chiarle, R. et al. S-phase kinase-associated protein 2 expression in non- Hodgkin's lymphoma inversely correlates with p27 expression and defines cells in S phase. Am. J. Pathol. 160, 1457–1466 (2002).
[63] Schmidt, M.H., Furnari, F.B., Cavenee, W.K., Bogler, O. Epidermal growth factor receptor signaling intensity determines intracellular protein interactions, ubiquitination, and internalization. Proc. Natl Acad. Sci. USA 100, 6505–6510 (2003).
[64] Heinemeyer, W., Fischer, M., Krimmer, T., Stachon, U., Wolf, D.H. The active sites of the eukaryotic 20S proteasome and their involvement in subunit precursor processing. J. Biol. Chem. 272, 25200−25209 (1997)
[65] Demo, S.D. et al. Antitumor activity of PR-171, a novel irreversible inhibitor of the proteasome. Cancer Res. 67, 6383–6391 (2007).
[66] Chauhan, D. et al. A novel orally active proteasome inhibitor induces apoptosis in multiple myeloma cells with mechanisms distinct from bortezomib. Cancer Cell 8, 407–419 (2005).
[67] Ling, Y.H. et al. PS-341, a novel proteasome inhibitor, induces Bcl-2 phosphorylation and cleavage in association with G2-M phase arrest and apoptosis. Mol. Cancer Ther. 1, 841–849 (2002).
[68] Karin, M., Cao, Y., Greten, F.R., Li, Z.W. NF- B in cancer: from innocent bystander to major culprit. Nature Rev. Cancer 2, 301–310 (2002).
[69] Hideshima, T. et al. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Res. 61, 3071–3076 (2001).
[70] Piva, R. et al. CEP-18770: a novel, orally active proteasome inhibitor with a tumor-selective pharmacologic profile competitive with bortezomib. Blood 111, 2765–2775 (2008).
[71] Dorsey, B.D. et al. Discovery of a potent, selective, and orally active proteasome inhibitor for the treatment of cancer. J. Med. Chem. 51, 1068– 1072 (2008).
[72] Yang, Y. et al. Inhibitors of ubiquitin-activating enzyme (E1), a new class of potential cancer therapeutics. Cancer Res. 67, 9472–9481 (2007).
[73] Kitagaki, J. et al. Nitric oxide prodrug JS-K inhibits ubiquitin E1 and kills tumor cells retaining wild-type p53. Oncogene 28, 619–624 (2009).
[74] Ceccarelli, D.F. et al. An allosteric inhibitor of the human Cdc34 ubiquitin‐conjugating enzyme. Cell 145, 1075–1087 (2011).
[75] Huang, D.T. et al. A unique E1–E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8. Nature Struct. Mol. Biol. 11, 927–935 (2004).
[76] Vassilev, L.T. et al. In vivo activation of the p53 pathway by small-molecular antagonists of MDM2. Science 303, 844–848 (2004).
[77] Issaeva, N. et al. Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors. Nature Med. 10, 1321–1328 (2004).
[78] Gopal, Y.N., Chanchorn, E., Van Dyke, M.W. Parthenolide promotes the ubiquitination of MDM2 and activates p53 cellular functions. Mol. Cancer Ther. 8, 552–562 (2009).
[79] Srinivasula, S.M., Ashwell, J.D. IAPs: what's in a name? Mol. Cell 30, 123– 135 (2008).
[80] Carter, B.Z. et al. Triptolide sensitizes AML cells to TRAIL-induced apoptosis via decrease of XIAP and p53-mediated increase of DR5. Blood 111, 3742–3750 (2008).
[81] Sanduja, S., Kaza, V., Dixon, D.A. The mRNA decay factor tristetraprolin (TTP) induces senescence in human papillomavirus-transformed cervical cancer cells by targeting E6-AP ubiquitin ligase. Aging (Albany NY). 1, 803–817 (2009).
[82] Nakamura, T., Lipton, S.A. Cell death: protein misfolding and neuro- degenerative diseases. Apoptosis 14, 455–468 (2009).
[83] Sufan, R.I. et al. Oxygen-independent degradation of HIF-alpha via bioengineered VHL tumour suppressor complex. EMBO Mol. Med. 1, 66–78 (2009).
[84] Tian, Z., et al. A novel small molecule inhibitor of deubiquitylating enzyme USP14 and UCHL5 induces apoptosis in multiple myeloma and overcomes bortezomib resistance. Blood 123, 706–716 (2014).
[85] Altun, M. et al. Activity-based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes. Chem. Biol. 18, 1401-1412(2011).
[86] Wick, A.N., Drury, D.R., Nakada, H.I., Wolfe, J.B. "Localization of the primary metabolic block produced by 2-deoxyglucose". J. Biol. Chem. 224, 963–969 (1957).
[87] Parniak, M., Kalant, N. Incorporation of glucose into glycogen in primary cultures of rat hepatocytes. Can. J. Biochem. Cell Biol. 63, 333-340 (1985).
[88] McComb, R.B., Yushok, W.D. Metabolism of ascites tumor cells: IV. Enzymatic reactions involved in adenosinetriphosphate degradation induced by 2-deoxyglucose. Cancer Res. 24, 198–205 (1964).
[89] Karczmar, G.S., Arbeit, J.M., Toy, B.J., Speder, A., Weiner M.W. Selective depletion of tumor ATP by 2-deoxyglucose and insulin, detected by 31P magnetic resonance spectroscopy. Cancer Res. 52, 71–76 (1992).
[90] Aft, R.L., Zhang, F.W., Gius, D. Evaluation of 2-deoxy-D-glucose as a chemotherapeutic agent: mechanism of cell death. Br. J. Cancer 87, 805–812 (2002).
[91] Kurtoglu, M. et al. Under normoxia, 2-deoxy-D-glucose elicits cell death in select tumor types not by inhibition of glycolysis but by interfering with N-linked glycosylation. Mol. Cancer Ther. 6, 3049–3058 (2007).
[92] Liu, H., Hu, Y.P., Savaraj, N., Priebe, W., Lampidis, T.J. Hypersensitization of tumor cells to glycolytic inhibitors. Biochemistry 40, 5542–5547 (2001).
[93] Johnathan, C.M., Awtar, K., Theodore, J.L. Greater cell cycle inhibition and cytotoxicity induced by 2-deoxy-D-glucose in tumor cells treated under hypoxic vs aerobic conditions. Cancer Chemother. Pharmacol. 53, 116-122 (2004)
[94] Liu, H., Savaraj, N., Priebe, W., Lampidis, T.J. Hypoxia increases tumor cell sensitivity to glycolytic inhibitors: a strategy for solid tumor therapy (Model C). Biochem. Pharmacol. 64, 1745–1751 (2002).
[95] Maschek, G. et al. 2-deoxy-D-glucose increases the efficacy of adriamycin and paclitaxel in human osteosarcoma and non-small cell lung cancers in vivo. Cancer Res. 64, 31–34 (2004).
[96] Aft, R.L., Lewis, J.S., Zhang F., Kim, J., Welch, M.J. Enhancing targeted radiotherapy by copper(II)diacetyl- bis(N4-methylthiosemicarbazone) using 2-deoxy-D-glucose. Cancer Res. 63, 5496–5504 (2003)
[97] Coleman, M.C. et al. 2-Deoxy-D-glucose causes cytotoxicity, oxidative stress, and radiosensitization in pancreatic cancer. Free Radic. Biol. Med. 44, 322–331 (2008)
[98] Stein, M. et al. Targeting tumor metabolism with 2-deoxyglucose in patients with castrate-resistant prostate cancer and advanced malignancies. Prostate 70, 1388–1394 (2010).
[99] Singh, D. et al. Optimizing cancer radiotherapy with 2-deoxy-D-glucose dose escalation studies in patients with glioblastoma multiforme. Strahlenther. Onkol. 181, 507–514 (2005).
[100] Raez, L.E. et al. A phase I dose-escalation trial of 2-deoxy-D-glucose alone or combined with docetaxel in patients with advanced solid tumors. Cancer Chemother Pharmacol 71,523–530 (2013).
[101] Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).
[102] Druker, B.J. et al. Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N. Engl. J. Med. 355, 2408–2417 (2006).
[103] Smith, I. et al. 2-year follow-up of trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer: a randomised controlled trial. Lancet 369, 29–36 (2007).
[104] Pecot, C.V., Calin, G.A., Coleman, R.L., Lopez-Berestein, G., Sood, A.K. RNA interference in the clinic: challenges and future directions. Nature Rev. Cancer 11, 59–67 (2011).
[105] Kim, D.H., Rossi, J.J. Strategies for silencing human disease using RNA interference. Nature Rev. Genet. 8, 173–184 (2007).
[106] Tabernero, J. et al. First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement. Cancer Discov. 3, 406–417 (2013).
[107] Strumberg, D. et al. Phase I Clinical Development of Atu027, a siRNA Formulation Targeting PKN3 in Patients with Advanced Solid Tumors. Int. J. Clin. Pharmacol. Ther. 50, 76– 78 (2012).
[108] Ganesh, S., Iyer, A.K., Morrissey, D.V., Amiji, M.M. Hyaluronic acid based self-assembling nanosystems for CD44 target mediated siRNA delivery to solid tumors. Biomaterials 34, 3489–3502 (2013).
[109] Karin, M., Ben-Neriah, Y. Phosphorylation meets ubiquitination: the control of NF- B activity. Annu. Rev. Immunol. 18, 621–663 (2000).
[110] Viatour, P., Merville, M.P., Bours, V., Chariot, A. Phosphorylation of NF- B and I B proteins: implications in cancer and inflammation. Trends Biochem. Sci. 30, 43–52 (2005).
[111] Rena, G., Pearson, E.R., Sakamoto, K. Molecular mechanism of action of metformin: old or new insights? Diabetologia 56, 1898–1906 (2013).
[112] Burcelin, R. The antidiabetic gutsy role of metformin uncovered? Gut 63, 706–707 (2014).
[113] Pommier, Y., Leo, E., Zhang, H., Marchand, C. DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem. Biol. 17, 421– 433 (2010).
[114] Pelzer, C., Groettrup, M. FAT10 : Activated by UBA6 and Functioning in Protein Degradation. Subcell Biochem. 54, 238–246(2010)
[115] Aichem, A. et al. USE1 is a bispecific conjugating enzyme for ubiquitin and FAT10, which FAT10ylates itself in cis. Nat. Commun. 1, 13 (2010).
[116] Hipp, M.S., Kalveram, B., Raasi, S., Groettrup, M., Schmidtke, G. FAT10, a ubiquitin-independent signal for proteasomal degradation. Mol. Cell Biol. 25, 3483–3491 (2005).
[117] Schmidtke, G., Kalveram, B., Groettrup, M. Degradation of FAT10 by the 26S proteasome is independent of ubiquitylation but relies on NUB1L. FEBS Lett. 583, 591–594 (2009).
[118] Bialas, J., Groettrup, M., Aichem, A. Conjugation of the ubiquitin activating enzyme UBE1 with the ubiquitin-like modifier FAT10 targets it for proteasomal degradation. PLoS One. 10, e0120329 (2015).
[119] Huang, H. et al. Physiological levels of ATP negatively regulate proteasome function. Cell Res. 20, 1372–1385 (2010).
[120] Wallace, D.C. Mitochondria and cancer. Nature Rev. Cancer 12, 685–698 (2012).
[121] Pouyssegur, J., Dayan, F., Mazure, N.M. Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature 441, 437–443 (2006).