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Mouse tumor susceptibility gene, tsg101, was discovered by Dr. Stanley Cohen at Stanford University in 1996. Subsequent studies have revealed multiple functions of tsg101. It participates in MDM2/p53 regulatory circuit, protein trafficking and regulation of cellular proliferation and differentiation. Knockout mouse experiment has demonstrated an essential role of tsg101 in early embryonic development, since tsg101 deficiency results in malformation of mesoderm, hence embryonic lethality. To further scrutinize the role of tsg101 in mouse tissues during the stages of organogenesis, we analyzed tsg101 protein steady state expression profile in the developing embryo. The data indicated that tsg101 was ubiquitously and diffusedly expressed in all early embryonic tissues, suggesting a role of tsg101 in rapid dividing early embryonic cells. Along the developmental path, tsg101 expressions became more specific in the epithelial cells of lung, intestine, kidney, thyroid, thymus and adrenal gland. This is concordant with previous report of tsg101 function in regulation of epithelial cell differentiation. In addition, tsg101 expression was found in pyramidal and stellate cells in cerebral cortex, substantia nigra cells of thalamus and Purkinje cells in cerebellum of adult mouse. Whether tsg101 participates in regulating the trafficking of neurotransmitters in these neuronal cells awaits further investigation. Surprisingly, we found congruous expression profile of thyroglobulin in these neuronal cells, except in stellate cells. Besides, the expression of TTF-1 was found in the VI layer of cerebral cortex and neuronal fiber in thalamus. Both thyroglobulin and TTF-1 are thyroid specific proteins, which are important for the development of brain tissues. Further investigation is urged to clarify whether these two proteins really present in these brain tissues and their functional role in these neuronal cells.

Inactivation of mouse tumor susceptibility gene tsg101 leads to neoplastic transformation which could reversed by restoration of tsg101 protein activity. In the varieties of human malignancies, no genomic defect could be identified questioning the role of TSG101as a classical tumor suppressor. Subsequent studies revealed presence of abnormal TSG101 transcripts in both tumor tissues and its normal counter parts, as well as in embryonic tissues. Hence, the relationship between TSG101 and human cancer development remains unclear. The previous studies have demonstrated that TSG101 has multiple biological functions, including regulations of protein degradation through ubiquitination, transcriptional, protein trafficking, cell survival and proliferation and epithelial cell differentiation. To further investigate the role of TSG101 in tumorigenesis, we employed immunohistochemistry and in situ hybridization analysis to study the expressions of TSG101 protein and mRNA in squamous cell carcinomas of different differentiation status. In addition, we scrutinized the relationship between TSG101 expression and the changs of cell cycle-related tumor suppressors and markers of epithelial differentiation, cell growth, tumor metastasis and apoptosis. The results reveal that TSG101 protein and mRNA are consistently expressed in the epidermal cells residing in the suprabasal, granular and cornified layers, but only weakly expressed in the cells of basal layer. The expressions of TSG101 are down regulated in poorly differentiated squamous cell carcinomas of various organs. Furthermore, TSG101 is also expressed in the tissue of squamous metaplasia, and the expression of TSG101 is positively related to that of cytokeratin. In addition, while TSG101 is down regulated, the expressions of p21Cip1/WAF1, p14ARF and MDM2 are also decreased ehereas that of p53 is conversely increased. Phospho-Rb and E-Cadherin were found to be down regulated in advanced cancers, but we failed to find their correlation with TSG101 on cell proliferation and tumor metastasis. Taken together, we hypothesize that TSG101 expression may influence and promote cell differentiation by regulating keratin expression, being involved in the MDM2-p53 circuit and interacting with p21Cip1/WAF1. Besides, by the integration of the studies of TSG101, keratin 10, and Rb protein expression, we infer that TSG101 may indirectly suppress expression of Rb by up-regulating keratin 10 in epithelial cells. The detailed mechanisms of the observation require further investigation. Nevertheless, our results have provided evidences to support the role of TSG101 on differentiation of squamous epithelial cells in addition to tumorigenesis.

TSG101 exhibits multiple functions, including vesicular trafficking, cell growth, differentiation, and transcriptional regulation. However, the cellular signaling that regulates TSG101 functions remains unexplored. Our previous result indicates that TSG101 can be phosphorylated by PKC and GSK3£]. In this thesis, we further investigate the detail phosphorylated amino acid residues by using in vitro kinase assay in conjunction with MALDI-TOF and peptide array analysis. The results indicate that S13, S48, S103 and S367, T383 residues could be phosphorylated by PKC, S182 and S212 by GSK3£]. Coiled-coil domain of TSG101, which contains 2 consensus CK¢º phosphorylation sites,could be phosphorylated by CK¢º. These results indicate the functions of TSG101 might be regulated by these kinases. Proteasome mediated protein degradation is important to maintain proper cellular functions including cell growth, differentiation and signaling associated with cell cycle control. Impaired function of this system has been implicated in human diseases associated with neurodegeneration and cancer. The inhibitor of proteasome has been successfully used in treatment of these related diseases. In this thesis, we successfully established Ub-X-GFP reporter cell lines, which could be used in the future study on the functional role of TSG101, an E2 variant, in the proteasome mediated degradation pathway. Furthermore, these cell lines will serve a useful cellular platform for screening new proteasome inhibitors.

Human tumor susceptibility gene 101, TSG101, exhibits a variety of functions including protein sorting, vesicular trafficking, and regulation of transcription, epithelial growth and differentiation. The upstream sequence of TSG101 gene shows a typical housekeeping TATA-less and Sp1 containing promoter. Our previous data indicated the essential role of TSG101 in skin keratinocyte differentiation that is under the regulation of PKC-Sp1 signaling. In this report, we investigated the correlation of TSG101 and Sp1 expression in the specimens of cervical intraepithelial neoplasia. Cervical intraepithelial neoplasia specimens used in this study were 129 paraffin blocks from 41 normal, 35 CIN I, 28 CIN II and 25 CIN III/CIS patients collected in Cancer Prevention and Screening Center at Kaohsiung from January 2005 to July 2007. The expression of TSG101 and Sp1 were analyzed by immunohistochemistry and digitally quantified by Image Pro-plus 6.1 Microimage software according to the method described by Eliane Pedra Dias et al. The quantified data were statistically analyzed using Spearman's rho coefficient and SPSS for Win, v.14 statistical software (SPSS Inc., Chicago, USA). Values were considered significantly different when the P value < 0.05. We found that TSG101 and Sp1 are expressed in cells of parabasal and intermediate layers in normal cervical epithelium, whereas their expressions in basal and superficial layers were either absence or reduced. Interestingly, the expressions of these two markers are significantly increased in more advanced progression stages (CIN II and CIN III/CIS) of cervical intraepithelial neoplastic specimens (P < 0.05). Congruous expression pattern of TSG 101 and Sp1 in normal cervical epithelium confirms the important of cellular Sp1 signaling in regulating TSG101 expression, which is essential during epithelial cell growth and differentiation. Our results also indicate upregulation of these two markers might be important for the progression of cervical intraepithelial neoplasia. Further analysis using more specimens should reveal the prognostic value of these two markers.

Abstract Tumor susceptibility gene TSG101 plays an important role in cellular functions including intracellular protein sorting, vesicular trafficking, and transcription regulation. Our previous results from yeast two-hybrid screening show that TSG101 interacts with a novel transcriptional repressor protein, KLIP1. In this study, we demonstrated in vivo interaction between TSG101 and KLIP1 in nucleus of 293 cells using co-immunoprecipitation assay and confocal imaging. In addition, we found KLIP1 could be modified in a modality of either poly- and mono-ubiquitination when exogenously expressed in 293 cells in conjunction with either wild type His-tagged ubiquitin or a mutant His-tagged ubiquitin (K0-Ub) which has no capability of forming polyubiquitin chain. Furthermore, we found that TSG101 could increase 60 kDa-KLIP1, but decrease 71 kDa-KLIP1 levels of monoubiquitinated KLIP1 protein species in a dose dependent manner. These results indicate that TSG101 might regulate KLIP1 protein function through affecting its monoubiquitin modification status. Further investigation using wildtype pHA-KLIP1 and mutant pHA-KLIP1-M6 containing mutation in its 6 lysine residues for possible ubiquitin modification revealed that wildtype HA-KLIP1, but not HA-KLIP1-M6, could inhibit transcription activity of thymidine kinase (TK) promoter. In conclusion, our results support that TSG101 interacts and acts as a transcriptional co-repressor of KLIP1 by keeping it in 60 kDa-monoubiquitinated status in the nucleus, where KLIP1 functions as a transcription repressor for TK promoter. Further experiment using mutant HA-KLIP1 expression plasmid containing single mutation in the 6 lysine sites should reveal the exact location of ubiquitin-modified lysine site for monoubiquitinated species of KLIP1 protein.

The TSG101 protein has been implicated in multiple biological functions including the regulation of gene transcription, vesicular trafficking, cellular growth and differentiation. Previous reports indicated the steady-state level of TSG101 must be maintained in a narrow range. Either deprivation or overexpression of TSG101 protein could result in neoplastic transformation. However, cellular signals that control TSG101 functions are not clear. TSG101 protein contains many kinase phosphorylation sites including two GSK-3£] phosphorylation sites, S-172 (S172-P-Y-P-S176 ) and S202 (S202-Q-Y-P-S206). Our previous in vitro kinase assay result indicated TSG101 could be phosphorylated by GSK-3£]. In the present study, we demonstrated that 47 kDa TSG101 is monoubiquitination of 42 kDa TSG101. The GSK-3£] inhibitors, LiCl and TDZD8 could decrease TSG101 level in both HeLa and HEp-2 cells. On the contrary, activation of GSK-3£] by serum starvation or by transfection of a plasmid encodes for constitutive active GSK-3£] led to the increase of TSG101 level in a dose-dependent manner. The effect of LiCl and TDZD8 could be blocked by MG132, implying the involvement of proteaosome mediated mechanism. Expression of constitutive active GSK-3bmutS9A led to a dose-dependent increases of wildtype and HA-TSG101mutS172176A, but decrease of HA-TSG101mutS202206A protein. In addition, either wildtype or mutant HA-TSG101 could complex with GFP-GSK-3b. The mutation of S202 GSK-3b phosphorylation site of TSG101 compromised its ability to for complex with GSK-3b. In summary, our data support that GSK-3b is an important cellular signaling in regulation of monoubiquitinated TSG101 steady-state level. Whether it also affects the subcellular localization of TSG101 awaits further investigation.

TSG101 is a tumor susceptibility gene, which exhibits many biological functions including the regulation of transcription, cell growth and differentation, protein trafficking and receptor recycling. Recent studies have revealed overexpression of TSG101 in human cancers of papillary thyroid carcinoma and breast cancer, whereas downregulation in the periphery of cancer tissue. These data indicated that the amount of TSG101 gene products might be relevant to tumor formation. Understanding the detail regulation of TSG101 gene expression might advance our knowledge on the processes of neoplastic conversion. In this regard, our lab have cloned and characterized upstream sequence of TSG101 transcription start site, and defined the region of -1~-436 as proximal promoter by luciferase reporter assay. Sequence analysis of this region using NSITE program on Softberry web site has revealed several transcription factor binding sites including MAZ, Sp1, ETF and ETS-1. Using EMSA and luciferase assay, we have demonstrated MAZ and Sp1 as major transcription factors regulate TSG101 proximal promoter. In this thesis, we advance our study to further explore the role of ETF and ETS-1 sites in regulation TSG101 promoter. We first cloned the region encompassing two ETS-1 sites in ¡V190~-1 region into pGL3-basic( -190/pGL3-basic ). The luciferase reporter assay of wildtype and mutant ¡V190/pGL3-basic plasmids further demonstrated important role of these ETS-1 sites. To explore detail contribution of the above mentioned transcription factor binding sites in regulation TSG101 promotor, we have made mutant reporter constructs containing different assortment of mutations in these binding sites, and assayed the effect of mutant on TSG101 promoter activity. The results showed that in cancer cell lines (COS-1, ARO and WRO) tested, Sp1, ETF, and ETS-1 binding sites are essential and they act co-operatively in regulating the activity of TSG101 proximal promoter. The results also indicated that Sp1 and ETS-1 were positive regulators, while ETF worked as a negative regulator.

Abstract¡G Tumor susceptibility gene 101, TSG101, is a protein exhibits multiple biological functions. For most protein, its specific function or structure stability can be regulated through protein phosphorylation or modification. The analysis of the amino acid sequence of TSG101 revealed that it has two GSK-3£] phosphorylation concensus sequences. Our previous data of in vitro kinase assay have demonstrated that TSG101 can be phosphorylated by GSK-3£], a wellknown protein kinase that regulates the stability and function of it¡¦s target protein. To investigate the effect of GSK-3£] phosphorylation on the stability and the function of TSG101 protein, we first exploited the effect of GSK-3£]inhibitor, LiCl, on endogenous TSG101 protein in COS1 cells. The results suggested that inhibition of GSK-3£] phosphorylation could impact on the stability of TSG101 protein. Upon the transfection of an active form GSK-3£] expression plasmid GSK-3£]/pEGFP, additional protein products of 40, 50-80 kD were detected, suggesting that GSK-3£] phosphorylation might induce modification or degradation of TSG101 protein. GSK-3£] phosphorylation site mutant TSG101 protein expression plasmids were constructed using site-directed mutagenesis, and were transfected into COS1 cells to evaluate the effect of GSK-3£] on TSG101 level. The results showed that GSK-3£] phosphorylation site mutant TSG101 protein is more stable then wild type TSG101 due to the lack of GSK-3£] phosphorylation site. The inhibition of GSK-3£] activity by LiCl treatment resulted in the increase of wildtype as well as the S172AS176 and S172AS176AS202AS206A mutant TSG101 proteins, whereas the S202AS206A mutant TSG101 protein level was not affected by LiCl treatment. The above data indicated that GSK-3£] might regulate the stability and biological activity of TSG101 protein through phosphorylation of serine residue at position 202, which is worthy of further investigation.

Tumor Susceptibility Gene, TSG101, has been identified as a tumor susceptibility gene with multiple functions. TSG101 encodes a 46 kDa protein composed of 390 amino acids. As previous studies reported, TSG101 participates in cell-cycle control, membrane proteins¡¦ trafficking, and transcriptional regulation. To identify the proteins that mediated function involved TSG101, we perform yeast two-hybrid cDNA library screening to search for TSG101-interacting proteins. A construct pAS2-1-TSG101 was used as a bait to screen a human testis cDNA library. This screening selected 68 TSG101 interacting clones, including 17 known proteins. These proteins were functionally classified as participating in cell-cycle alteration, protein sorting, transcriptional regulation, modification, signal transduction and other functions. Our results provide the evidences which not only confirm the results of previous studies, but also provide further information related to TSG101 biological functions worth intensive study. Among these clones, we choose KLIP1 gene, which encodes a transcription factor, for further study to elucidate the functional role of TSG101 in nucleus. In vitro GST pull-down assay and in vivo co-immunoprecipitation assay were performed using GST-KLIP1 and HA-tagged KLIP1, respectively, have demonstrated that TSG101 and KLIP1 indeed interact with each other within mammalian cells. Detailed biological function mediated through this TSG101 and KLIP1 interaction awaits further investigation.