Tumor Susceptibility Gene 101, TSG101, exhibits multiple biological functions including the regulation of gene transcription, vesicular trafficking, cellular growth and differentiation. However, the signals involve in the regulation of TSG101 gene functions are unclear. In this present study, we observed congruous TSG101 up-regulation and the differentiation status of keratinocyte in both human foreskin tissue and reconstructed organotypic skin culture. In addition, we found an essential and downstream role of TSG101 in calcium-induced early keratinocyte differentiation since TSG101 siRNA inhibits this process. Our results also indicate a PKC-dependent mechanism is involved based on the following findings. First, a PKC agonist, TPA up-regulates TSG101 and keratin 10 under low calcium condition. Second, co-treatment of keratinocytes with GF 109203X, a PKC inhibitor, blocks TPA-induced TSG101 and keratin 10 up-regulation. Previous report indicates TSG101 gene exhibits a TATA-less and Sp1-containing promoter. Our analysis further shows that both calcium and TPA stimulate phosphorylation of Sp1 and the corresponding TSG101 wild type promoter activity, but not the activity of Sp1 site mutant TSG101 promoter. The co-treatment with GF 109203X blocks the above effects of calcium and TPA, implying that this is a PKC signaling-dependent process. Taken together, these data suggest a PKC-Sp1 signaling is involved in early differentiation switch of keratinocyte through up-regulation of TSG101. Functional inactivation experiment indicates that tsg101 is a tumor suppressor in mouse model. However, many studies using human tumor specimens or conditional knockout mouse give discrepant and contradictive results. Therefore, the role of TSG101 in human cancer remains illusive. Here we demonstrate an inverse correlation between TSG101 and p16INK4a or acetylated- histone H4 protein expression profiles in human head and neck squamous cell carcinomas (HNSCC) (N=98, p<0.001). Using conditioned human HEp2 cells, we confirm that TSG101 negatively modulates p16INK4a expression. Chromatin immunoprecipitation and the subsequent PCR analysis reveal that TSG101 dose-dependently decreases the amount of acetylated histone H4-associated chromatin on p16INK4a promoter. In addition, TSG101 interacts and colocalizes with HDAC1 and SUMO-1 in the nucleus. Furthermore, TSG101 confers a dose-dependent effect on promoting HDAC1 SUMOylation, hence its activity. Taken together, our data demonstrate for the first time that TSG101 can promote SUMO-1 modification of HDAC1, which impacts on down-regulation of p16INK4a gene expression, providing evidence whereby TSG101 might participate in the epigenetic silencing of p16INK4a during the development of HNSCC.

The aim of this thesis is to evaluate the expression of both TSG101 tumor susceptibility gene and ret oncogene in human thyroid carcinoma specimens. Functional inactivation of TSG101 in mouse fibroblast leads to cellular transformation and the ability to form metastatic tumors in nude mice. No genomic deletion of TSG101 gene has been reported in human cancer, casting a doubt on the role of TSG101 as a classical tumor suppressor. Subsequent studies reveal that TSG101 is a frequent target of spilicing defects, which is correlated with cellular stress and p53 status, and might reflect the cellular environment during the cancer development. Furthermore, recent reports demonstrate TSG101 as a part of the MDM2/p53 regulatory circuitry, a well recognized circuitry that upon deregulation results in tumorigenesis. In this study we have analyzed TSG101 gene expression in 85 specimens of thyroid carcinomas. The results indicated that 100% of papillary carcinomas (48/48), 85% of follicular carcinomas (18/21), 91% of medullary carcinomas (10/11) and 60% of undifferentiated carcinomas (3/5) showed strong to moderate cytoplasmic staining, whereas the staining was completely negative, or cytoplasmic dot-staining in the adjacent non-neoplastic follicular cells. Occasionally, the staining could be found in the nucleus. Subsequently, sequence analysis of 17 papillary carcinoma specimens revealed no mutation in steadiness box region, indicating that it might not be the cause of TSG101 protein overexpression. In summary, our results indicate strong correlation of TSG101 overexpression and thyroid carcinomas. Further experiments are urged to clarify the relationship of TSG101 overexpression and thyroid tumorigenesis. Rearrangement of ret proto-oncogene is unique to papillary thyroid carcinoma (PTC). These rearrangements consist of the fusion of ret tyrosine kinase domain to a variety of heterologous genes, thus generating chimeric transforming oncogenes termed, ret/PTC. The frequency of ret/PTC activation in non-radiation exposured adult populations has been reported to vary from 0-55% depending on the geographic distribution. To detect ret rearrangement and to identify candidate of novel ret/PTC in 62 specimens of PTC collected from southern Taiwan, a RT-multiplex PCR method was used to reveal the possible specimens that harbor ret rearrangements. Type specific-PCR amplification and subsequent sequence analysis of PCR product were performed to identify the known types of ret/PTC. We have identified two cases of ret/PTC1, two cases of ret/PTC3 and one case of ELKS-RET. Excitingly, four cases of unknown ret/PTC type were identified. Hence, 5¡¦-RACE strategy will be employed to identify novel ret/PTC in these four specimens.

Functional inactivation of tumor susceptibility gene tsg101 leads to cellular transformation and tumorigenesis in mice. No genomic DNA deletion in TSG101gene in human cancer indicated TSG101 is not a typical tumor suppressive gene. TSG101 participates in the MDM2/p53 feedback control loop and the regulation of the cellular membrane trafficking. However, detail functional characteristics remains to be elucidate. In this study, we explored the tsg101 expression in adult mouse tissues from various organs using immunohistochemistry and in situ hybrid- ization. The results indicated that tsg101 expression was ubiquitous but in differential steady-state level in various cell types. The expression of tsg101 mainly found in epithelial cells¡Bsecretory cells and nerve cells. The second topic of this study was to characterize the phosphorylation status of TSG101 protein. Endogeneously expressed TSG101 and exogeneously expressed HA-tag TSG101 protein were purified by immunoprecipiation with #820 antiserum against TSG101, and were subjected for western blot analysis using anti-phosphoserine and anti-phosphothreonine antibodies. This experiment had confirmed that TSG101 protein contained both phosphoserine and phosphthreonine residues. In vitro kianse assay using GST-tag and his-tag TSG101 funsion proteins was exploited to investigate the kinase responsible for TSG101 phosphorylation. The results clearly indicated that cdc2¡BGSK3£] and PKC kinases could phosphorylate TSG101 fusion Protein, implying that the function of TSG101 might be regulated by the signaling involving these kinases.

Abstract: TSG101 is a tumor susceptibility gene exhibits multiple biological function, including the regulation of cell progression, intracellular protein sorting and membrane trafficking, and transcription activity of nuclear recptor such as estrogen recptor. TSG101 contains an UBC domain which is homologous to that in ubiquitin conjugating E2 enzyme. However, it lacks an essential cysteine residue, which is essential for catalytic activity. Cellular protein ubiquitination serves as a signal for protein degradation or sorting into multivesicular body. UBC domain of TSG101 was proved to contain amino acid residues that are important for its interaction with ubquitin (residues V43, N46, D46 and F88) and PTAP sequence found in the late domain of HIV gag protein (residues Y63, M95, V141). SUMO is an ubquitin-like modifier which can modify cellular protein harbors £ZKXE amino acid sequence, thereby change its subcellular localization and biological activities. TSG101 protein contains K98, K243, K264 and K269 residues that localize in potential SUMO modification site. Our preliminary data indicated that TSG101 colocalize with SUMO in nucleus. It is interesting to know whether TSG101 is sumoylated, and its functional significance. In this thesis, a series of site-directed mutageneic mutant HA and GFP-tagged expression plasmids which contain mutation of the above mentioned functional related amino acid residues were constructed for future TSG101 functional studies.

TSG101 was identified as a tumor susceptibility gene by Stanley Cohen. In a variety of human cancers, no genomic deletion in TSG101 gene has been reported but many aberrant TSG101 transcripts has been found. Some studies have revealed that TSG101 participates in MDM2/p53 regulatory circuitry¡Bmembrance trafficking and receptor recycling. Other reports also showed that TSG101 might be a transcription regulatory factor. However, mechanism of these TSG101 function awaits further characterization. To further scrutinize the function of TSG101 and its subcellular localization, a varieties of GFP-based recombinant plasmids which contain various length of TSG101 cDNA have been constructed and transfected into cells. Western blot analysis had shown that these constructs could express GFP-TSG101 fusion protein of expected size. The fluorescence and confocal microscopy have shown that wild type TSG101 localized in ER, Golgi and endosome compartments, also amino acid residues 136-233 and 316-390 of TSG101 are two important regions for its subcellular localization. Previous reports had shown that TSG101 interact with OP18 which is an important regulator for spindle formation in M phase. To elucidate the localization of TSG101 and OP18 in M phase cell, we have cloned OP18 and generate GST-OP18 fusion protein for anti-OP18 antiserum production.Then, pDsRed-OP18 fusion protein expressed in OP18/pDsRed recombinant plasmid transfected cell was detected by western blotting analysis using this anti-OP18 antiserum. The subcellular localization of DsRed-OP18 and GFP-TSG(1-390) fluorescence were recorded in double transfected cells which were arrested in M phase by nocodzole treatment. We observed the evenly distribution of pDsRed-OP18 red fluorescence and punctate vesicular localization of GFP-TSG(1-390) green fluorescence. Whether these two protein interact functionally awaits further investigation.

TSG101 is a tumor susceptibility gene exhibits multiple biological functions, including the regulation of vesicular trafficking, transcription, cellular growth and differentiation. The intracellular steady-state level of TSG101 was shown to under stringent control in a narrow range. Either deprivation or overexpression of mouse tsg101 in NIH3T3 cells leads to neoplastic transformation and subsequent tumorigenic potential of the transformed cells. However, the detail mechanism for regulation of TSG101 gene promoter activity is not clear. Our results indicated TSG101 is a housekeeping gene and contains a TATA-less and Sp1 binding site promoter. Here, we demonstrate in vivo binding of Sp1 transcription factor on TSG101 promoter region by chromatin immunoprecipitation(ChIP). In addition, Sp1-associated transcription regulators were purified using DNA affinity precipitation assay (DAPA) method and subjected to two-dimensional gel electrophoresis and the subsequent MALDI-TOF analysis. Our results verify the biding of Sp1 transcription on the DAPA probe containing wildtype but not the mutant Sp1 biding sequence by subsequent western blotting. Our MALDI-TOF analysis of protein spots from two-dimensional gel did not reveal the binding of Sp1 protein, instead the identified a number of cellular proteins, such as U5 small nuclear RNP¡BATP-dependent DNA helicase 2 and actin of unknown significance.

Inactivating EGF signalling is key to modulating cell growth and avoiding cancer. To do this, the EGF receptor is ubiquitinated, internalized and sorted to lysosome for degradation. This latter process is coordinated by the endosomal sorting complex required for transport (ESCRT) machinery, a multi-complex protein machinery divided into four groups: ESCRT-0, I, II, III. ESCRTs recognise ubiquitinated cargoes and sort them from the limiting membrane of intermediate vesicles of maturing endosomes. In mammalian cells, the ESCRT machinery is also involved in other membrane related events, such as cytokinesis and viral budding. Certain ESCRTs, such as ESCRT-0, seem to be specifically required for cargo sorting to lysosomes, whereas other downstream ESCRTs, such as ESCRT-I, are required for all the cellular processes where the ESCRT machinery is involved. The existence of multiple variants of ESCRT-I components may suggest that ESCRT-I itself exists in different variants, each specific for a different membrane-based event. A bioinformatic study suggested Ubiquitin Associated Protein1 (UBAP1)as a novel variant of the ESCRT-I component MVB12. Moreover, a preliminary Y2H study identified UBAP1 as a potential binding partner of the ESCRT machinery regulator, HDPTP. This study aims to characterise UBAP1 as a variant MVB12 and a novel member of ESCRT-I. The results show that loss of UBAP1 impairs EGFR trafficking to lysosomes and causes the accumulation of ubiquitinated proteins on aberrant vacuolar structures. In cells, UBAP1 is incorporated in a complex with the ESCRT-I members TSG101, VPS28 and VPS37A. Importantly, UBAP1 uses three tandem ubiquitin associated (UBA) domains to bind ubiquitin and this activity is key for UBAP1 to function in cells. UBAP1 binds HDPTP via a peptide motif located about 100 aa. proximal to the tandem UBA domains. Altogether, the data shown in this thesis suggest that UBAP1 represents a subunit of an endosome-specific ESCRT-I complex, whose function may be coordinated by the ESCRT machinery regulator HDPTP.

O vírus da Zika (ZIKV) é um arbovírus emergente da família Flaviviridae, do gênero Flavivirus transmitido por mosquitos Aedes. Apesar da sua importância emergente na saúde pública, ainda pouco se conhece sobre os mecanismos moleculares envolvidos no ciclo replicativo do ZIKV em célula humanas. Assim, o objetivo geral deste estudo foi caracterizar a distribuição subcelular do ZIKV na célula hospedeira e elucidar fatores celulares que regulam o tráfego intracelular de proteínas envolvidos nesses processos. Mais especificamente, determinar os compartimentos celulares que servem de plataforma de montagem para o ZIKV. Além disso, também verificar se o funcionamento da maquinaria Endosomal Sorting Complexes Required for Transport (ESCRT) é requerido no ciclo replicativo de ZIKV. Para identificar a localização subcelular do ZIKV, foram utilizados diferentes marcadores celulares, e, de acordo com os resultados, foi demonstrado que com 3 horas pós infecção (h. p. i.) ocorre colocalização de proteínas do ZIKV com um marcador de endossomo primário, enquanto que com 15h p.i. já é possível detectar proteínas virais no Retículo Endoplasmático (RE). Subsequentemente, com 27h p.i. o ZIKV direciona-se para o complexo de Golgi. Juntos, esses resultados indicam o direcionamento do ZIKV através da via secretória ao longo do tempo. Além disso, foi testado o envolvimento da maquinaria dos ESCRTs por meio do silenciamento da expressão da proteína TSG101 de ESCRT-I em células infectadas com ZIKV. Os resultados obtidos, sugerem que ESCRT-I tem participação importante na replicação do ZIKV, ocorrendo a diminuição dos títulos virais quando TSG101 é depletada da célula. Em conjunto, os resultados permitem concluir que ao longo da infecção o ZIKV encontrase associado aos compartimentos da via secretória inicial (RE e complexo de Golgi), e que a proteína TSG101 de ESCRT-I exerce papel importante na replicação viral. Sendo assim, esse estudo possibilitou um melhor entendimento sobre a dinâmica de replicação do ZIKV em células humanas. / Zika virus (ZIKV) is an arbovirus of the Flaviviridae family, of the genus Flavivirus that is transmitted by Aedes mosquitoes. Despite its emerging importance in public health, little is known about the molecular mechanisms involved in the replicative cycle of ZIKV in human cells. Thus, the general objective of this study was to characterize the subcellular distribution of the ZIKV in the host cell and to elucidate cellular factors that regulate the intracellular trafficking of proteins involved in these processes. More specifically, to determine the cellular compartments that serve as assembly platforms for the ZIKV. In addition, the study aimed to verify if the functioning of the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery is required in the replicative cycle of ZIKV. In order to identify the subcellular localization of ZIKV, different intracellular markers were used, and, according to the results, it was demonstrated that at 3 hours post infection (h. p. i.) ZIKV proteins colocalize with an early endosome marker, whereas within 15h p.i. it is already possible to detect newlysynthesized viral proteins in the endoplasmic reticulum (ER). Subsequently, within 27h p.i., the ZIKV is directed to the Golgi complex. Together, these results delineate the targeting of ZIKV proteins through the secretory pathway over time. In addition, the involvement of the ESCRT machinery was tested by knocking down the expression of ESCRT-I protein TSG101 in ZIKV-infected cells. The results obtained suggest that ESCRT-I plays an important role in ZIKV replication, with viral titers decreasing when TSG101 levels are depleted in the cell. Together, the results allow us to conclude that ZIKV is associated with the initial secretory pathways (RE and Golgi complex) throughout the infection, and that the ESCRT-I TSG101 protein plays an important role in viral replication. Thus, this study contributes to a better understanding of the dynamics of ZIKV replication in human cells.

O vírus da Zika (ZIKV) é um arbovírus emergente da família Flaviviridae, do gênero Flavivirus transmitido por mosquitos Aedes. Apesar da sua importância emergente na saúde pública, ainda pouco se conhece sobre os mecanismos moleculares envolvidos no ciclo replicativo do ZIKV em célula humanas. Assim, o objetivo geral deste estudo foi caracterizar a distribuição subcelular do ZIKV na célula hospedeira e elucidar fatores celulares que regulam o tráfego intracelular de proteínas envolvidos nesses processos. Mais especificamente, determinar os compartimentos celulares que servem de plataforma de montagem para o ZIKV. Além disso, também verificar se o funcionamento da maquinaria Endosomal Sorting Complexes Required for Transport (ESCRT) é requerido no ciclo replicativo de ZIKV. Para identificar a localização subcelular do ZIKV, foram utilizados diferentes marcadores celulares, e, de acordo com os resultados, foi demonstrado que com 3 horas pós infecção (h. p. i.) ocorre colocalização de proteínas do ZIKV com um marcador de endossomo primário, enquanto que com 15h p.i. já é possível detectar proteínas virais no Retículo Endoplasmático (RE). Subsequentemente, com 27h p.i. o ZIKV direciona-se para o complexo de Golgi. Juntos, esses resultados indicam o direcionamento do ZIKV através da via secretória ao longo do tempo. Além disso, foi testado o envolvimento da maquinaria dos ESCRTs por meio do silenciamento da expressão da proteína TSG101 de ESCRT-I em células infectadas com ZIKV. Os resultados obtidos, sugerem que ESCRT-I tem participação importante na replicação do ZIKV, ocorrendo a diminuição dos títulos virais quando TSG101 é depletada da célula. Em conjunto, os resultados permitem concluir que ao longo da infecção o ZIKV encontrase associado aos compartimentos da via secretória inicial (RE e complexo de Golgi), e que a proteína TSG101 de ESCRT-I exerce papel importante na replicação viral. Sendo assim, esse estudo possibilitou um melhor entendimento sobre a dinâmica de replicação do ZIKV em células humanas. / Zika virus (ZIKV) is an arbovirus of the Flaviviridae family, of the genus Flavivirus that is transmitted by Aedes mosquitoes. Despite its emerging importance in public health, little is known about the molecular mechanisms involved in the replicative cycle of ZIKV in human cells. Thus, the general objective of this study was to characterize the subcellular distribution of the ZIKV in the host cell and to elucidate cellular factors that regulate the intracellular trafficking of proteins involved in these processes. More specifically, to determine the cellular compartments that serve as assembly platforms for the ZIKV. In addition, the study aimed to verify if the functioning of the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery is required in the replicative cycle of ZIKV. In order to identify the subcellular localization of ZIKV, different intracellular markers were used, and, according to the results, it was demonstrated that at 3 hours post infection (h. p. i.) ZIKV proteins colocalize with an early endosome marker, whereas within 15h p.i. it is already possible to detect newlysynthesized viral proteins in the endoplasmic reticulum (ER). Subsequently, within 27h p.i., the ZIKV is directed to the Golgi complex. Together, these results delineate the targeting of ZIKV proteins through the secretory pathway over time. In addition, the involvement of the ESCRT machinery was tested by knocking down the expression of ESCRT-I protein TSG101 in ZIKV-infected cells. The results obtained suggest that ESCRT-I plays an important role in ZIKV replication, with viral titers decreasing when TSG101 levels are depleted in the cell. Together, the results allow us to conclude that ZIKV is associated with the initial secretory pathways (RE and Golgi complex) throughout the infection, and that the ESCRT-I TSG101 protein plays an important role in viral replication. Thus, this study contributes to a better understanding of the dynamics of ZIKV replication in human cells.

La polyprotéine de structure Gag du VIH-1 est responsable de l’assemblage des particules virales dans les cellules infectées. Au niveau moléculaire, cette protéine s’oligomérise en formant des complexes Gag-Gag autour de deux plates-formes moléculaires, d'une part l'ARN génomique via son domaine NCp7 (NucleoCapsid protein 7) et d'autre part, la membrane plasmique via son domaine MA (Matrice). De plus, lors du trafic de Gag dans la cellule, Gag détourne les protéines ESCRT comme TSG101 et ALIX de la machinerie cellulaire afin de bourgeonner et d’être libérées dans le milieu extracellulaire. Dans cette thèse, nous avons étudié le rôle du domaine NCp7 seul ou au sein de Gag (GagNC) dans les interactions Gag-Gag et Gag-TSG101 en utilisant des approches biochimiques et de la microscopie de fluorescence quantitative. Les résultats ont montré que l'absence du domaine NCp7 affecte l’oligomerisation de Gag qui s’accumule alors dans le cytoplasme sous forme d’agrégats de taille importante. Par ailleurs, le trafic intracellulaire de Gag est affecté par les mutations dans le domaine GagNC avec une augmentation importante de temps nécessaire à Gag pour arriver à la membrane plasmique. Enfin, nous avons montré que GagNC i) renforce l’interaction entre le domaine p6 de Gag et TSG101 et ii) par sa fonction dans le trafic de Gag, est responsable de la localisation de TSG101 à la PM. Sur la base de ces résultats, des études sont maintenant en cours pour développer des tests afin d’identifier des molécules possédant un potentiel anti virale. / The Gag structural polyprotein of HIV-1 orchestrates viral particle assembly in producer cells, in a process that requires two platforms, the genomic RNA on the one hand and a membrane with a lipid bilayer, on the other. During its transportation from translating ribosomes to plasma membrane, Gag hijacks cellular proteins of the cytoskeleton and the ESCRT proteins like TSG101, Alix, etc., to egress viral particles. However, a number of questions remain to be answered before they are clearly apprehended. In this thesis, , we studied the role of the NC domain alone or as part of Gag (GagNC) in Gag-Gag and Gag-TSG101 interactions, which are essential for the assembly and budding of HIV-1 particles using quantitative fluorescent microscopy and biochemical approach. Results, showed that the absence of NC domain lead to (1) an accumulation of Gag as large aggregates that are dispersed in the cytoplasm, (2) a decrease of Gag-Gag condensation and (3) a delay for Gag-Gag complexes in reaching the PM, (4) improved interaction between Gag and TSG101, and (5) by its virtue in Gag trafficking docks TSG101 to the PM. This regulatory effect of NCp7 domain in either TSG101 or Gag or both protein- regulated pathways during virus budding can be exploited to develop inhibitors targeting HIV-1.