No description available.

Ulm, Univ., Diss., 2007.

Marburg, Univ., Diss., 2009

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.

Understanding the signaling mechanisms that determine juvenile-to-mature transition and bud fate is vital for controlling tree reproduction. FD-like proteins also appear to be important for initiating reproductive development. In this study, phylogenetic analysis showed that three FD-like genes (FDL1, FDL2, and FDL3) are present in the poplar genome. FDL1 and FDL2 are products of a recent whole genome duplication event while FDL3 escaped such duplication. Yeast two-hybrid assays demonstrated that FT1 and FT2 proteins interact with FDL3 protein, but not with FDL1 or FDL2 protein. Analysis of the expression levels of FD-like transcripts in Populus deltoides via quantitative real-time PCR showed that FDL3 abundantly expresses in the shoot apex where it probably interacts with FT1 in late winter and early spring. Following the duplication event, FDL1 and FDL2 appear to have diverged in function as they express in a number of tissues in the fall, winter, and spring.

Dans le thyrocyte normal, la TSH active une voie dépendante de l’adénylyl cyclase/AMPc, qui représente l’une des trois voies mitogéniques de la thyroïde. La cascade de signalisation de la TSH diffère des deux autres voies dans sa capacité à induire à la fois la prolifération et la différenciation, comprenant la synthèse et la sécrétion des hormones thyroïdiennes. Identifier les acteurs de cette cascade de signalisation, ainsi que les interactions entre effecteurs, est donc très important pour la compréhension de la fonction de la cellule thyroïdienne. C’est dans ce cadre que s’insère notre travail au cours duquel nous nous sommes intéressés au récepteur de la TSH ainsi qu’à une protéine récemment identifiée dans le laboratoire et dont l’expression est modulée en réponse à la TSH dans la thyroïde : C3VS. C3VS est une protéine qui présente six motifs ankyrine et une tirette à leucine et dont la fonction était inconnue à l'époque. Dans un premier temps, nous avons contribué à l’obtention de la séquence codante complète du C3VS de chien, puis, l'identification des partenaires d'une protéine pouvant aider à caractériser sa fonction, nous nous sommes proposé de rechercher les partenaires potentiels de la région N-terminale de C3VS par la méthode double-hybride. Nous avons étudié la distribution tissulaire et la régulation par la TSH de différents partenaires isolés. Parmi eux, SUG1, une ATPase du protéasome 26S, a été étudiée plus avant mais l’interaction n’a pas pu être confirmée par "GST-pulldown assay". Simultanément, une remise en question de la position de la méthionine initiale de C3VS, couplée à une impossibilité d’exprimer la protéine en cellules COS par transfection mettait en péril le travail. En l’absence de plus d’arguments fonctionnels permettant d’orienter l’étude des positifs, cette partie du travail a été suspendue au profit de notre étude sur le récepteur de la TSH. L'activation de cascades différentes dans le thyrocyte humain et canin pouvant être due à l'action de protéines intracellulaires, nous avons tenté de rechercher par double-hybride des partenaires protéiques autres que les protéines G pour le récepteur de la TSH. Nous avons ainsi identifié PRA1 mais nous n’avons pas pu confirmer l'interaction entre les deux protéines par "GST-pulldown assay". Pour tenter de comprendre le rôle de cette interaction, nous avons réalisé des essais fonctionnels en transfectant des cellules pour évaluer l'implication de PRA1 sur la synthèse d’AMPc. Ces expériences ne nous ont pas permis de montrer un rôle pour PRA1 au niveau de la cascade, mais en revanche, nous avons mis en évidence le fait que la co-transfection de deux ADNc codant pour des protéines membranaires sature la machinerie de traduction et diminue l'expression du RTSH. Dans une deuxième partie de notre travail, nous avons étudié la 5-phosphatase SHIP2, dont l’implication dans la cascade de réponse à l’insuline était suggérée, entre autres, par le travail d’Isabelle Vandenbroere qui avait montré l’interaction de cette protéine avec CAP et c-Cbl. Nous avons développé au laboratoire la culture de la lignée pré-adipocytaire 3T3-L1 et étudié la localisation de SHIP2 au niveau des rafts de ces cellules. Nous avons montré que SHIP2 n’y est pas recrutée. CAP et c-Cbl ne semblent pas non plus y être recrutées, tandis que nous y avons détecté le récepteur de l'insuline. La localisation de différentes protéines impliquées dans la cascade de l'insuline dans les rafts est une question controversée à l’heure actuelle et notre étude montre que l’implication fonctionnelle de SHIP2 dans la cascade de l'insuline n'est probablement pas dépendante des rafts.

The plasminogen activator (PA) system plays an important role in cell adhesion, migration and invasion, and may require the coordinated expression of various proteins. The human urokinase-type plasminogen activator (uPA) receptor (uPAR) is a central protein component of the PA system. By binding its ligand uPA, uPAR can direct proteolysis of the extracellular matrix. Also, it is now apparent that uPAR can initiate proteolytic independent signal transduction to influence angiogenesis, inflammation, wound repair and tumour progression. To determine whether any novel proteins interacted with uPAR, a yeast two-hybrid screening analysis was undertaken using alternate uPAR domain constructs as baits. These included full-length three domain uPAR (uPAR-DIDIIDIII), two domain uPAR (uPAR-DIIDIII), and each individual uPAR domain (uPAR-DI, uPAR-DII and uPAR-DIII). A number of proteins were identified as putative candidate interactors for the alternate constructs, with two of special interest for uPAR-DIDIIDIII. These were the heat shock protein Mrj, and the extracellular matrix protein fibulin-2. The protein Mrj was shown to bind uPAR both in vitro and in vivo using GST-pull down and co-immunoprecipitation assays respectively. The GST-pull down assay identified the interaction between Mrj and uPAR dependent on the C-terminal domain of Mrj and DI of uPAR. Using in vivo co-immunoprecipitation analysis, Mrj also bound to uPAR. Preliminary data suggest the association between uPAR and Mrj may play a role in the regulation of apoptosis. In regard to the uPAR interactor of fibulin-2, a calcium dependent binding interaction with uPAR was identified using the GST-pull down assay. However due to the large molecular weight and stringent conditions needed to solubilise fibulin-2, it was not possible to co-immunoprecipitate both uPAR and fibulin-2. Together, the identification of both Mrj and fibulin-2 amongst other candidate interactors of uPAR presented here provides further insight into the intricate relationship between uPAR and other proteins which may influence a range of biological functions.

DNA post-replication repair (PRR) is a cellular tolerance mechanism by which eukaryotic cells survive lethal lesions during or after DNA synthesis. In the yeast Saccharomyces cerevisiae, modification of proliferating cell nuclear antigen (PCNA) by ubiquitin and by small ubiquitin-like modifier (SUMO) plays an important role in PRR. PCNA ubiquitination is dependent on Rad6, a ubiquitin-conjugating enzyme (E2) and Rad18, a ubiquitin ligase (E3). Rad6 and Rad18 form a stable complex. PCNA sumoylation is dependent on Ubc9, an E2 specific to SUMO modification. <p>PRR in mammalian cells is less well understood. However, human Rad18 (hRad18) has been found to interact with human Rad6 (HHR6A/B). In this study, we detected physical interaction between hRad18 and human Ubc9 (hUbc9) through yeast two-hybrid assays. In order to define the domain(s) of hRad18 involved in the formation of a complex with HHR6B or hUbc9, a series of yeast two-hybrid constructs containing various hRAD18 gene deletions and mutations were made. A C-terminal region of hRad18, containing the putative HHR6A/B binding domain (amino acids 340 to 395), interacts with HHR6A/B while the N-terminus (amino acids 1-93) does not. Yeast Rad18 has a homologous fragment of the HHR6A/B binding domain and this fragment is sufficient to interact with yeast Rad6 in yeast two-hybrid assays, so we infer that hRad18 interacts with HHR6B through the same domain. Surprisingly, both the N-terminal and C-terminal fragments of hRad18 can interact with hUbc9, suggesting the existence of two separate domains in hRad18 interacting with hUbc9. The N-terminal fragment of hRad18 contains only a RING finger domain (amino acids 25-64), which is probably responsible for binding to hUbc9. The C-terminal fragment of hRad18 with HHR6A/B binding domain deletion can still interact with hUbc9, suggesting that the HHR6A/B binding domain is not involved in hUbc9 interaction. A key cysteine mutation (C28F) in the RING finger domain abolished the interactions of hRad18 with both HHR6A/B and hUbc9. This amino acid substitution is likely to alter the three-dimensional structure of the protein, thus making the protein unstable. Taken together, results obtained from this study suggest that hRad18 may regulate the modification status of PCNA by interacting with two different E2s, HHR6A/B and hUbc9, through distinct domains.

The goal of this research is to understand how three different parameters affect single chain variable fragment (scFv) binding capacity. The parameters that were varied include the number of variable complementarity determining regions (CDRs), the number amino acids used to diversify CDRs, and configuration of the structure. How the parameters affect the binding capacity will be tested using the yeast two hybrid assay against five different protein domains. Eight scFv libraries were generated; the genes expressing the scFvs were constructed and the CDRs were randomized using PCR amplification. Genes expressing scFvs were cloned, using the homologous gap repair mechanism in <i>Saccharomyces cerevisiae</I>. Representative members of scFv libraries were sequenced to confirm correct construction.<p> Library diversity was calculated from the library transformation efficiency. Transformation efficiency refers to the number of cells that grew at the time of transformation of the scFv gene into yeast cells. There were significant differences in the diversity of the scFv libraries, which created difficulty in comparing the library binding capacities. Sequencing the scFv libraries revealed that on average 50% of each library contained correct scFv sequences. The percent of correct sequences within each library was then used to calculate the functional diversity.<p> The yeast two-hybrid assay was used to screen the scFv libraries for interactions and to test binding capacity. The binding capacity of the scFv libraries was tested and compared in five different yeast two-hybrid assays using five protein domains as the targets for each screen. The screening results showed that in all cases cyclic scFv libraries had a statistically significant higher binding capacity than linear scFv libraries despite a diversity bias against the cyclic libraries. There was no clear trend in binding capacity with the other two parameters; however, the four amino acid three CDR libraries dominated over the other libraries in almost every screen.<p> Some of the scFvs isolated from the screens were expressed in <i>E. coli</i> and <i>S. cerevisiae</i>to analyze for proper expression and correct size. All the scFvs that were isolated and analyzed were the correct size and could be purified using a poly histidine tag.<p> Due to its bioaffinity and specificity, scFvs were constructed to profile disease patterns, and to identify potential drug targets. In addition to its original application to health-related studies, scFvs could also be extended to locate potential metabolic bottlenecks, to alter metabolic flux to enhance productivity, and regulate metabolic bionetworks. Industrial microorganisms are generally carrying more than two sets of chromosomes, making it difficult to be genetically engineered when conventional approaches are employed. With the availability of scFvs as reported in this thesis, we are able to design specific scFvs that selectively bind to target proteins, resulting in re-routing of metabolic flux within the microorganism, toward a high productivity of desired product. ScFvs can be applied to industrial microorganisms directly, leading to the development of new fermentation processes.

DNA post-replication repair (PRR) is a cellular tolerance mechanism by which eukaryotic cells survive lethal lesions during or after DNA synthesis. In the yeast Saccharomyces cerevisiae, modification of proliferating cell nuclear antigen (PCNA) by ubiquitin and by small ubiquitin-like modifier (SUMO) plays an important role in PRR. PCNA ubiquitination is dependent on Rad6, a ubiquitin-conjugating enzyme (E2) and Rad18, a ubiquitin ligase (E3). Rad6 and Rad18 form a stable complex. PCNA sumoylation is dependent on Ubc9, an E2 specific to SUMO modification. <p>PRR in mammalian cells is less well understood. However, human Rad18 (hRad18) has been found to interact with human Rad6 (HHR6A/B). In this study, we detected physical interaction between hRad18 and human Ubc9 (hUbc9) through yeast two-hybrid assays. In order to define the domain(s) of hRad18 involved in the formation of a complex with HHR6B or hUbc9, a series of yeast two-hybrid constructs containing various hRAD18 gene deletions and mutations were made. A C-terminal region of hRad18, containing the putative HHR6A/B binding domain (amino acids 340 to 395), interacts with HHR6A/B while the N-terminus (amino acids 1-93) does not. Yeast Rad18 has a homologous fragment of the HHR6A/B binding domain and this fragment is sufficient to interact with yeast Rad6 in yeast two-hybrid assays, so we infer that hRad18 interacts with HHR6B through the same domain. Surprisingly, both the N-terminal and C-terminal fragments of hRad18 can interact with hUbc9, suggesting the existence of two separate domains in hRad18 interacting with hUbc9. The N-terminal fragment of hRad18 contains only a RING finger domain (amino acids 25-64), which is probably responsible for binding to hUbc9. The C-terminal fragment of hRad18 with HHR6A/B binding domain deletion can still interact with hUbc9, suggesting that the HHR6A/B binding domain is not involved in hUbc9 interaction. A key cysteine mutation (C28F) in the RING finger domain abolished the interactions of hRad18 with both HHR6A/B and hUbc9. This amino acid substitution is likely to alter the three-dimensional structure of the protein, thus making the protein unstable. Taken together, results obtained from this study suggest that hRad18 may regulate the modification status of PCNA by interacting with two different E2s, HHR6A/B and hUbc9, through distinct domains.