THAP5 is a protein which was recently isolated in the Zervos Lab as an interactor of a pro-apoptotic protein, Omi/HtrA2. THAP5 is unique because it shares no homology with mouse or rat and can only be found in humans. The only homology it shares with any other protein is its THAP domain. THAP proteins are zinc-dependent sequence specific DNA-binding factors belonging to the zinc-finger family of proteins (2). There are 12 identified members of TIIAP proteins in humans, THAP0-THAP 11. The roles of these THAP proteins include proliferation, apoptosis, cell cycle, chromosome segregation, chromosome modification, and transcriptional regulation (2). The function of THAP5 is still unclear and thus, a Yeast Two-Hybrid experiment will be done to further determine its function. The Yeast Two-Hybrid System is a common molecular biology technique used to identify interactors of a certain protein of interest. By identifying the protein interactors of THAP5 and their functions, it is possible to further determine the function of THAP5.

Melanome stellen die gefährlichste Form von Hautkrebs mit der höchsten Mortalitätsrate dar. Der Transformation normaler Melanozyten zu malignen Melanomen liegen komplexe molekulare und biochemische Veränderungen zu Grunde. Im Xiphophorus-Melanom-Modell ist die onkogene Rezeptortyrosinkinase "Xiphophorus melanoma receptor kinase" (Xmrk) der alleinige Auslöser der Melanominitiation und -progression. Die Aufklärung der Xmrk-vermittelten Signaltransduktion kann zum besseren Verständnis von Ereignissen, die auch bei der humanen Melanomentwicklung eine Rolle spielen, beitragen. In der vorliegenden Arbeit wurde mit Hilfe der Microarray-Technologie die Regulation der Genexpression durch Xmrk analysiert. Zu den nach Rezeptoraktivierung am stärksten herabregulierten Genen gehörten "son of sevenless homolog 1" (Sos1) und "ubiquitin-conjugating enzyme E2I" (Ube2i); stark hochreguliert waren "early growth response 1" (Egr1), "cysteine-rich protein 61" (Cyr61), "dual-specificity phosphatase 4" (Dusp4), "fos-like antigen 1" (Fosl1), "epithelial membrane protein" (Emp1), Osteopontin (Opn), "insulin-like growth factor binding protein 3" (Igfbp3) und "tumor-associated antigen L6" (Taal6). Die für die Regulation dieser Gene verantwortlichen Signalwege wurden durch die Anwendung von niedermolekularen Inhibitoren und siRNA identifiziert, wobei für die SRC-Kinase FYN eine zentrale Bedeutung bei der Xmrk-abhängigen Regulation der Genexpression festgestellt wurde. Darüber hinaus wurde die Expression der Gene in humanen Melanomzelllinien im Vergleich zu normalen humanen Melanozyten untersucht. Als besonders vielversprechende Kandidaten stellten sich dabei DUSP4 und TAAL6 heraus, deren Rolle in der humanen Melanominduktion und -progression Gegenstand zukünftiger Studien sein wird. In einem anderen Ansatz zur Aufklärung des Signalnetzwerkes sollten Zielproteine von Xmrk durch Protein-Protein-Interaktionsstudien mit Hilfe des Split-Ubiquitin-Systems ermittelt werden. Aufgrund ungünstiger Expressions- oder Faltungseigenschaften von Xmrk in diesem System war es aber nicht möglich, den Rezeptor als Köderprotein einzusetzen. Das für die Xmrk-vermittelte Melanomentstehung zentrale Protein FYN konnte jedoch als Köder etabliert und seine Wechselwirkung mit der Tyrosinkinase FAK analysiert werden. Es wurde gezeigt, dass der phosphorylierte Tyrosinrest an Position 397 von FAK für die Interaktion einer N-terminal trunkierten FAK-Variante mit FYN notwendig ist und dass diese Phosphorylierung in Hefe gewährleistet zu sein scheint. Die Suche nach neuen Interaktionspartnern von FYN mittels der Split-Ubiquitin-Technologie könnte Einblicke in weitere FYN-abhängige Ereignisse bieten, die zur Aufklärung seiner zentralen Rolle bei der Tumorentstehung dienen könnte. / Melanoma is the most aggressive type of skin cancer with the highest mortality rate. The transformation of melanocytes to malignant melanoma is based on complex molecular and biochemical alterations. In the Xiphophorus melanoma model, the oncogenic receptor tyrosine kinase Xiphophorus melanoma receptor kinase (Xmrk) is the sole trigger of melanoma initiation and progression. Elucidating Xmrk-dependent signaling pathways may contribute to a better understanding of processes that play a role in human melanomagenesis, too. Here, the regulation of gene expression by Xmrk was analyzed using a microarray approach. The genes with the strongest down-regulation in response to receptor activation included son of sevenless homolog 1 (Sos1) and ubiquitin-conjugating enzyme E2I (Ube2i), whereas early growth response 1 (Egr1), cysteine-rich protein 61 (Cyr61), dual-specificity phosphatase 4 (Dusp4), fos-like antigen 1 (Fosl1), epithelial membrane protein (Emp1), Osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), and tumor-associated antigen L6 (Taal6) were strongly up-regulated. The pathways regulating expression of these genes were identified by applying small molecule inhibitors and siRNA. Interestingly, the SRC-family kinase FYN was found to be a key-player in Xmrk-dependent gene regulation. Furthermore, expression of the genes in human melanoma cell lines compared to normal human melanocytes was investigated. The most promising candidates, which might be important for melanoma induction and progression, were DUSP4 and TAAL6. Their potential suitability as diagnostic and prognostic melanoma markers will be addressed in future studies. In addition to gene expression analysis, protein-protein interactions were to be assayed by the split-ubiquitin-system in order to identify novel Xmrk targets. Unfortunately, inappropriate expression or folding of the receptor in this system precluded it from working as bait. However, the FYN protein, which has a central role in Xmrk-mediated signaling, was established as bait and its association with FAK was analyzed in more detail. A phosphorylated tyrosine residue at position 397 of FAK was demonstrated to be necessary for the interaction of an N-terminally truncated FAK variant with FYN, and this phosphorylation event seems to be feasible in yeast. In future, a split-ubiquitin based screen for novel interaction partners of FYN might provide insights into FYN-dependent processes and help to understand its central role in tumor development.

Septinas são proteínas que pertencem a super família das GTPases e que foram inicialmente identificadas em Saccharomyces cerevisae, mas logo em seguida, também em eucariotos superiores, exceto em plantas. Estas proteínas estão envolvidas em uma variedade de processos celulares tais como segregação de cromossomos, polaridade celular, dinâmica da membrana, tráfego de vesículas, exocitose, apoptose, entre outros. Mutações ou alterações no padrão de expressão de septinas são associadas com vários cânceres e doenças neurológicas. Objetivando contribuir com informações funcionais sobre tais proteínas, as septinas humanas 1, 5 e 7 foram usadas como iscas em ensaios de duplo híbrido em leveduras visando à identificação de seus parceiros protéicos. Após a varredura de bibliotecas de cDNA de leucócitos e cérebro fetal humano, os parceiros protéicos predominantemente encontrados foram outras septinas de grupos diferentes aos das iscas. As interações septina-septina envolveram o domínio de ligação a GTP. Ainda, outros parceiros, diferentes de septinas, foram também identificados nas bibliotecas e estes se mostraram funcionalmente relacionados à endocitose, à regulação da atividade de GTPases, ao tráfego intracelular, aos ciclos de sumoilação, à manutenção da placa metafásica e à maturação do centrossomo. Algumas destas funções são inéditas e foram pela primeira vez relacionadas às septinas. Este trabalho também esteve voltado à caracterização biofísica das septinas 3 e 5 (SEPT3 e SEPT5). Muitos protocolos diferentes foram desenvolvidos na tentativa de obter amostras homogêneas de SEPT5, mas não foram bem sucedidos. Por outro lado, SEPT3 recombinante, destituída do domínio amino-terminal (SEPT3GC) foi eficientemente produzida em E. coli. O estado monomérico de SEPT3GC em solução foi confirmado por cromatografia de exclusão molecular e espalhamento de raios-X a baixos ângulos (SAXS). SEPT3GC mostrou-se ativa e capaz de hidrolizar GTP in vitro. A afinidade de SEPT3GC por GTPγS e GDP foi avaliada por calorimetria de titulação isotérmica (ITC), sendo que o KD de SEPT3GC para GTPγS foi de 5,43 μM e a ligação para este nucleotídeo foi dependente de Mg2+. A ligação para GDP não foi detectável. Agregados de SEPT3GC induzidos por temperatura foram capazes de ligar a sonda fluorescente tioflavina-T, sugerindo uma natureza amilóide para tais estruturas. / Septins are proteins that belong to the superfamily of GTPases, which were initially identified in Saccharomyces cerevisiae, and then in higher eukaryotes, except plants. These proteins are involved in a variety of cellular processes such as chromosome segregation, cell polarity, membrane dynamics, vesicle trafficking, exocytosis, apoptosis, among others. Mutations or changes in the expression of septins have been associated with various cancers and neurological diseases. Aiming to provide functional information about these proteins, the human septins 1, 5 and 7 were used as baits in yeast two-hybrid assay in order to identify their protein partners. After screening cDNA libraries from human leukocytes and fetal brain, the protein partners predominantly found were septins from others groups. The septin-septin interactions involved the GTP binding domain. Others non-septins interactors have also been identified in the libraries and were functionally related to endocytosis, the regulation of the GTPase activity, intracellular trafficking, sumoylation, maintenance of metaphase plate and centrosome maturation. Some of these functions are new and were related to the septins for the first time. This work also focused on the biophysical characterization of the septins 3 and 5 (SEPT3 and SEPT5). Many different protocols were developed aiming to obtain homogeneous samples of SEPT5, but were not successful. On the other hand, recombinant SEPT3, without the amino-terminal domain (SEPT3GC), was produced in a homogeneous form in E. coli. SEPT3GC is monomeric in solution as confirmed by size exclusion chromatography and small-angle X-ray scattering (SAXS). Also, SEPT3GC has shown to be active and able to hydrolyze GTP in vitro. The SEPT3GC affinity by GTPγS and GDP were evaluated by isothermal titration calorimetry (ITC). The KD for GTPγS was about 5,43 μM and it was observed to be Mg2+ dependent. The binding to GDP was not detectable. SEPT3GC aggregates induced by temperature were able to bind the thioflavin-T fluorescent probe, suggesting an amyloid nature for such structures.

Neuronal migration is an essential aspect of nervous system development; improper or incomplete neuronal migration can lead to debilitating disorders. The model organism Caenorhabditis elegans has 302 neurons and is ideal for studying nervous system development. The cytoplasmic adaptor protein, MIG-10, is necessary for the long range anteroposterior migration during embryogenesis of the neurons CAN, ALM, and HSN. Mutations in the mig-10 gene result in incomplete migrations of all three neurons. MIG-10 is a homologue of the vertebrate proteins lamellipodin and RIAM-1, which are involved in directing actin polymerization during axon outgrowth and guidance. RIAM-1 is known to interact with proteins from the Ras GTPase family. The MIG-10 protein has a pleckstrin homology (PH) domain, a Ras-associating (RA) domain, and a proline-rich region. We used a yeast two-hybrid system to investigate which Ras family proteins MIG-10 interacts with. Three isoforms of MIG-10, MIG-10A, MIG-10B, and MIG-10C, as well as the RAPH domain alone, were used as baits. No evidence of interaction was observed for any of the baits used. These results do not reject our hypothesis as the constitutively active Ras clones may need to be used or there may not be a direct interaction between MIG-10 and the Ras family members. We are currently screening a C. elegans cDNA library for interactions with all three isoforms of MIG-10. In the future we plan to investigate how MIG-10 may be involved in the WAVE/SCAR actin nucleation pathway.

The type II secretion system is used by many pathogenic and non-pathogenic bacteria for the extracellular secretion of enzymes and toxins. <i>Aeromonas hydrophila</i> is a Gram-negative pathogen that secretes proteins via the type II secretion system.<p> In the studies described here, a series of yeast two-hybrid assays was performed to identify protein-protein interactions in the type II secretion system of <i>A. hydrophila</i>. The periplasmic domains of ExeA and ExeB were assayed for interactions with the periplasmic domains of Exe A, B, C, D, K, L, M, and N. Interactions were observed for both ExeA and ExeB with the secretin ExeD in one orientation. In addition, a previously identified interaction between ExeC and ExeD was observed. In order to further examine and map these interactions, a series of eight two-codon insertion mutations in the amino terminal domain of ExeD was screened against the periplasmic domains of ExeA and ExeB. As a result, the interactions were verified and mapped to subdomains of the ExeD periplasmic domain. To positively identify the region of ExeD involved in the interactions with ExeA, B, C and D, deletion mutants of ExeD were constructed based on the two-codon insertion mutation mapping of subdomains of the ExeD periplasmic domain, and yeast two-hybrid assays were carried out. The results showed that a fragment of the periplasmic domain of ExeD, from amino acid residue 26 to 200 of ExeD, was involved in the interactions with ExeA, B and C. As an independent assay for interactions between ExeAB and the secretin, His-tagged derivatives of the periplasmic domains of ExeA and ExeB were constructed and co-purification on Ni-NTA agarose columns was used to test for interactions with untagged ExeD. These experiments confirmed the interaction between ExeA and ExeD, although there was background in the co-purification test.<p> These results provide support for the hypothesis that the ExeAB complex functions to organize the assembly of the secretin through interactions between both peptidoglycan and the secretin that result in its multimerization into the peptidoglycan and outer membrane layers of the envelope.

Reagents that bind proteins are applicable in biology for detection of molecules, perturbation of signaling pathways and development of small-molecule pharmaceuticals. Protein ligands interact with proteins, inhibiting or altering their function. They are isolated from combinatorial libraries to interact with a specific target, using selection techniques such as phage display or yeast-two-hybrid assay. For the latter, one inconvenience is the detection of false positives, which can be solved by screening pools containing the samples to be tested, instead of individual samples. Samples are distributed in the pools following a pooling design. The PI-deconvolution pooling design was developed to screen cDNA libraries using the yeast-two-hybrid assay, which are smaller in size than protein ligand combinatorial libraries. Modifications to the PI-deconvolution screening technique were developed to adapt it to the screening of protein ligand combinatorial libraries using the yeast-two-hybrid assay. Every spot of the array containing the combinatorial library was randomly pooled. However, the yeast-two-hybrid assay loses sensitivity when strains are pooled. As PI-deconvolution requires detecting every interaction, we determined the optimal amount of library members that can be pooled in a spot, and the optimal number of replicates to ensure the detection of an interaction.<p> The yeast-two-hybrid assay was used to perform a screening of a combinatorial library with seven domains of BCR-ABL, which were pooled according to PI-deconvolution. BCR-ABL is a chimeric protein with unregulated kinase activity that is responsible for chronic myelogenous leukemia. The scaffold used in the combinatorial library was an engineered intein that forms lariat peptides. After a screening of this library was performed, positive interactions were detected in 775 spots of the arrays that contained 1432 positive hits. Only 53 spots were deconvoluted. The coding sequences of the lariat peptides were determined for 23 lariat peptides interacted with the GEF domain of BCR, and for ABL, two with the FABD domain, one with the SH1 domain, and one with the SH3 domain. Finally, a &beta;-galactosidase assay was performed to assess the affinity of the lariat peptides for their target.<p> The isolated lariat peptides are potential inhibitors of BCR-ABL that can have therapeutic potential. This study will improve other screenings of combinatorial libraries with the yeast-two-hybrid assay.

The type II secretion system is used by many pathogenic and non-pathogenic bacteria for the extracellular secretion of enzymes and toxins. <i>Aeromonas hydrophila</i> is a Gram-negative pathogen that secretes proteins via the type II secretion system.<p> In the studies described here, a series of yeast two-hybrid assays was performed to identify protein-protein interactions in the type II secretion system of <i>A. hydrophila</i>. The periplasmic domains of ExeA and ExeB were assayed for interactions with the periplasmic domains of Exe A, B, C, D, K, L, M, and N. Interactions were observed for both ExeA and ExeB with the secretin ExeD in one orientation. In addition, a previously identified interaction between ExeC and ExeD was observed. In order to further examine and map these interactions, a series of eight two-codon insertion mutations in the amino terminal domain of ExeD was screened against the periplasmic domains of ExeA and ExeB. As a result, the interactions were verified and mapped to subdomains of the ExeD periplasmic domain. To positively identify the region of ExeD involved in the interactions with ExeA, B, C and D, deletion mutants of ExeD were constructed based on the two-codon insertion mutation mapping of subdomains of the ExeD periplasmic domain, and yeast two-hybrid assays were carried out. The results showed that a fragment of the periplasmic domain of ExeD, from amino acid residue 26 to 200 of ExeD, was involved in the interactions with ExeA, B and C. As an independent assay for interactions between ExeAB and the secretin, His-tagged derivatives of the periplasmic domains of ExeA and ExeB were constructed and co-purification on Ni-NTA agarose columns was used to test for interactions with untagged ExeD. These experiments confirmed the interaction between ExeA and ExeD, although there was background in the co-purification test.<p> These results provide support for the hypothesis that the ExeAB complex functions to organize the assembly of the secretin through interactions between both peptidoglycan and the secretin that result in its multimerization into the peptidoglycan and outer membrane layers of the envelope.

Reagents that bind proteins are applicable in biology for detection of molecules, perturbation of signaling pathways and development of small-molecule pharmaceuticals. Protein ligands interact with proteins, inhibiting or altering their function. They are isolated from combinatorial libraries to interact with a specific target, using selection techniques such as phage display or yeast-two-hybrid assay. For the latter, one inconvenience is the detection of false positives, which can be solved by screening pools containing the samples to be tested, instead of individual samples. Samples are distributed in the pools following a pooling design. The PI-deconvolution pooling design was developed to screen cDNA libraries using the yeast-two-hybrid assay, which are smaller in size than protein ligand combinatorial libraries. Modifications to the PI-deconvolution screening technique were developed to adapt it to the screening of protein ligand combinatorial libraries using the yeast-two-hybrid assay. Every spot of the array containing the combinatorial library was randomly pooled. However, the yeast-two-hybrid assay loses sensitivity when strains are pooled. As PI-deconvolution requires detecting every interaction, we determined the optimal amount of library members that can be pooled in a spot, and the optimal number of replicates to ensure the detection of an interaction.<p> The yeast-two-hybrid assay was used to perform a screening of a combinatorial library with seven domains of BCR-ABL, which were pooled according to PI-deconvolution. BCR-ABL is a chimeric protein with unregulated kinase activity that is responsible for chronic myelogenous leukemia. The scaffold used in the combinatorial library was an engineered intein that forms lariat peptides. After a screening of this library was performed, positive interactions were detected in 775 spots of the arrays that contained 1432 positive hits. Only 53 spots were deconvoluted. The coding sequences of the lariat peptides were determined for 23 lariat peptides interacted with the GEF domain of BCR, and for ABL, two with the FABD domain, one with the SH1 domain, and one with the SH3 domain. Finally, a &beta;-galactosidase assay was performed to assess the affinity of the lariat peptides for their target.<p> The isolated lariat peptides are potential inhibitors of BCR-ABL that can have therapeutic potential. This study will improve other screenings of combinatorial libraries with the yeast-two-hybrid assay.

Equine infectious anemia virus (EIAV) is an important animal model to study the contribution of macrophages in viral persistence during lentiviral infections. EIAV is unique amongst the lentiviruses in that it causes a rapid, rather than the very slow disease progression, characteristic of other lentiviral infections. The accessory gene, S2, unique to EIAV, is an important determinant in viral pathogenesis. A functional S2 gene is required to achieve high-titer viremia and the development of disease in infected horses. Despite its essential role, the mechanisms by which S2 influences EIAV pathogenesis remain elusive. The goal of this research was to gain insight into the role of S2 in pathogenesis. To accomplish this goal we: (i) Examined the effects of EIAV and its S2 protein in the regulation of the cytokine and chemokine responses in macrophages, (ii) Assessed the influence of EIAV infection and the effect of S2 on global gene expression in macrophages and (iii) Identified host cellular proteins that interact with S2 as a starting point for the identification of host factors implicated in S2 function. The results from this study provide evidence for a role of S2 in enhancing a proinflammatory cytokine and chemokine response in infected macrophages. Specifically, S2 enhances the expression of IL-1 alpha, IL-1 beta IL-8, MCP-2, MIP-1 beta, IP-10 and a newly discovered cytokine, IL-34. Involvement of S2 in cytokine and chemokine dysregulation may contribute to disease development by optimizing the host cell environment to promote viral dissemination and replication. Microarray analyses revealed an interesting set of differentially expressed genes upon EIAV infection. Genes affected by EIAV were involved in the immune response, transcription, translation, cell cycle and cell survival. Finally, we used the yeast two-hybrid system to identify S2 host cellular interacting proteins. We identified osteosarcoma amplified 9 (OS-9) and proteasome 26S ATPase subunit 3 (PSMC3) proteins as interacting partners of S2. Additional evidence is needed to demonstrate the physiological relevance of these interactions in vivo. In summary, the results from this study contribute towards our understanding of the role S2 in disease expression and allow the formulation of new hypotheses as to the potential mechanisms of action of S2 during EIAV infection.

Loss of the promyelocytic leukemia (PML) protein is associated with genomic instability/cancer. There are several isoforms of the PML protein that localize in PML nuclear bodies (PML NBs). How each individual isoform contributes to the functions of PML NBs is unknown. The objective of this study was to identify and characterize PML isoform-I (PML-I) specific protein-protein interactions. Using yeast two-hybrid screens, several interacting partners of PML-I were identified that play roles in translational regulation, including eukaryotic initiation factor 3 subunit K (eIF3K). Our studies demonstrated that eIF3K interacts with PML-I in vitro and in vivo. Through its interaction with eIF3K, overexpression of PML-I resulted in the concomitant increase in eIF3K protein levels in mammalian cells. This suggests that PML-I may be involved in regulating eIF3K protein translation or stability, which in turn could affect translation of specific mRNAs or global translation in cancer cells with reduced expression of PML-I.