Oncoprotein18 (Op18) is a 19 kDa cytosolic phosphoprotein critical for cell growth and differentiation. Unphosphorylated Op18 associates with £\£]-tubulin heterodimer to form 2 tubulin-1 stathmin T2S complex and promotes microtubule catastrophe in interphase. Numerous cellular signals activate downstream protein kinases result in the phosphorylation of Ser16, Ser25, Ser38 and Ser 63 residues in Op18 that lowers its affinity for tubulin thereby increases the stability of microtubule and facilitates the formation of spindle during mitosis. Here, we found in addition to phosphorylation, Op18 could also be ubiquitin modified in vivo. An expression plasmid encodes for mutant EGFP-Op18-M5K protein whose potential lysine residues K42, K53, K75, K104, and K119 were mutated to arginines was generated to investigate the effect of ubiquitin modification of Op18 on the tubulin polymerization. Our results revealed a decrease of ubiquitin modification of mutant EGFP-Op18-M5K in comparison with that of wildtype EGFP-Op18. The expression of mutant but not the wildtype Op18 resulted in a significant increase of polymerized tubulin in mitotic cell implying that they might exhibit differential tubulin binding affinity. Moreover, the result of western blotting showed that the mutant Op18 detected in the mitotic cell corresponds to the phosphorylated version of Op18. In summary, these results imply the ubiquitination of Op18 might interfere with its phosphorylation and decrease its tubulin binding potential, thereby facilitates the polymerization of tubulin in mitotic cells. The in vitro tubulin polymerization assay will be performed to further confirm the above finding.

The genetic defects that results in TAL1 oncogene activation are commonly seen in leukemic cells of the patient with T-cell Acute Lymphoblastic Leukemia ( T-ALL ). The ectopic expression of TAL1 oncoprotein perturbs the development of T-cell, hence promotes the formation of leukemia. TAL1 gene encodes proteins with basic helix-loop-helix ( bHLH ) domain, a protein dimerization and DNA binding domain. In T-ALL cells, two Tal1 proteins, pp42(1-331 amino acids) and pp22(176-331 amino acids) are produced that both contain bHLH domain. Both proteins interact with immunoglobulin gene enhancer binding protein, E12/E47 to form DNA-binding heterodimers, that can bind to consensus E-box DNA sequence AACAGATGGT. Phosphorylation of S122 residue modulates the trans-activation potential of Tal1 protein. In addition, S172 is an inducible c-AMP dependent protein kinase (PKA) phosphorylation site in vivo. The phosphorylation of TAL1 S172 upon stimulation by forskolin can increase the DNA binding of E12-Tal1 heterodimer. We used site-directed mutagenesis to investigate the effect of S194,S224 mutation on the function of truncated Tal1 oncoprotein.Mutant Tal1 and E12 proteins expression plasmids were constructed and introduced into COS-1 cells by cotransfection. Tal1 and E12 protein expression in transfected cell were evaluated by Western blotting. The protein-DNA interaction were evaluated by electrophorectic mobility shift assay. The mutation of S194 and S224 of Tal1 protein all resulted in the loss of DNA-binding complex formation. This data indicated that these serine residues are essential for bHLH function. However, the phosphorylation status of these two residues in vivo, and what kinase is responsible for the phosphorylation of these residues, await further investigation.

Abstract¡G TAL1 gene was originally discovered as a result of its activation by chromosome rearrangements in T cell acute lymphoblastic leukaemia(T-ALL). Further studies have shown that TAL1 expression is aberrantly activated by several mechanisms including chromosome translocations, interstitial deletion and transactivation without detectable chromosomal alteration. TAL1 gene encodes a bHLH transcription factor, that is essential for the development of all haematopoietic lineages and its expression is maintained during differentiation along erythroid, mast and megakaryocytic cell lineages, but not in normal peripheral T-lymphocytes. The bHLH motif of these protein is responsible for DNA binding and dimerization with other bHLH proteins involved in transcription regulation. TAL1 protein is able to form heterodimers with the ubiquitously expressed E2A gene products, E47 and E12, and the heterodimers bind to E-box motif with the general sequence CANNTG. But the target genes for TAL1 oncoprotein have not yet been identified. We have previously isolated TAL1/E12 heterodimer bound genomic fragments by chromatin immunoprecipitation from K562 cells, and selected 6 fragments with one to four E-box CANNTG sequences. In order to determine if these fragments could be the regulatory elements of potential target genes of TAL1 oncoprotein, we inserted these 6 DNA fragments individually into pGL3 to generate recombinant reporter plasmids. The transfection experiments indicated that K34 and K94 DNA fragments behaved as a transcriptional transactivating sequence, and TAL1 and E12 proteins are required for efficient transcriptional activity. We also showed that transfection of these two recombinant constructs into K562 cells generated positive transcriptional activity, in a level similar to that in TAL1 and E12 co-transfected COS1 cells. These results established that both K34 and K94 DNA fragments are likely to contain a promoter of potential TAL1 target genes.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-04-03T14:39:58Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese - Carolina Amaral (2).pdf: 3097271 bytes, checksum: a09eebe935773ec4b3446a95c4507f5c (MD5) / Made available in DSpace on 2017-04-03T14:39:58Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese - Carolina Amaral (2).pdf: 3097271 bytes, checksum: a09eebe935773ec4b3446a95c4507f5c (MD5) Previous issue date: 2015-12-15 / FACEPE / Os Papilomavírus humano (HPV) infectam mucosas e epitélio contribuindo para o desenvolvimento de tumores benignos como também malignos. São amplamente conhecidos como causadores do câncer cervical, contudo, atualmente, vem apresentando evidências de associação com diversos outros tipos de canceres, como o câncer de pulmão. Sendo assim, o presente estudo avaliou a presença do DNA do HPV em tumores de pulmão de pacientes do Estado de Pernambuco bem como a expressão de suas oncoproteínas E6 e E7. Para isto, a detecção foi feita em amostras de tecidos de tumores frescos e parafinados de 63 pacientes. HPV estava presente em 52% das amostras, sendo detectados os tipos 16 e 18 com frequências de 81 e 19%, respectivamente. Quanto a presença do vírus nos diferentes tipos histológicos dos tumores, foi detectado HPV em 40% dos carcinomas escamosos, 33% dos adenocarcinomas, 18% dos carcinomas de células pequenas e 9% em carcinoma de células grandes. Através da técnica de imunohistoquimica detectou-se a presença das oncoproteinas virais E6 (anticorpo anti-HPV 16 e anti-HPV 18) e E7 (anticorpo anti-HPV 16 e anti-HPV 18) com frequências de 85 e 75%, respectivamente. Tal resultado confirma os resultados obtidos molecularmente quanto à presença do HPV e é sugestivo de que o vírus esteja em atividade nas células tumorais e provavelmente esteja desempenhando um papel na carcinogênese de pulmão. No entanto, mais estudos são necessários para se ter um maior esclarecimento sobre interação de E6 e E7 com proteínas celulares na tumorigenese pulmonar. / Small DNA viruses - Human Papillomavirus (HPV) - infect oral mucosa and the epthelium, which leads to the development of both benign and malign tumors. They are widely known as the principal causes of cervical cancer although currently there is evidence to show that they are associated with several other types of cancer, such as lung cancer. In the light of this, this study evaluated the presence of HPV in the tumors of lungs of patients from the State of Pernambuco, as well as the E6 and E7 oncoproteins expression. This involved carrying out the detection in tumor tissue samples that were fresh and paraffin-embedded and taken from 63 patients. HPV was found to be present in 52% of the samples, and types 16 and 18 were detected with frequencies of 81% and 19% respectively. With regard to the presence of the vírus in different histological types of tumors, HPV was detected in 40% of the squamous carcinomas, 33% of the adenocarcinomas, 18% of the small cell carcinomas and 9% in large cell carcinomas. The presence of the E6 (antibody anti-HPV 16 and anti-HPV 18) and E7 (antibody anti-HPV 16 and anti-HPV 18) oncoproteins was detected by means of the immunohistochemical technique and this confirmed the results obtained from a molecular analysis with regard to the presence of the virus and it is suggestive that the virus is active in tumor cells and is probably playing a role in lung carcinogenesis. However, further studies are required to have a clearer understanding of the interaction of E6 and E7 with the cell proteins in pulmonary tumorigenesis.

Thesis advisor: Junona Moroianu / Some non melanoma skin cancers (NMSC) have been associated with human papillomavirus (HPV) pathogenesis, like epidermodysplasia verruciformis (EV) and squamous cell carcinoma (SCC). EV is a genetically inherited skin disease that develops when the individuals are infected with cutaneous HPV types belonging to the β-genus, especially types 5 and 8. Transgenic mouse lineages expressing all early genes of cutaneous HPV8 develop papillomas, dysplasias and SCC after UV irradiation and this correlates with enhanced HPV8 oncogenes expression. We have previously discovered that the nuclear localization of mucosal HPV16 E7 and HPV11 E7 proteins is mediated by their zinc-binding domain via a Ran-dependent pathway and independent of nuclear import receptors and that a patch of hydrophobic residues within the zinc-binding domain of HPV16 E7 and HPV11 E7 proteins is responsible for their nuclear import via hydrophobic interactions with FG nucleoporins. Here we investigated the nucleocytoplasmic traffic of cutaneous HPV8 E7 protein using confocal microscopy to analyze the intracellular localization of EGFP-8E7, its subdomains and its mutants after transient transfections. We also investigated the nuclear import ability of GST-8E7, its subdomains and mutants using in vitro nuclear import assays in digitonin-permeabilized HeLa cells. In addition, we performed isolation assays to study the direct interaction between HPV8 E7 and two FG nucleoporins, Nup62 and Nup153 or the nuclear export receptor, CRM1. We found that the nuclear import of cutaneous HPV8 E7 is mediated by a nuclear localization signal (NLS) located within its zinc-binding domain. Furthermore, we determined that the hydrophobic residues within the 65LRLFV69 patch are responsible for the nuclear import and nuclear localization of HPV8 E7 via direct hydrophobic interactions with FG nucleoporins, Nup62 and Nup153, whereas the positively charged arginine 66 plays no significant role in the function of the NLS. In addition, we examined the nuclear export mechanism of cutaneous HPV8 E7 protein and showed that it has a leucine-rich nuclear export signal (NES) in its C-terminal domain that is recognized by the CRM1 nuclear export receptor. These studies are essential for understanding the nucleocytoplasmic traffic of cutaneous HPV8 E7 protein. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Junona Moroianu / Human papillomaviruses (HPVs) have been estimated to be the most common sexually transmitted infection in the United States. In addition to condyloma accuminata, infection of the squamous basal epithelium by high-risk HPVs, notably type 16 (HPV16), has been shown to be the primary etiological agent in the majority of cervical carcinomas. The E7 major transforming protein of HPV16, along with E6, has been linked to tumorigenesis and malignancy. While the E7 protein itself possesses no enzymatic activity, its ability to bind a number of nuclear and cytoplasmic targets subverts a variety of cellular regulatory complexes and facilitates viral replication. Previous studies in the Moroianu Lab have shown the HPV16 E7 oncoprotein to translocate across the nuclear pore complex (NPC) in a facilitated manner dependent on a non-canonical, c-terminal, nuclear localization signal (cNLS) for import, and a consensus leucine-rich nuclear export sequence (NES) for export (28). While the leucine-rich NES has been characterized, a full examination of the cNLS has yet to be performed. Here we present evidence that the karyopherin independent nuclear import mediated by the cNLS of 16E7 is dependent on its c-terminal Zn binding domain. Furthermore, we demonstrate that nuclear import is mediated by the direct interaction of a small patch of hydrophobic residues, 65LRLCV69, with the FG domain of the central FG-nucleoporin Nup62. In addition, we examined a potential regulatory mechanism of 16E7 nucleocytoplasmic translocation. Previous work has shown that a serine conserved in the high-risk HPVs at position 71 is phosphorylated by an unknown kinase. Here we present evidence that while phosphorylation of S71 is not required for either 16E7 nuclear localization or nuclear export in HeLa cells, mimicking phosphorylation of the S71 residue results in a statistically significant shift in the distribution of localization phenotypes of the resultant cell population toward a larger percentage exhibiting more nuclear localization. These data suggest that nucleocytoplasmic transport of 16E7 is, at least in part, a regulated process. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

MDM2 has been shown to induce G0-Gl/S phase arrest. To determine the cell cycle step targeted by MDM2, flow cytometry was employed to detect induction of events during the G1-S phase transition in MDM2-arrested cells. MDM2 overexpression does not prevent expression of cyclin D, cyclin D-CDK mediated phosphorylation of Rb or cyclin E in normal, immortal or tumor-derived cells. However, MDM2 down-regulates cyclin A expression specifically in normal cells, which is associated with G1 arrest. The domain of MDM2 capable of this function is located within its N-terminal 58-109 amino acids. To down-regulate cyclin A, MDM2 requires a functional pl6/Brg 1 pathway, as silencing of either of these proteins disables this function of MDM2. Bromodeoxyuridine incorporation studies suggest that another inhibitory domain, ID2, inhibits DNA replication, while an MDM2 deletion mutant containing the N-terminal 1-220 amino acids including inhibitory domain ID1 does not effectively prevent BrdU incorporation in an immortal cell line that is non-responsive to growth arrest by the cyclin A inhibitory domain. This suggests that induction of MDM2 leads to G1 arrest by at least two independent mechanisms, and multiple genetic damages are necessary to overcome MDM2-mediated growth arrest. To determine novel interacting partners of MDM2, proteomic analysis of MDM2 overexpressed in tumor-derived H1299 cells was carried out. This analysis revealed interaction of MDM2 with the translation elongation factor efl-α, and was validated by immunoprecipitation and Western blotting and shown to colocalize with MDM2 in the cytoplasm. To interact with efl-α, MDM2 was determined to require two domains, one of which is located within amino acids 221-325 and another within the N-terminal 58 amino acids of MDM2.

A proteína jun é um dos principais integrantes do complexo AP-1 e está envolvido nos processos inflamatórios, diferenciação, apoptose e migração celular. Esta proteína pode formar homodímeros e heterodímeros por meio da dimerização que ocorre pelo sítio de sequências de leucinas. Existem evidências de que a proteína jun pode ser inibida pela proteína RPL10 mediante a ligação destas proteínas, no mesmo sítio de sequências de leucinas no núcleo celular, parando a progressão de tumores. O objetivo deste trabalho foi expressar, isolar e caracterizar a região de ligação das sequências de leucinas (região AP-1), para estudos posteriores de ligação com a proteína RPL10. O cDNA para proteína jun foi amplificado por PCR e clonado nos vetores de expressão pET 26b(+), pET 28a-c(+) e p1813 e expressa em E.coli BL21 (DE3). A proteína expressa em vetor pET28_AP1 foi eficientemente purificada pela técnica de cromatografia de afinidade a íons metálicos, por possuir uma sequência (poli)histidina que facilitou a purificação, apresentando um excelente grau de pureza. A identidade da proteína foi confirmada através de análise feita por western blotting e dot blotting e também por analise por espectrometria de massas. / The jun protein is one of the main AP-1 complex members and is involved in the inflammatory process, differentiation, apoptosis and cell migration. The Jun protein may form homodimers and heterodimers by dimerization in the leucines sequences site. There are evidences that jun protein can be inhibited by RPL10 protein through these protein binding in the same leucine sequences site, in the cell nucleus, stopping the tumor progress. The objective of this study was to express, isolate and characterize the binding region of the leucine sequences (AP-1 region) for subsequent binding studies with RPL10 protein. The jun protein cDNA was amplified by PCR and cloned into pET 26b(+), pET 28a-c(+) and p1813 expression vectors, and was expressed in E.coli BL21 (DE3). The protein expressed in pET28_AP1 vector was efficiently purified by the affinity chromatography technique to metal ions because have a (poly)histidine sequence which facilitate the protein purification, showing an excellent high purity. The protein identity was confirmed by western blotting and dot blotting and also by mass spectrometry analysis.

A proteína jun é um dos principais integrantes do complexo AP-1 e está envolvido nos processos inflamatórios, diferenciação, apoptose e migração celular. Esta proteína pode formar homodímeros e heterodímeros por meio da dimerização que ocorre pelo sítio de sequências de leucinas. Existem evidências de que a proteína jun pode ser inibida pela proteína RPL10 mediante a ligação destas proteínas, no mesmo sítio de sequências de leucinas no núcleo celular, parando a progressão de tumores. O objetivo deste trabalho foi expressar, isolar e caracterizar a região de ligação das sequências de leucinas (região AP-1), para estudos posteriores de ligação com a proteína RPL10. O cDNA para proteína jun foi amplificado por PCR e clonado nos vetores de expressão pET 26b(+), pET 28a-c(+) e p1813 e expressa em E.coli BL21 (DE3). A proteína expressa em vetor pET28_AP1 foi eficientemente purificada pela técnica de cromatografia de afinidade a íons metálicos, por possuir uma sequência (poli)histidina que facilitou a purificação, apresentando um excelente grau de pureza. A identidade da proteína foi confirmada através de análise feita por western blotting e dot blotting e também por analise por espectrometria de massas. / The jun protein is one of the main AP-1 complex members and is involved in the inflammatory process, differentiation, apoptosis and cell migration. The Jun protein may form homodimers and heterodimers by dimerization in the leucines sequences site. There are evidences that jun protein can be inhibited by RPL10 protein through these protein binding in the same leucine sequences site, in the cell nucleus, stopping the tumor progress. The objective of this study was to express, isolate and characterize the binding region of the leucine sequences (AP-1 region) for subsequent binding studies with RPL10 protein. The jun protein cDNA was amplified by PCR and cloned into pET 26b(+), pET 28a-c(+) and p1813 expression vectors, and was expressed in E.coli BL21 (DE3). The protein expressed in pET28_AP1 vector was efficiently purified by the affinity chromatography technique to metal ions because have a (poly)histidine sequence which facilitate the protein purification, showing an excellent high purity. The protein identity was confirmed by western blotting and dot blotting and also by mass spectrometry analysis.

Cancer is the main cause of death in economically developed countries and the second leading cause of death in developing countries. Along with today’s knowledge that more than two hundred different diseases lie in the category of this prognosis there is an urge for more detailed and case-specific treatments to replace the dramatic actions of available radiation- and chemotherapy, which in many cases do not make a difference between healthy and cancer cells. The transcription factor and onco-protein c-Myc has, after being extensively studied during the past decades, become a prognostic marker for almost all cancer forms known. Still, many questions remain regarding how c-Myc interacts with its many different target proteins involved in cell-cycle regulation, proliferation and apoptosis. Current cell biology states that one of the regulating proteins, hPin1, interacts with c-Myc in a phosphorylation-dependent manner which appears to direct the correct timing of c-Myc activation and degradation through the ubiquitin/proteasome-pathway. The critical phosphorylation sites, T58 and S62, are located in the Myc-Box-I (MBI) region, a highly conserved sequence strongly coupled to aggressive tumourigenesis by hotspot mutations. Interestingly, preliminary results in the Sunnerhagen group suggested that MBI alone did not bind hPin1, suggesting hPin1 targeting a site distal from the residues to be phosphorylated. In this thesis, results from Surface Plasmon Resonance (SPR) and Nuclear Magnetic Resonance (NMR) show that the docking WW-domain of hPin1 binds unphosphorylated c-Myc at a region distal from the phosphorylation site, including residues 13-34. Furthermore, SPR experiments revealed that hPin1 binds unphosphorylated c-Myc with apparently greater affinity and with much slower kinetics than phosphorylated c-Myc. Thus, hPin1 recognition and interaction with c-Myc appears not to be dependent on phosphorylation of c-Myc prior binding. The newly identified binding region of c-Myc, located N-terminal of MBI, may further increase the understanding of protein degradation control and c-Myc function. The studies presented in this thesis provide a brick in the puzzle of c-Myc and hPin1 coupled oncogenesis for further development of new therapeutic strategies.