The MDM2 homolog MDMX is an important regulator of p53 during mouse embryonic development. DNA damage promotes MDMX phosphorylation, nuclear translocation, and degradation by MDM2. Here we show that MDMX copurifies with 14-3-3, and DNA damage stimulates MDMX binding to 14-3-3. Chk2-mediated phosphorylation of MDMX on S367 is important for stimulating 14-3-3 binding, MDMX nuclear import by a cryptic NLS, and degradation by MDM2. Mutation of MDMX S367 inhibits ubiquitination and degradation by MDM2, and prevents MDMX nuclear import. Expression of 14-3-3 stimulates the degradation of phosphorylated MDMX. Chk2 and 14-3-3 cooperatively stimulate MDMX ubiquitination and overcome the inhibition of p53 by MDMX. These results suggest that MDMX-14-3-3 interaction plays a role in p53 response to DNA damage by regulating MDMX localization and stability. We also show the identification of a cryptic nuclear localization sequence within the C-terminus RING finger domain MDMX. Mutation of MDMX on one lysine residue at position 468 to glutamic acid completely abrogates the nuclear import after DNA damage. This mutation had no effect on MDM2-mediated nuclear import of MDMX in cotransfection assays, suggesting that it is specifically required for the MDM2-independent nuclear import. Interestingly, the MDMX- K468E mutant induces the expression of p21 more efficiently than the wild-type MDMX after ionizing radiation (IR). Furthermore, the K468E mutant induction of p21 is associated with enhanced G1 arrest after DNA damage. These results indicate an important function of MDMX nuclear import in regulating p53 activity after DNA damage.

No description available.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 171 pages. Includes vita. Includes bibliographical references.

No description available.

Recent approaches to constraining peptide sequences into more structurally-defined α- helical secondary structures, so-called peptide stapling, are discussed. Stapled peptides are a class of therapeutics that have been shown to more effectively target protein-protein interactions, which are harder to target using a classical small-molecule therapeutic approach. Stapling a peptide constrains it into a well-defined secondary structure. This more accurately mimics the protein-protein interaction making the peptide a more viable therapeutic. Starting from the p53-MDM2 interaction, a protein-protein interaction with important implications in cell health, a known peptidyl inhibitor of this interaction was stapled and analysed for increased α-helicity. This was achieved by using monomers that utilise the copper (I) alkyne azide cycloaddition as a cross-linking methodology, which has been less well researched in the context of peptide stapling. The viability of a novel stapled peptomer inhibitor approach, accomplished using a new, optimised monomer synthesis, is investigated. Additionally, the synthesis of a ligand series designed for use in the copper(I) alkyne azide cycloaddition is also discussed.

No description available.

Made available in DSpace on 2014-06-11T19:23:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-06-01Bitstream added on 2014-06-13T19:30:16Z : No. of bitstreams: 1 mazon_rc_me_arafcf.pdf: 520188 bytes, checksum: 4af452f70d4ec4f1806399a4a3c1661c (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / O mdm2 parece controlar o tráfego da proteína p53 do núcleo-citoplasma enquanto a proteína p27 é um inibidor de CDK que controla a fase G1 para S do ciclo celular. Em contraste, a catepsina B parece induzir um sinal de apoptose independente de caspase na presença do HPV. Entretanto, a influência da infecção pelo HPV na carcinogênese oral e o envolvimento na desregulação das proteínas do ciclo celular não são claras. Assim, o objetivo deste estudo foi avaliar a associação entre a expressão de proteínas do ciclo celular, p27, mdm2 e catepsina B e câncer oral na presença do HPV. Cinqüenta e cinco biópsias orais representando lesões benignas (LB), lesões potencialmente malignas (LPM) e lesões malignas (LM). A detecção e tipagem do HPV (6/11,16,18, 31 e 33) foi realizada pela reação da polimerase em cadeia (PCR). A expressão quantitativa de p27, mdm2 e catepsina B foi analisada pela reação imunohistoquímica. Vinte e uma (38%) de 55 lesões orais foram positivas para HPV. O DNA do HPV 6/11 foi encontrado em 6 (33%), HPV 16 em 1(5%) e o HPV 18 em 14 (72%). Não foi encontrado HPV 31 e 33. De 55 biópsias, foi encontrada alta expressão de todas as proteínas: o p27 foi encontrado em 37 (67,2%) lesões, mdm2 em 17 (30,9%) e a catepsina B em 37 (67,2%). Entre lesões orais de HPV positivo, uma superexpressão de p27, mdm2 e catepsina B foi encontrada, respectivamente, em 6 (33%) de 18 lesões benignas, 4 (22%) de 18 lesões potencialmente malignas e 11 (57,9%) de 19 lesões malignas. Adicionalmente, o HPV16/18 (11/58%) foi detectado em lesões malignas com alta expressão de todas as proteínas. Nós concluímos que os tipos de HPVs de alto risco devem estar associados com carcinoma oral, bem como, com a superexpressão de p27, mdm2 e catepsina B. Estes resultados sugerem que os tipos de HPVs de alto risco podem desregular o controle do ciclo celular, contribuindo para a carcinogênese oral. / Mdm2 seems to control p53 nucleus-cytoplasm traffic while p27 is a CDK inhibitor which control G1 to S phase of the cell-cycle. In contrast, cathepsin B is reported in HPVinduced apoptotic signalling caspase-independent. However, the influence of HPV infection in oral carcinogenesis and its involvement to this cell-cycle proteins dysfunction remain still unclear. The purpose of this study was to clarify the association between the expression of cell-cycle proteins, p27, mdm2 and cathepsin B and oral cancer HPV-related. Fifty-five oral biopsies presenting benign oral lesions (BL), potentially malignant lesions (PML) and malignant oral lesions (ML). HPV detection and typing (6/11, 16, 18, 31 and 33) was performed using polymerase chain reaction. p27, mdm2 and cathepsin B quantitative expression were performed by immunohistochemistry. Twenty one (38%) of the 55 oral lesions were positive for HPV, of which HPV6/11 DNA was found in 6 (33%), HPV 16 in 1(5%) e o HPV 18 in 14 (72%). No HPV positivity was found to HPV31 and 33. Of the 55 biopsies, a high expression of all proteins was found: to p27 in 37 (67,2%), mdm2 was observed in 17 (30,9%) slides, and cathepsin B in 37 (67,2%). Among HPV-positive oral lesions, an overexpression for mdm2, p27 and cathepsin B was found, respectively, in 6 (33%) out of 18 BL, 4 (22%) out of 18 PML and 11 (57,9%) out of 19 ML. In addition, HPV16/18 (11/58%) was detected in OSSC with high expression of all proteins. We conclude that high-risk HPV types might be associated with oral carcinoma, as well as, with the overexpression of mdm2, p27 and cathepsin B. These results suggest that high-risk HPV types might deregulate cell-cycle control, contribuiting to oral carcinogenesis.

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-06T18:52:51Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-CarolinaAmaral.pdf: 1292331 bytes, checksum: dd6bdfd9f0916002036086fe27d37cbf (MD5) / Made available in DSpace on 2017-04-06T18:52:51Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-CarolinaAmaral.pdf: 1292331 bytes, checksum: dd6bdfd9f0916002036086fe27d37cbf (MD5) Previous issue date: 2012-03-02 / Pequenos vírus de DNA, os Papilomavírus humano (HPV), são transmitidos por via sexual e infectam mucosas e epitélios levando ao desenvolvimento de tumores benignos, os quais podem evoluir para um processo carcinogênico. Este vírus apresenta seu genoma dividido em regiões de expressão precoce e tardia. Dentre as de expressão precoce, estão as regiões E6 e E7 as quais codificam para proteínas que interferem no ciclo celular do hospedeiro. A proteína viral E6 interage com a proteína celular p53 inibindo sua função supressora de tumor. Enquanto isso, a proteína E7 interage com a proteína pRb, também supressora de tumor, possibilitando a passagem do ciclo celular da fase G1 para a fase S. Dessa forma, o HPV é responsável pela transformação celular. Considerada o principal agente etiológico do câncer cervical, a infecção por HPV é necessária, porém não suficiente nesse processo. Fatores ambientais e genéticos têm sido apontados como co-fatores, sendo esse último responsável por 37% do risco total. Polimorfismos no gene TP53 bem como naqueles envolvidos na via da p53 têm sido relacionados com a susceptibilidade a lesões e câncer cervicais. Este trabalho teve como objetivo avaliar, em mulheres infectadas com HPV, a possível associação de polimorfismos em dois genes envolvidos na via da p53, MDM2(SNPT309G) e WAF-1(SNPC590T) com o aumento da susceptibilidade a lesões e câncer cervicais. Para tanto, foi utilizada a técnica de RFLP (Restriction Fragment Length Polymorphism). Os dados foram analisados através de testes estatísticos qui-quadrado de Pearson, teste G de aderência, equilibro de Hardy-Weinberg e teste exato de Fisher. Não se observou diferenças significativas entre as freqüências alélicas e genotípicas entre grupos de casos e de controles nos dois genes analisados. Os resultados obtidos nesse estudo sugerem que os polimorfismos MDM2(SNPT309G) e WAF-1(SNPC590T) não estão associados com lesões e câncer cervicais em pacientes da região nordeste do Brasil. / Small DNA viruses, the human papillomavirus (HPV) are sexually transmitted and infect mucosal and epithelia tissue leading to the development of benign tumors, wich can progress to a carcinogenic process. Its genome is divided into regions with early and late expression. Among the early expression, E6 and E7 encoded proteins that interfere in host cellular cycle. The viral E6 protein interacts with the cellular protein p53 and inhibits its tumor suppressor function. The E7 protein interacts with the pRb protein, also tumor suppressor, allowing the passage of the cell cycle form G1 phase to S phase. Thus, HPV is responsible for cell transformation. Considered the main etiological agent of cervical cancer, HPV infection is necessary but not sufficient in this process. Environmental and genetic factors have been implicated as cofactors, the latter being responsible for 37% of total risk. Polymorphisms in TP53 gene as well as those involved in the p53 pathway have been linked with susceptibility to cervical lesions and cancer. The aim of thisworkwas analysewomen infected with HPV to evaluate the possible association of polymorphisms in two genes involved in the p53 pathway, MDM2(SNPT309G) and WAF1(SNPC590T) with increasing susceptibility to cervical lesions and cancer. To this, technique Restriction Fragment Length Polymorphism(RFLP) was used. The dada was analyse by Pearson chi-square test, G adhesiontest, Hardy-Weinberg equilibriumand Fisher’s exact test. No significant differences were observed between the allelic and genotypic frequencies between groups of cases and controls in the two genes analyzed. The results of this study suggest that the MDM2polymorphism (SNPT309G) and WAF1(SNPC590T) are not associated with cervical lesions and cancer in patients from the Northeasternof Brazil.

Made available in DSpace on 2014-08-20T13:32:57Z (GMT). No. of bitstreams: 1 tese_helena_strelow_thurow.pdf: 807973 bytes, checksum: 9ce5f5a742c1cda1d087be4eaaf27eae (MD5) Previous issue date: 2011-07-15 / A proteína p53,codificada pelo gene TP53, vem sendo estudada há mais de 30 anos e já foi denominada de "guardiã do genoma.

No description available.