For proteins the link between their structure and their function is a central tenet of biology. A common approach to understanding protein function is to ‘solve’ its structure and subsequently probe interactions between the protein and its binding partners. The first part of this approach is non-trivial for proteins where localised regions or even their entire structure fail to fold into a three-dimensional structure and yet they possess function. These so called intrinsically or inherently disordered proteins (IDP’s) or intrinsically disordered regions (IDR’s) constitute up to 40% of all expressed proteins. IDPs which have crucial roles in molecular recognition, assembly, protein modification and entropic chain activities, are often dynamic with respect to both conformation and interaction, so in the course of a protein’s ‘lifespan’ it will sample many configurations and bind to several targets. For these proteins, there is a need to develop new methods for structure characterization which exploit their biophysical properties. The solvent free environment of a mass spectrometer is ideally suited to the study of intrinsic interactions and how they contribute to structure. Ion mobility mass spectrometry is uniquely able to observe the range of structures an IDP can occupy, and also the effect of selected binding partners on altering this conformational space. This thesis details the technique of ion mobility mass spectrometry and illustrates its use in assessing the relative disorder of p53 protein. The tumour suppressor p53 is at the hub of a plethora of signalling pathways that maintain the integrity of the human genome and regulate the cell cycle. Deregulation of this protein has a great effect on carcinogenesis as mutated p53 can induce an amplified epigenetic instability of tumour cells, facilitating and accelerating the evolution of the tumour. Herein mass spectrometry provides a compelling, detailed insight into the conformational flexibility of the p53 DNA-binding domain. The plasticity of the p53 DNA-binding domain is reflected in the existence of more than one conformation, independent of any conformational changes prompted by binding. The in vacuo conformational phenotypes exhibited by common cancer-associated mutations are determined and the second-site suppressor mutation from loop L1, H115N, is probed whether it could trigger conformational changes in p53 hotspot cancer mutations. The structural basis of the binding promiscuity of p53 protein is investigated; of particular interest is the molecular interaction of the p53 N-terminus with the oncoprotein murine double minute 2, as well as with the antiapoptotic factor B-cell lymphoma-extralarge.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 161-195). Also available in electronic version.

Thesis (M.S.)--University of Kentucky, 2004. / Title from document title page (viewed June 21, 2004). Document formatted into pages; contains viii, 76 p. : ill. Includes abstract and vita. Includes bibliographical references (p. 69-74).

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Ovarian cancer is a leading cause of death among of gynecological cancers. Current therapies are ineffective with a poor 5-year survival of only ~25%. p70 S6 kinase (p70 S6K) is a downstream target of the phosphatidylinositol 3-kinase pathway and is frequently activated in human ovarian cancer. However, the molecular targets and signaling pathways by which p70 S6K may affect tumor development and progression are poorly understood. Interestingly, in the laboratory, Mdm2, an important negative regulator of the p53 tumor suppressor, was identified in a yeast two hybrid screening of potential interacting partners for p70 S6K. In this study, I aimed to investigate the specific interaction of p70 S6K and Mdm2 and determine how this may contribute to ovarian tumorigenesis. Using a co-immunoprecipitation assay, the in vivo interaction of p70 S6K and Mdm2 in human ovarian cancer cells was confirmed. Upon UV-induced genotoxic stress, p70 S6K activation was associated with Mdm2 phosphorylation on S166 and subsequent p53 accumulation. This could be reversed by the use of rapamycin and p70 S6K siRNA to inhibit its kinase activity and expression respectively, confirming that the effect was p70 S6K specific. Conversely, ectopic expression of wildtype p70 S6K or a constitutively active mutant of p70 S6K, D3E-E389 (D3E) was sufficient to induce phosphorylation of Mdm2. Moreover, the p70 S6K mediated activation of Mdm2 was independent of p53 mutations. Similar results were observed upon other stress challenges such as hypoxia using hypoxia mimicking agent desferrioxamine (DFX). These findings identify Mdm2 as a new target of p70 S6K and reveal that p70 S6K intervenes the Mdm2-p53 regulatory loop in ovarian cancer, which may provide a survival advantage to cancer cells under stress conditions. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

Endoplasmic Reticulum (ER) stress signal is a cellular response to various insults including abnormal protein folding load, activating the unfolded protein response. Under severe ER stress, apoptosis will occur in most cell types. Interestingly, this does not happen in a disease model for Metaphyseal chondrodysplasia type Schmid (MCDS), where ER stress was activated in the hypertrophic zone of the growth plate where mutant collagen X proteins that cannot be folded correctly is expressed. Instead of normal progression from proliferating chondrocytes (PCs) to hypertrophic chondrocytes (HCs) and conversion to bone, HCs in MCDS mice undergo re-differentiation to PCs as a survival strategy due to an activation of ER stress. Transcription factors are known to be important in regulating differentiation. p53 family members, as transcription factors, are known to play important roles in developmental processes including cellular reprogramming, thus, we hypothesize that the ectopic expression of key transcription factors, p53 and TAp63, which are activated by ER stress is involved in HC re-differentiation. p53 is normally expressed in late PCs, Pre-HCs, and upper HCs, while TAp63 is expressed in PCs and Pre-HCs suggesting they may have roles in chondrocyte differentiation. p53 activated under ER stress in HCs are nuclear localized in MCDS mice, but did not invoke the apoptotic programme. In this project, using quantitative analyse to study the expression level of p53 and p63 isoforms, it was confirmed that p53 and TAp63γ are in part transcriptionally activated upon ER stress. From functional study by inactivating p53 in MCDS mice, it was shown that p53 alone was not sufficient to mediate re-differentiation. Given that TAp63γ isoforms is also highly upregulated upon ER stress, and the negative regulator, ΔNp63, is downregulated, this combination of change in gene expression also need to be considered. Furthermore, known regulators of p53 and p63 activity such as ASPP1 and iASPP are also differentially expressed in HCs, and are altered upon activation of ER stress favouring cell survival. Thus, it would be important to evaluate the combination of TAp63 in the re-differentiation process from conditional inactivation of p63 or in combination with p53 to gain a clearer understanding of the contribution and relationship of these transcription factors in the survival strategy of stressed HCs. / published_or_final_version / Biochemistry / Master / Master of Philosophy

Thesis (Ph. D.)--State University of New York at Binghamton, Department of Biological Sciences, 2009. / Includes bibliographical references (leaves 128-145).

Orientador: Antônio Carlos Alessi / Banca: Karin Werther / Banca: Felipe Augusto Ruiz Sueiro / Resumo: Os tumores de mama são a principal causa de morte em cães e isso vem despertando maior interesse no desenvolvimento de estudos relacionados a este distúrbio. A proximidade com os seres humanos, tanto na convivência quanto aos hábitos, podem influenciar o aparecimento das neoplasias. A semelhança dos tumores mamários caninos com os tumores de mama da mulher leva a um interesse no estudo da patologia comparada, estimulando o uso de modelos animais. Apesar dos muitos estudos, pouco se conhece sobre o prognóstico e as causas dos tumores mamários caninos, observando-se um esforço crescente na tentativa de acrescentar aos fatores prognósticos clássicos novos parâmetros, de natureza molecular, que auxiliem a decisão clínica, à semelhança do verificado em Medicina Humana, estando entre eles os marcadores moleculares, como a proteína P53 e as proteínas de estresse. A expressão de proteína P53 e das proteínas de estresse tem sido observada em muitas neoplasias, incluindo o câncer de mama. Nesse sentido, o objetivo do presente estudo foi investigar a imunomarcação de HSP 27, HSP 72, HSP 90 e proteína P53 em tecido mamário normal e neoplásico de cadelas e estabelecer uma relação entre a expressão destas proteínas e o grau histológico das neoplasias. Foi realizada análise estatística e o nível de significância (α) adotado foi de 5%. Dentre os tumores malignos, os carcinomas simples foram o tipo histológico predominante. Para a proteína P53, não houve diferença significativa em sua expressão entre os grupos de tumores malignos avaliados, ocorrendo o mesmo para as HSPs 27, 72 e 90. A sensibilidade do teste de imuno-histoquímica para a proteína P53, nesta amostra, foi de 67,5%, a especificidade foi de 100%, o valor preditivo positivo foi de 100%, o valor preditivo negativo foi de 25% e acurácia do teste foi de 92%. Ainda para a proteína P53, comparando-se o grupo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Breast tumors are the leading cause of death in dogs and this has aroused great interest in developing studies related to this disease. The proximity with humans, much as in living habits, may influence the onset of tumors. The similarity of canine mammary tumors with breast tumors of women take an interest in the study of comparative pathology, stimulating the use of animal models. Despite many studies, little is known about the prognosis and causes of canine mammary tumors, observing a growing effort in trying to add to the classic prognostic factors new parameters of molecular nature, that help the clinical decision, like that seen in Human Medicine, and among them the molecular markers such as P53 and stress proteins. The expression of P53 protein and the stress proteins has been observed in many cancers, including breast cancer. Accordingly, the purpose of this study was to investigate the immunostaining of HSP 27, HSP 72, HSP 90 and P53 protein in normal and neoplastic breast tissue of female dogs and establish a relationship between the expression of these proteins and the histological grade of tumors. Statistical analysis was performed and significance level (α) was 5%. Among the malignant tumors, simple carcinomas were the predominant histologic type. Protein P53, presented no significant difference in expression between the evaluated groups of malignant tumors, the same occurring for HSPs 27, 72 and 90. The test sensitivity of immunohistochemistry for protein P53 in this sample was 67,5%, specificity was 100%, positive predictive value was 100%, negative predictive value was 25% and accuracy of test was 92%. Although protein P53, compared to the control group with other groups in relation to staining intensity and proportion score, there was a significant difference only between the group of simple carcinomas. With regard to staining intensity, we tested the correlation between... (Complete abstract click electronic access below) / Mestre

The p53 tumor suppressor protein has been well-characterized for its role in inducing growth arrest, senescence, and apoptosis upon various types of stresses. Recently, however, roles of p53 have expanded beyond the canonical functions, and now include cellular processes such as metabolism, oxidative balance, and ferroptosis. Through RNA-seq screening, we first identified phosphoglycerate dehydrogenase (PHGDH), a rate-limiting enzyme in the serine biosynthesis pathway, as a novel metabolic target of p53. p53 suppresses PHGDH expression and inhibits de novo serine biosynthesis. Notably, upon serine starvation, p53-mediated cell death is significantly enhanced in response to Nutlin-3 treatment. Moreover, PHGDH has been demonstrated to be frequently amplified in human melanomas. We found that PHGDH overexpression significantly suppresses the apoptotic response, whereas RNAi-mediated knock-down of endogenous PHGDH promotes apoptosis under the same treatment. Together, our findings demonstrate an important role of p53 in regulating serine biosynthesis through suppressing PHGDH expression, and reveal serine deprivation as a novel approach to sensitize p53-mediated apoptotic responses in human melanoma cells. In addition, we also identified spermidine/spermine N1-acetyltransferase 1 (SAT1) as a novel metabolic target of p53. SAT1 is a rate-limiting enzyme in polyamine catabolism critically involved in the conversion of spermidine and spermine back to putrescine. Surprisingly, we found that activation of SAT1 expression induces lipid peroxidation and sensitizes cells to undergo ferroptosis upon reactive oxygen species (ROS)-induced stress, which also leads to suppression of tumor growth in xenograft tumor models. Notably, SAT1 expression is down-regulated in human tumors, and CRISPR-cas9-mediated knockout of SAT1 partially abrogates p53-mediated ferroptosis. Moreover, SAT1 induction is correlated with the expression levels of arachidonate 15-lipoxygenase (ALOX15), and SAT1-induced ferroptosis is significantly abrogated in the presence of PD146176, a specific inhibitor of ALOX15. Together, these data indicate a novel regulatory role of p53 in polyamine metabolism and provide insight into the regulation of p53-mediated ferroptotic responses. Our studies on PHGDH and SAT1 led us to the question of whether these unconventional functions of p53 contribute to its role as a tumor suppressor. In fact, previous view regarding the mechanism of p53-mediated tumor suppression, which was long thought to be growth arrest, apoptosis, and senescence, has recently been challenged by several knockout and knock-in mouse studies. Previously, we established mice (p533KR/3KR) in which p53 acetylation at lysine residues K117, K161, and K162 were abolished by replacing lysine with arginine. p533KR/3KR mice completely lost p53-mediated cell cycle arrest, apoptosis, and senescence functions in response to stresses. However, unlike p53-null mice which rapidly develop spontaneous thymic lymphomas, all of the p533KR/3KR mice remain tumor-free, indicating that other aspects of p53 functions are sufficient to prevent tumor formation. Notably, p533KR retains the ability to regulate metabolic targets including TIGAR and SAT1, as well as ferroptosis regulator SLC7A11. In this study, we have identified two novel acetylation sites- K98 and K136, in the mouse p53 DNA-binding domain. Whereas loss of K98 or K136 acetylation (p53K98R, p53K136R) alone has modest effect on p53 transcriptional activity, simultaneous mutations at all of these acetylation sites (p534KR98: K98R+3KR, p534KR136: K136R+3KR, p535KR: K98R+K136R+3KR) completely abolish the ability of p53 to regulate TIGAR, SAT1, and SLC7A11. In addition, p534KR98, p534KR136, and p535KR are defective in Erastin-induced ferroptosis. Notably, p534KR98/4KR98, p534KR136/4KR136, and p535KR/5KR knock-in mice lost intact tumor suppression and developed spontaneous tumors. This suggests that p53-mediated ferroptosis may function as a critical barrier to prevent tumor formation independently from growth arrest, apoptosis, and senescence. Interestingly, both p534KR98/4KR98 and p534KR136/4KR136 mice displayed significantly delayed tumorigenesis comparing with p53-null and p535KR/5KR mice. We found that unlike p535KR, p534KR98 retains the capacity to inhibit mammalian target of rapamycin (mTOR) signaling pathway through activating the expression of two mTOR negative regulators, Sestrin2 and DDIT4. Altogether, our findings underscore the extensive scope of p53 functions in metabolic regulation, oxidative stress response, and ferroptosis, and provide novel insights into the tumor suppression mechanism of p53.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-05-17T21:24:47Z No. of bitstreams: 1 NadjaCruzGaido.pdf: 2766927 bytes, checksum: 6fdfd1a4a2beb11f1da8d3669b11750a (MD5) / Made available in DSpace on 2017-05-17T21:24:47Z (GMT). No. of bitstreams: 1 NadjaCruzGaido.pdf: 2766927 bytes, checksum: 6fdfd1a4a2beb11f1da8d3669b11750a (MD5) Previous issue date: 2016-09-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) / The pituitary adenomas represent about 10 to 15% of intracranial tumors and are usually benign, comprising a cell population of monoclonal origin. They are classified as macroscopic and radiological aspects, functional status, dyeing, immunohistochemistry and microscopic. About 70% of human cancers have mainly a deficiency in the function gene. The p53 gene is extensively studied in tumors, showing that patients with mutations have a worse prognosis, as well, the p53 protein is critical in preventing tumor development performing change detection function in DNA and thereby fix or apoptosis. It is possible that the increase in expression of this protein is the result of an attempt to stop the cell cycle in response to deregulation by a stimulus from another source. This way, the study seeks to determine the expression of p53 protein in pituitary adenomas. an analytical study, with crosssectional was held in which were included 62 patients older than 16 years of both sexes, who were diagnosed with pituitary adenoma, from 2008 to 2016, arising from the Endocrinology Service University hospital of the Federal University of Maranhão - HUUFMA. These patients underwent surgical resection of the tumor, obtaining the samples of tumor tissue. After conventional histopathology, they were embedded in paraffin, for carrying out the immuno-histoqumico study aimed to identify the expression of p53 protein and implications correlated with biological behavior of pituitary adenomas. The analysis for p53 protein expression in adenomas was obtained by immunoenzymatic of streptoavidin-biotin or immunohistochemistry. The p53 protein immunostaining revealed no statistically significant correlation, as the clinical and demographic characteristics such as gender, age, p53 expression in tumor subtypes and tumor volume. It is necessary to obtain greater numbers of patients, so that p53 expression is effective when the real prognostic value in pituitary adenomas. / Os adenomas hipófisários representam cerca de 10 a 15% das neoplasias intracranianas e são geralmente benignos e compostos por uma população celular de origem monoclonal. São classificados conforme aspectos macroscópicos e radiológicos, status funcional, tintoriais, imuno-histoquímica e microscópicas. Cerca de 70% dos cânceres humanos possuem principalmente uma deficiência na função do gene. O gene p53 é extensivamente estudado nas neoplasias mostrando que pacientes com mutações apresentam um pior prognóstico, assim, a proteína p53 é fundamental na prevenção do desenvolvimento de tumores exercendo a função de detecção de alterações no DNA e, consequentemente, correção ou apoptose. É possível que o aumento na expressão desta proteína seja decorrente de uma tentativa de frear o ciclo celular como resposta à desregulação por um estímulo de outra origem. Dessa forma, o estudo busca avaliar a imunoexpressão da proteína p53 em adenomas hipofisários. Foi realizado um estudo analítico com delineamento do tipo transversal, no qual foram incluídos 62 pacientes com idade superior 16 anos de ambos os sexos, que apresentaram diagnóstico de adenoma hipofisário no período de 2008 a 2016, oriundos do Serviço de Endocrinologia do Hospital Universitário da Universidade Federal do Maranhão – HUUFMA. Esses pacientes foram submetidos à ressecção cirúrgica do tumor, com obtenção das amostras do tecido tumoral. Após diagnóstico histopatológico convencional, foram emblocadas em parafina, para realização do estudo imuno-histoqumico com o objetivo de identificar a imunoexpressão para proteína p53 e implicações correlacionadas ao comportamento biológico dos adenomas hipofisários. A análise para expressão da proteína p53 em adenomas hipofisários foi obtida através da reação imunoenzimática da streptoavidina-biotina-peroxidase ou imuno-histoquimica. A imunoexpressão da proteína p53 não revelou correlação estatisticamente significante, quanto aos achados clínicos e características demográficas, tais como sexo, idade, imunoexpressão da p53 para os subtipos tumorais e volume tumoral. Torna-se necessário obter números mais expressivos de pacientes para que a imunoexpressão da p53 seja efetiva, quanto ao real valor prognóstico em adenomas hipofisários.