O espalhamento de raios X a baixo ângulo tem se mostrado uma poderosa ferramenta na ánalise estrutural de proteínas em solução. Estudos em condições próximas ao estado nativo podem ser realizados, permitindo a visualização tridimensional de proteínas ou complexos formados. A tese apresentada aborda a teoria envolvida para utilização desta ferramenta. Uma nova metodologia foi proposta para a determinação da massa molecular de proteínas em solução, utilizando apenas uma curva de SAXS em unidades arbitrárias, visto que até o momento, este procedimento era realizado em comparação com outra proteína padrão de peso molecular conhecido. Com relação à instrumentação científica, um equipamento de SAXS foi desenvolvido no Instituto de Física de São Carlos, permitindo agora que medidas de SAXS em proteínas em solução sejam realizadas no instituto. Clonagem, expressão e purificação foram realizadas para o domínio de ligação ao DNA da isoforma do receptor tireoideano humano, a caracterização experimental desta proteína foi realizada por anisotropia de fluorescência, crosslink e SAXS. Após formação do complexo DNA-proteína, F2-DBD hTR, o mesmo foi submetido a cristalização, os cristais obtidos para o complexo não apresentaram padrão de difração e modelos de baixa resolução foram gerados utilizando SAXS. Além disso, estudos de baixo ângulo foram realizados linha de SAXS do LNLS para a enzima ferredoxina redutase de leptospira interrogans e para o complexo formado por interleucina-22 e pelo receptor interleucina-22, sendo seus modelos tridimensionais resolvidos. / Small angle X-ray scattering has been proven to be a powerful tool in the structural analysis of proteins in solution. This technique permits the three-dimensional visualization of native proteins envelop at the level of nanometers. In this study we discuss the small angle X-ray scattering theory and we proposed a new methodology to determine the molecular weight of proteins in solution, using only SAXS curve in arbitrary units. Prior the development of this method, the proteins molecular weighs were calculated by comparison with another of known size, usually bovine serum albumin. We also assembled SAXS equipment at the Physics Institute of São Carlos, which will permits in house measurements; as well as the cloning, expression and purification of DBD hTR, followed by the characterization of this protein by fluorescence anisotropy, crosslink and SAXS. The DNA-protein complex, F2-DBD hTR, was subjected to crystallization assays. Although, the crystals obtained for the complex showed no pattern of diffraction we were able to generate low-resolution models for the F2-DBD hTR using SAXS analysis. Moreover, the studies of the protein LepFNR and the complex IL-22/IL-22R1 by small angle X-ray scattering were performed in the line of SAXS of the LNLS, and their threedimensional models were resolved

Orientador: Munir Salomão Skaf / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-20T04:47:12Z (GMT). No. of bitstreams: 1 Mottin_Melina_M.pdf: 3350054 bytes, checksum: 28a03021273db312bbcba2e77fe7b2eb (MD5) Previous issue date: 2012 / Resumo: O Receptor Ativador da Proliferação de Peroxissomos g (PPARg) e membro de uma família de receptores nucleares cuja atividade é regulada por ligantes. O PPARg atua no metabolismo de lipídios e promove a sensibilização sistêmica à insulina, sendo, portanto, um alvo em potencial para o desenvolvimento de fármacos antidiabéticos. Dentre os ligantes desse receptor, o mais conhecido é a rosiglitazona (RSG), um agonista total que tem a utilização limitada por seus efeitos adversos: toxicidade cardiovascular, ganho de peso e retenção hídrica. A utilização de agonistas parciais é uma alternativa promissora para a redução desses efeitos, já que, apesar de apresentarem menor efeito farmacológico, conseguem desacoplar a sensibilização à insulina do acúmulo de triglicerídeos. Neste trabalho, realizamos simulações de dinâmica molecular do PPARg ligado a um agonista parcial recentemente descoberto, o GQ16. O complexo PPARg-RSG também foi simulado e utilizado como comparativo, para estudar os modos de ligação dos ligantes e suas influências sobre a dinâmica do PPARg. O mecanismo clássico de ativação do receptor é através da estabilização de uma de suas hélices (H12). Analisando-se a estabilidade da H12 durante as simulações observamos que esta permaneceu mais estável em presença da RSG em relação ao GQ16. As simulações revelaram que enquanto a RSG interage diretamente com um resíduo da H12, o GQ16 interage através de uma molécula de água, estabilizando mais fracamente a H12. Essa diferença de comportamento entre os ligantes pode estar por trás da menor ativação promovida pelo GQ16 em relação a RSG, o que está de acordo com os estudos funcionais que mostraram que o GQ16 atua como agonista parcial. Estudos recentes mostram que características estruturais do receptor são importantes na ativação ligante-específica. Um desses fatores auxiliares da ativação do PPARg está relacionado a fosforilação de uma serina (S245) do receptor, mediada por uma proteína quinase, a Cdk5. As simulações revelaram que há uma maior estabilização do loop que contém a S245 em presença do GQ16. Além disso, vimos que o resíduo K244, vizinho ao alvo da fosforilação pela Cdk5, varre um espaço conformacional menor em relação ao complexo PPARg-RSG. Esses resultados sugerem que o GQ16 possibilitaria um bloqueio mais efetivo da fosforilação, ao estabilizar esse loop como um todo, incluindo o resíduo K244 e deixando a S245 menos suscetível a acção da Cdk5 / Abstract: The Peroxisome Proliferator-Activated Receptors gamma (PPARg) is a member of a family of nuclear receptors whose activity is regulated by ligands. The PPARg acts on lipid metabolism and promotes systemic insulin sensitization, therefore being a potential target for the development of antidiabetics agents. Among the ligands of this receptor, the most popular is rosiglitazone (RSG), a full agonist which has restricted use by its side effects: cardiovascular toxicity, weight gain and water retention. The use of partial agonists is a promising alternative to reduce these effects because they can uncouple insulin sensitization from the triglyceride accumulation. In this work, we performed molecular dynamics simulation to investigate the dynamics of PPARg in presence of a partial agonist recently discovered, GQ16 and rosiglitazone (RSG). The classic mechanism of receptor activation is through the stabilization of one of its helixes (H12). Analyzing the stability of H12 during the simulations we found that it remained more stable in the presence of RSG in relation to GQ16. The simulations revealed that while RSG interacts directly with a residue of H12, GQ16 interacts through a water molecule, thus destabilizing somewhat the productive conformation of H12. This difference in behavior promoted by the ligands may underlie the lower GQ16-induced activation in relation to the RSG, which is consistent with the functional studies that showed that GQ16 acts as partial agonist. Recent studies show that structural characteristics are important in receptor ligand-specific activation. One of these auxiliary factors of the activation of PPARg is related to the phosphorylation of a serine (S245) receptor mediated by a protein kinase, Cdk5. The simulations revealed that there is a greater stabilization of the loop containing the S245 in the presence of GQ16. In addition, we found that the residue K244, neighboring the target of phosphorylation by Cdk5, sweeps a narrower conformational space in relation to complex PPARg-RSG. These results suggest that GQ16 enable a more effective blocking of the phosphorylation by stabilizing this loop as a whole, including the residue K244, letting S245 less susceptible to the action of Cdk5 / Mestrado / Físico-Química / Mestre em Química

Orientador: Munir Salomão Skaf / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-21T17:39:27Z (GMT). No. of bitstreams: 1 daSilva_JulioCesarAraujo_D.pdf: 43361733 bytes, checksum: 212cdef85174d1daf286133ff839cfb2 (MD5) Previous issue date: 2012 / Resumo: Receptores de androgênio (AR) são membros da superfamília de receptores nucleares que incluem os receptores de esteroides, entre outros. O AR liga os esteroides sexuais endógenos diidrotestosterona e testosterona. O desenvolvimento normal do fenótipo masculino e do sistema reprodutivo necessita de ações pré- e pósnatais promovidas pela interação do AR com esses hormônios. Mutações no gene do receptor de androgênio podem levar a várias doenças como o câncer de próstata e a síndrome de insensibilidade ao androgênio (AIS). Substituições diferentes no mesmo resíduo de aminoácido podem resultar em impactos variáveis na atividade do receptor levando a diferentes graus de AIS. Um grande número de mutações tem sido reportado para o AR envolvendo AIS e células tumorais de câncer de próstata e sua localização e função podem ajudar a entender como essas doenças devem ser tratadas. Entretanto, pouco se sabe sobre como as mutações mudam a estrutura e a dinâmica do AR, uma vez que apenas poucas estruturas cristalográficas de mutantes foram obtidas. Neste trabalho, apresentamos estudos de simulação de dinâmica molecular de algumas estruturas do AR humano com mutações localizadas no domínio de ligação do ligante (LBD) em comparação com a estrutura nativa complexadas com o ligante sintético metiltrienolona (R1881). Nosso objetivo é investigar as bases moleculares das mudanças sutis no receptor causadas pelas mutações que afetam sua afinidade pelo ligante R1881. Embora nenhum dos resíduos mutados deste estudo interajam diretamente com o ligante, os resultados das simulações indicaram que as mutações causam mudanças estruturais e dinâmicas no AR-LBD na região onde se localiza a mutação e na cavidade de ligação do ligante (LBP). A principal mudança observada foi o deslocamento do resíduo Arg752, facilitando a entrada de moléculas de água no LBP e o reposicionamento de cadeias laterais dos resíduos do domínio F, uma importante região que contribui para a estabilidade da estrutura do AR-LBD e da conformação ativa da hélice 12. Os resultados obtidos mostram que essas mutações, que ocorrem naturalmente, são exemplos de resíduos que não estão em contato direto com o ligante e não pertencem à região de recrutamento do coativador, mas que possuem um importante papel na ligação do ligante e na ativação do receptor por estabilizar a hélice 12 e o domínio F na conformação ativa / Abstract: Androgen receptors (AR) are members of the superfamily of nuclear receptors that includes the steroid receptors, among others. AR binds the male endogenous sex steroids, dihydrotestosterone and testosterone. Normal development of the male phenotype and reproductive system requires pre- and postnatal actions promoted by AR interaction with these hormones. Mutations in the androgen receptor gene may lead to several diseases like prostate cancer (PCa) and the androgen insensitivity syndrome (AIS). Different substitutions at the same amino acid residue may result in variable impact on the activity of the receptor leading to different degrees of AIS. A number of mutations have been reported for the AR in AIS and PCa tumor cells and their location and function may help us to understand how these diseases should be treated. Nevertheless, not much is known about how the mutations change the structure and dynamics of the AR since only a few crystallographic structures of mutants were obtained. In this work we present molecular dynamics simulation (MD) studies of some human AR mutations located in the ligand binding domain (LBD) in comparison with wild type (WT) structure in complex with the synthetic ligand methyltrienolone (R1881). Our goal is to investigate the molecular basis of subtle changes in the receptor caused by mutations in the AR-LBD/R1881 affinity. Although the mutated residues do not interact with the ligand, the simulations results indicated that the mutants cause structural and dynamical changes in the AR-LBD in the region in which the mutation is placed and in the binding pocket (LBP). The principal change observed was the displacement of residue Arg752, facilitating water penetration in LBP, and the repositioning of F-domain side chains, which makes important contributions to the stability of the AR-LBD structure and helix 12 active conformation. The results obtained show that these naturally occurring mutations are examples of residues that are not in contact with the ligand and do not belong to coactivator recruitment region, but which do have an important role in ligand binding and receptor activation by stabilizing the helix H12 and the F-domain in the active conformation / Doutorado / Físico-Química / Doutor em Ciências

La récidive tumorale est l'un des principaux obstacles à surmonter à l'avenir pour améliorer la survie globale des patients atteints de cancer du côlon (CCR). Les échecs thérapeutiques observés chez les patients sont compatibles avec une accumulation de cellules souches cancéreuses (CSCs) résistantes aux médicaments. Dans cette étude, nous démontrons que le récepteur nucléaire PXR (NR1I2) agit comme un régulateur important de la chimiorésistance des CSCs coliques et de leur capacité à initier la rechute tumorale après traitement. Nous avons d'abord montré que l'expression de PXR augmente avec celle de certains marqueurs des CSCs dans des cellules cancéreuses de patients CCR traitées par chimiothérapies. Nous avons constaté que PXR est préférentiellement exprimé dans les CSCs coliques et qu'il contribue à l'enrichissement des CSCs après chimiothérapies in vitro et in vivo. Par des approches de transcriptomiques, nous avons observé qu'au sein des CSCs coliques, PXR contrôle l'expression d'un large réseau de gènes marqueurs des CSCs coliques, ainsi que des gènes impliqués dans la résistance aux médicaments ou à l'apoptose, ou impliqués dans la dissémination métastatique. Enfin, l'inhibition de PXR par interférence à ARN diminue la survie et auto-renouvèlement des cellules souches cancéreuses du côlon in vitro, ainsi que leur capacité à résister à la chimiothérapie après xénogreffes, conduisant à des retards importants de rechute tumorale après traitements par chimiothérapies in vivo. Cette étude suggère fortement que l'inhibition ciblée de PXR peut représenter une stratégie de traitement néo-adjuvant afin de diminuer la résistance aux médicaments et la récidive des patients CCR via la sensibilisation des cellules souches cancéreuses aux chimiothérapies classiques. / Tumor recurrence is one of the major obstacles to overcome in the future to improve overall survival of patients with colon cancer. High rates and patterns of therapeutic failure seen in patients are consistent with a steady accumulation of drug-resistant cancer stem cells (CSCs). Here, we demonstrate that the nuclear receptor PXR (NR1I2) acts as a key regulator of colon CSC chemoresistance and of their ability to generate post-treatment tumor relapse. We first determined that the enrichment of PXR paralleled that of CSC markers upon treatment of colon cancer cells with standard of care chemotherapy. We found that PXR was highly expressed in colorectal cancer cells displaying CSC markers and function and that it was instrumental for the emergence of CSCs following chemotherapy in vitro and in vivo. mRNA profiling experiments in colon CSCs indicated that PXR transcriptionally controls a large network of genes including markers of stemness, genes involved in resistance to drug/apoptosis or migration/invasion. Finally, PXR down-regulation altered the survival and self-renewal of colon CSCs in vitro and hampered their capacity to resist chemotherapy in vivo, leading to significant delays of post-chemotherapy tumor relapse. This study strongly suggests that targeting PXR may represent a novel treatment strategy to prevent drug resistance and recurrence through the sensitization of CSCs to standard chemotherapy. Taken together, our data strongly suggest that PXR plays an instrumental role in the so-called "intrinsic" pan-resistance of CSCs against therapy.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第18924号 / 薬科博第38号 / 新制||薬||5(附属図書館) / 31875 / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 岡村 均, 教授 中山 和久, 教授 竹島 浩 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DGAM

Glucocorticoids bind with high affinity to intracellular receptors located in high density within discrete regions of the rodent and primate brain. The binding of [3H]corticosterone was compared in the brains of male vs female rats. The number and affinity of cytosol receptors in the hippocampus and hypothalamus were examined in vitro. The cytosolic binding capacity of the hippocampus is greater in the female than in the male. This difference in binding capacity is not dependent on the presence of gonadal steroids: the effect of gonadectomy was not significant for either sex. The difference is not due to transcortin since the binding capacity of [3H]dexamethasone is also greater in the female hippocampus. Receptor affinity in the female hippocampus is half that of the male value. In the hypothalamus, the dimorphism is in the opposite direction: the number of [3H]corticosterone cytosolic binding sites was found to be greater in the male. The male hypothalamus also showed a greater affinity for [3H]corticosterone than did the female. Ovariectomy increased the number of binding sites in the female hypothalamus. In vivo nuclear uptake of a tracer dose of [3H]corticosterone was determined in animals having intact gonads. The percent of tissue [3H]corticosterone present in cell nuclei from 4 brain regions, including the hippocampus and hypothalamus, was calculated per unit DNA. The concentrations of [3H]corticosterone in nuclei relative to tissue homogenates were higher in females than males for the 4 brain regions, but not for the pituitary or liver. The data are interpreted as suggesting that glucocorticoid secretion under basal conditions and during stress may differentially effect specific brain structures in male vs female rats.

<p> The Liver X receptor (LXR) is a type II nuclear receptor that is known to be a master regulator of cholesterol levels in the body through its transcriptional control of target genes involved in the handling of cholesterol. The regulation of LXR occurs at multiple levels including ligand and protein availability, post-translational modifications, protein-protein interactions with various cofactors and/or chaperones and a new concept of regulation that involves compartmentalization. This involves the establishment of regions where proteins can be active or inactive. Type II nuclear receptors have recently been found to shuttle between the cytoplasm and the nucleus, thus a compartmentalization component is likely to be involved. It was recently implicated that the centrosome-associated protein 350 (CAP350) can sequester PPARa. into nuclear bodies, and to regions in the cytoplasm. The significance of this appears to be the control of PPAR action. CAP350 is a large protein that has the ability to interact with nuclear receptors via an LXXLL motif, and with the cytoskeleton via a CAP-Giy motif. CAP350 is suggested to play a role in the organization of nuclear receptors in the nucleus, and their retention in compartments. In this report, LXRa. was confirmed to interact with CAP350 in vitro, using a GST-binding assay. Utilizing fluorescent protein chimeras with both nuclear receptors and CAP350 allowed the monitoring of this interaction in vivo. CAP350 was observed to form nuclear bodies that were capable of recruiting LXRa.. This recruitment was dependant on the integrity of the LXXLL motif. The mutated LXXLL motif of CAP350 was not able to colocalize with LXRa.. The significance of this interaction remains unknown. It is likely to be similar to that observed with PPARa., since the nuclear bodies formed by CAP350 seem to correspond to transcriptionally silent regions in the nucleus. </p> / Thesis / Master of Science (MSc)

Yes / Seasonal animals undergo changes in physiology and behavior between summer and winter conditions. These changes are in part driven by a switch in a series of hypothalamic genes under transcriptional control by hormones and, of recent interest, inflammatory factors. Crucial to the control of transcription are histone deacetylases (HDACs), generally acting to repress transcription by local histone modification. Seasonal changes in hypothalamic HDAC transcripts were investigated in photoperiod-sensitive F344 rats by altering the day-length (photoperiod). HDAC4, 6 and 9 were found to change in expression. The potential influence of HDACs on two hypothalamic signaling pathways that regulate transcription, inflammatory and nuclear receptor signaling, was investigated. For inflammatory signaling the focus was on NF-κB because of the novel finding made that its expression is seasonally regulated in the rat hypothalamus. For nuclear receptor signaling it was discovered that expression of retinoic acid receptor beta was regulated seasonally. HDAC modulation of NF-κB-induced pathways was examined in a hypothalamic neuronal cell line and primary hypothalamic tanycytes. HDAC4/5/6 inhibition altered the control of gene expression (Fos, Prkca, Prkcd and Ptp1b) by inducers of NF-κB that activate inflammation. These inhibitors also modified the action of nuclear receptor ligands thyroid hormone and retinoic acid. Thus seasonal changes in HDAC4 and 6 have the potential to epigenetically modify multiple gene regulatory pathways in the hypothalamus that could act to limit inflammatory pathways in the hypothalamus during long-day summer-like conditions. / Biotechnology and Biological Sciences Research Council (BBSRC)

In multicellular organism, bioenergetic metabolism is strictly regulated toward efficient generation of ATP. However, in certain situations, such as in limiting oxygen or in the rapidly proliferating system like growing juvenile or cancer cells, organisms apply the metabolic strategy that favors the production of biomass (e.g., nucleotides, amino acids, and lipids) over efficiency of ATP generation. The conserved estrogen-related receptors (ERRs) are master regulators in controlling metabolic homeostasis, and good candidates for mediating the metabolic transition induced by hypoxia and development. First, we investigate how dERR influences hypoxic adaptation in Drosophila melanogaster. We find that dERR is required for a competent hypoxic response alone, or together with hypoxia inducible factor (HIF), which is the main transcription factor modulating the hypoxic adaptation. We show that dERR binds to dHIFα and participates in the HIF-dependent transcriptional program in hypoxia. In addition, dERR acts in the absence of dHIFα in hypoxia and a significant portion of HIF-independent transcriptional responses can be attributed to dERR actions, including up-regulation of glycolytic transcripts. These results indicate that competent hypoxic responses arise from complex interactions between HIF-dependent and -independent mechanisms, and that dERR plays a central role in both of these programs. Secondly, we examine how dERR modulates metabolic transition toward the fatty acid oxidation at late L3 larva stage. We show that dERR is essential for the expression of an uncharacterized long-chain-fatty-acid acyl-CoA synthetase, CG4500, which is subject to induction by starvation. Furthermore, late L3 larvae of dERR mutants exhibit altered lipid profiles with elevated medium-chain and long-chain fatty acids. Together, with the previous finding that ERR directs an early switch toward glycolysis in the embryo, our studies indicate that ERR is a master regulator of programmed metabolic shifts through Drosophila development.

Le récepteur ERRα (Estrogen Receptor-Related Receptor alpha) appartient à la superfamille des récepteurs nucléaires. Une forte expression de ERRα est corrélée à un mauvais pronostic, suggérant l’implication de ce récepteur dans le processus métastatique. Mon projet est d'analyser le rôle de ERRα dans les mouvements cellulaires. J’ai montré qu’inhiber ERRα perturbe la migration cellulaire. L’étude des mouvements montre que l’absence de ERRα induit une perturbation de l’orientation cellulaire, du nombre des fibres de stress et des protrusions membranaires. Les cellules migrent de façon désorientée. J’ai démontré l’existence d’une cascade de régulation où ERRα stimule transcriptionnellement l'expression de la protéine BACURD2/TNFAIP1, elle-même régulant la stabilité de RhoA. Inhiber ERRα induit également une surexpression de RhoA, sa suractivation et une perturbation de la migration orientée. Cette cascade a été confirmée par des expériences de complémentation. J’ai vérifié ces résultats par des expériences in vivo et ex vivo, chez les souris KO pour ERRα. L’absence de ce récepteur induit une diminution de l’expression de TNFAIP1 inhibant la dégradation de RhoA et entrainant finalement une perturbation des mouvements cellulaires. Ainsi ERRα régule positivement la migration cellulaire conduisant à une forte potentialité métastatique dans les tumeurs surexprimant ce récepteur.L’ensemble de mes résultats pourrait faire de ERRα une nouvelle cible en vue de nouvelles thérapies anticancéreuses et nous pourrions proposer BACURD2/ TNFAIP1 comme un nouveau marqueur de pronostic dans les cancers. / High expression of the orphan nuclear receptor ERRα is strongly correlated with poor prognosis in various types of tumors, including those of the breast. The fact that high ERRα expression in tumors is also correlated with elevated invasiveness suggests that this nuclear receptor positively regulates cell migration and invasiveness.This possibility was investigated using MDA-MB231 breast cancer cell line as a model. Inactivating ERRα impairs cell migration. Using time-lapse-based cell tracking analysis and Golgi positioning, we show that this impairment is not due to reduced migration speed but rather to cell disorientation. The enhanced number of cell protrusions present in migrating cells and disorganized actin fibers confirm this. In summary cells do migrate but do not sustain persistent linear movement. We observed that upon ERRα inactivation, RhoA, which is instrumental in oriented movement, is overexpressed at the protein level. Further analysis showed that the stability and proteasome-dependent degradation of the protein is affected. To analyze the relationship between ERRα (as a transcription factor) and RhoA protein stability we performed a transcriptomic analysis comparing (by RNA-Seq) wt cells to ERRα-depleted ones. We identified genes regulated by ERRα that are involved in both cell migration (as a biological process) and in protein stability and degradation, more specifically that of RhoA protein (as a molecular process). TNFAIP1/Bacurd2 is stimulated by ERRα and fits these criteria: this protein mediates the Culin3-based, proteasome-dependent of RhoA and its inactivation leads to defects in cell migration.TNFAIP1/RhoA cascade is a major downstream effector of ERRα in cell migration