Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 132-165.

Thesis (Ph.D. in Cell Biology, Stem Cells, and Development) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 105-116).

Orientadores: Nilson Ivo Tonin Zanchin, Beatriz Gomes Guimaraes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T09:39:45Z (GMT). No. of bitstreams: 1 Scapin_SandraMaraNaressi_D.pdf: 11335048 bytes, checksum: 153f9eea9142fb7f3cb17de59a608da6 (MD5) Previous issue date: 2007 / Resumo: As GTPases da família Rab regulam o transporte intracelular de vesículas em eucariotos. Cada Rab atua em uma via de transporte específica e seu mecanismo de ação se dá através da realização de um ciclo de ligação e hidrólise de GTP. Neste trabalho, foi determinada a estrutura cristalográfica das formas inativa (ligada a GDP) e ativa (ligada a GppNHp) da GTPase Rab11b, um membro da subfamília Rab11 que está envolvida na reciclagem de proteínas dos endossomos para a membrana plasmática, no tráfego de vesículas da rede trans-Golgi para a membrana plasmática e na fagocitose. Os resultados foram confrontados com os dados estruturais da Rab11a descritos anteriormente. A Rab11b inativa cristalizou como um monômero, o que gera conflitos a respeito da formação de dímeros funcionais pela Rab11a. A Rab11b e a Rab11a ativas divergiram em relação à posição e à interação da serina 20, que é importante na hidrólise de GTP, mas apresentaram taxas hidrolíticas semelhantes in vitro. Visando uma investigação mais ampla da família Rab, a GTPase Rab21 também foi cristalizada, mas os cristais difrataram até 2.90 Å de resolução. Ensaios de desnaturação térmica revelaram que a Rab21 é estruturalmente mais instável do que a Rab11, talvez pela presença de cisteínas que estão susceptíveis à oxidação, contribuindo para a agregação e precipitação da proteína. A Rab11 é bastante estável, e possivelmente forma estruturas do tipo beta-amilóide em altas temperaturas. Este trabalho envolveu também o estudo funcional da interação entre a proteína TIP41 humana (TIPRL) e o fator de transcrição MafB. A TIPRL é uma proteína conservada que foi identificada como uma ativadora de MAP quinases enquanto sua homóloga em levedura foi caracterizada como um antagonista da via de sinalização da quinase TOR que regula o crescimento celular. A MafB está envolvida no controle transcricional em diversos processos de desenvolvimento, mas seus reguladores ainda não estão bem estabelecidos. A interação direta entre a TIPRL e a MafB inteira, ou seu domínio bZIP isolado, foi confirmada através de ensaios de ligação in vitro. As proteínas co-localizaram no núcleo de células HEK293 e nossos resultados preliminares mostram que a TIPRL inibe a atividade transcricional da MafB in vivo, embora apenas interfira na ligação in vitro do domínio bZIP da MafB ao seu DNA-alvo mediante a estabilização do complexo TIPRL-bZIP. A TIPRL pode, portanto, constituir um novo regulador da atividade de MafB / Abstract: GTPases of the Rab family are responsible for the intracellular transport of vesicles. Each family member acts on a specific transport pathway and their function is regulated by GTP binding and hydrolysis, cycling between inactive (GDP-bound) and active (GTP-bound) forms. In this work, we describe the crystal structure of inactive and active forms of the GTPase Rab11b, a member of the Rab11 subfamily which is involved in recycling of proteins from endosomes to the plasma membrane, in polarized transport in epithelial cells, in the transport of molecules of the trans-Golgi network to the plasma membrane and in phagocytosis. The Rab11b structure showed several differences from the Rab11a isoform previously described. Inactive Rab11b crystallized as a monomer, contradicting the hypothesis about functional dimers formed by Rab11a. Active Rab11b differ from Rab11a relative to the position of the serine 20 sidechain, which is involved in GTP hydrolysis, although both GTPases show similar GTP hydrolysis rates in vitro. In order to obtain structural information on Rab GTPases, Rab21 was also crystallized, but the crystals diffracted to a relatively low resolution (2.90 Å). Rab21 is a cysteine rich protein, showing a higher instability relative to Rab11b. Thermal unfolding followed by circular dicroism confirmed this hypothesis. Both Rab11b and Rab11a show a relatively high thermal stability and circular dicroism analysis indicate that they undergo conversion to structures rich in beta-strands upon thermal denaturation. This work includes also studies on the function of TIPRL in regard to its interaction with the transcription factor MafB. TIPRL is a conserved human protein identified as an activator of MAP kinases whereas its yeast counterpart Tip41 functions as an antagonist of the TOR kinase pathway. MafB is a large member of the Maf family of bZIP transcription factors controlling developmental processes in vertebrates. Regulation of MafB is critical, for example, during erythroid differentiation. A direct interaction between TIPRL and full length MafB and the bZIP domain of MafB was confirmed by in vitro interaction assays. TIPRL is localized throughout the whole cell and overlaps with MafB in the nucleus of HEK293 cells. Preliminary assays showed that TIPRL inhibits transcriptional activation mediated by MafB in HEK293 cells, although MafB shows a higher binding affinity to its target DNA relative to TIPRL in vitro. This evidence indicates that TIPRL may control MafB activity in vivo / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 106-117.

Vesicle budding, membrane trafficking, and lipid metabolism depend on the switching of Arf and Rab GTPases from the inactive GDP bound state to the active GTP bound state. However, Arf and Rab GTPases have intrinsic rates of GDP to GTP exchange that are much slower (hours to days) than the time scale of the relevant trafficking processes (seconds or less). In cells, the activation of Arf and Rab GTPases is tightly regulated by guanine nucleotide exchange factors (GEFs) with Sec7 or Vps9 domains, respectively. Full length Cytohesins, which have a domain architecture consisting of heptad repeats, a Sec7 domain, a pleckstrin homology (PH) domain, and a polybasic motif, have 100-fold lower exchange activity than the isolated Sec7 domain. Insights into the low exchange activity were obtained by structural, biochemical and kinetic analyses. It was found that the Sec7-PH domain linker and a C-terminal amphipathic helix physically block the docking sites for the switch regions of Arf GTPases. Mutations within either element result in partial or complete relief of autoinhibition. Autohibition is also strongly relieved by phosphorylation of protein kinase C (PKC) sites in the polybasic motif of Cytohesin-1 or by phosphoinositide head group-dependent binding of active Arf6. Despite unrelated folds, Sec7 and Vps9 domains engage cognate GTPases in a strikingly similar manner and supply a critical acidic residue that interacts with an invariant lysine residues from phosphate binding (P) loop of the GTPase in the nucleotide free complex. The key acidic residues have also been proposed to disrupt the Mg2+ binding site; however, it is not known whether disruption of Mg2+ binding contributes to the rate limiting step for nucleotide release. To investigate the kinetic mechanism for catalysis of nucleotide exchange in the absence of autoinhibitory interactions, a detailed stopped flow kinetic analysis of the intrinsic and GEF mediated exchange reactions was conducted for the isolated catalytic cores. Using three different fluorescence methods to monitor Mg2+ dissociation, formation of the nucleotide free intermediate, and subsequent nucleotide binding, the catalytic cores of Cytohesin-1 and Rabex-5 were found to robustly accelerate nucleotide exchange on Arf1 and Rab5, respectively, by at least 105- fold at physiological concentrations of Mg2+. The acceleration of nucleotide exchange was reduced by roughly an order of magnitude at sub-micromolar concentrations of Mg2+. In addition, the Cytohesin-1 and Rabex-5 catalytic cores have similarly high catalytic efficiencies (kcat/KM) as well as high lower limits on both the rate (kcat) and steady state (KM) constants for GDP release at physiological as well as low Mg2+ concentration. The limits on kcat and KM are comparable to the highest values reported for other well characterized GEFs and likely reflect dual requirements of membrane targeting and autoregulatory mechanisms for tight control of catalytic output. These results provide a solid structural and mechanistic foundation for future experiments to investigate the spatial-temporal dynamics of Cytohesin and Rabex-5 activation in cellular contexts.

O desenvolvimento das doenças neurodegenerativas, como a doença de Alzheimer, está associado à presença de agregados proteicos contendo Tau hiperfosforilada (p-Tau). Esta disfunção da Tau leva a prejuízos na homeostase celular. Um mecanismo chave para diminuir e/ou prevenir os danos promovidos pelos agregados contendo Tau seria o estímulo de sua degradação. Neste sentido, a proposta do presente estudo foi analisar a degradação da proteína Tau após aumento da expressão exógena da cochaperona Bag-2, a qual influencia o sistema proteassomal de degradação; bem como avaliar a ativação dos sistemas de degradação, a fim de correlacionar estes sistemas em cultura de células primárias e organotípica do hipocampo de ratos. Os resultados mostraram que a rotenona foi capaz de aumentar os níveis de p-Tau e que a superexpressão de Bag-2, foi eficiente em prevenir e degradar a p-Tau. O mecanismo envolvido neste processo envolve a coordenação dos sistemas proteassomal e lisossomal, já que a Rab7 e a Rab24 (envolvidas na via lisossomal) mostraram-se diminuídas na fase que antecede a agregação proteica, enquanto houve aumento da Rab24 na presença dos agregados proteicos. Com relação ao peptídeo beta amiloide, foi demonstrado tendência de aumento de p-Tau acompanhado de diminuição da atividade proteassomal e lisossomal. O tratamento com PADK (ativador lisossomal) foi capaz de reverter este efeito nestas diferentes condições. A análise da interrelação entre os sistemas mostrou que uma inibição do proteassoma favorece a via lisossomal e que o inverso não se repete. Os resultados sugerem que a modulação das vias de degradação pode ser interessante para o estudo, prevenção e tratamento das doenças neurodegenerativas associadas à agregação de proteínas / Neurodegenerative diseases, such as Alzheimer\'s, are associated to protein inclusions containing hyperphosphorylated Tau (p-Tau). It is well established that Tau dysfunction impairs cell homeostasis. A key mechanism to prevent and/or reduce the damage promoted by aggregates of Tau might be its degradation. In view of this, the aims of the present study are to evaluate p- Tau clearance following exogenous expression of Bag-2, which stimulates proteasome; as well as to analyze the activation of both lysosome and proteasome pathways in order to understand the crosstalk between these two systems in primary and organotypic cultures of rat hippocampus. Results showed that rotenone was able of increasing p-Tau that was prevented and degraded by Bag-2 overexpression. Mechanisms involved in this process involve the coordination of cell degradation systems, depending upon aggregation status, since Rab7 and Rab24 (involved in lysosomal pathway) were decreased before protein aggregation, while Rab24 increased in the presence of protein inclusions. Amyloid-beta peptide also increased p-Tau accompanied by decreased proteasome and lysosome activity. PADK (lysosomal activator) treatment reverted the inhibition promoted by amyloidbeta peptide. Inhibition of proteasome leads to activation of lysosome, but lysosome inhibition does not affect proteasome. Overall, results suggest that targeting degradation pathways might be useful to understand, prevent and treat neurodegenerative diseases associated with protein deposits

O desenvolvimento das doenças neurodegenerativas, como a doença de Alzheimer, está associado à presença de agregados proteicos contendo Tau hiperfosforilada (p-Tau). Esta disfunção da Tau leva a prejuízos na homeostase celular. Um mecanismo chave para diminuir e/ou prevenir os danos promovidos pelos agregados contendo Tau seria o estímulo de sua degradação. Neste sentido, a proposta do presente estudo foi analisar a degradação da proteína Tau após aumento da expressão exógena da cochaperona Bag-2, a qual influencia o sistema proteassomal de degradação; bem como avaliar a ativação dos sistemas de degradação, a fim de correlacionar estes sistemas em cultura de células primárias e organotípica do hipocampo de ratos. Os resultados mostraram que a rotenona foi capaz de aumentar os níveis de p-Tau e que a superexpressão de Bag-2, foi eficiente em prevenir e degradar a p-Tau. O mecanismo envolvido neste processo envolve a coordenação dos sistemas proteassomal e lisossomal, já que a Rab7 e a Rab24 (envolvidas na via lisossomal) mostraram-se diminuídas na fase que antecede a agregação proteica, enquanto houve aumento da Rab24 na presença dos agregados proteicos. Com relação ao peptídeo beta amiloide, foi demonstrado tendência de aumento de p-Tau acompanhado de diminuição da atividade proteassomal e lisossomal. O tratamento com PADK (ativador lisossomal) foi capaz de reverter este efeito nestas diferentes condições. A análise da interrelação entre os sistemas mostrou que uma inibição do proteassoma favorece a via lisossomal e que o inverso não se repete. Os resultados sugerem que a modulação das vias de degradação pode ser interessante para o estudo, prevenção e tratamento das doenças neurodegenerativas associadas à agregação de proteínas / Neurodegenerative diseases, such as Alzheimer\'s, are associated to protein inclusions containing hyperphosphorylated Tau (p-Tau). It is well established that Tau dysfunction impairs cell homeostasis. A key mechanism to prevent and/or reduce the damage promoted by aggregates of Tau might be its degradation. In view of this, the aims of the present study are to evaluate p- Tau clearance following exogenous expression of Bag-2, which stimulates proteasome; as well as to analyze the activation of both lysosome and proteasome pathways in order to understand the crosstalk between these two systems in primary and organotypic cultures of rat hippocampus. Results showed that rotenone was able of increasing p-Tau that was prevented and degraded by Bag-2 overexpression. Mechanisms involved in this process involve the coordination of cell degradation systems, depending upon aggregation status, since Rab7 and Rab24 (involved in lysosomal pathway) were decreased before protein aggregation, while Rab24 increased in the presence of protein inclusions. Amyloid-beta peptide also increased p-Tau accompanied by decreased proteasome and lysosome activity. PADK (lysosomal activator) treatment reverted the inhibition promoted by amyloidbeta peptide. Inhibition of proteasome leads to activation of lysosome, but lysosome inhibition does not affect proteasome. Overall, results suggest that targeting degradation pathways might be useful to understand, prevent and treat neurodegenerative diseases associated with protein deposits