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Phylogenetic Inference for Multidomain ProteinsStolzer, Maureen 01 August 2011 (has links)
In this thesis, I present a model of multidomain evolution with associated algorithms and software for phylogenetic analysis of multidomain families, as well as applications of this novel methodology to case-studies and the human genome.
Phylogenetic analysis is of central importance to understanding the origins and evolution of life on earth. In biomedical research, molecular phylogenetics has proved an essential tool for practical applications. Current molecular phylogenetic methods are not equipped, however, to model many of the unique characteristics of multidomain families. Genes that encode this large and important class of proteins are characterized by a mosaic of sequence fragments that encode structural or functional modules, called domains. Multidomain families evolve via domain shuffling, a process that includes insertion, internal duplication, and deletion of domains. This versatile evolutionary mechanism played a transformative role in major evolutionary transitions, including the emergence of multicellular animals and the vertebrate immune system.
Multidomain families are ill-suited to current methods for phylogeny reconstruction due to their mosaic composition. Different regions of the same protein may have different evolutionary histories. Moreover, a protein may contain domains that also occur in otherwise unrelated proteins. These attributes pose substantial obstacles for phylogenetic methods that require a multiple sequence alignment as input. In addition, current methods do not incorporate a model of domain shuffling and hence, cannot infer the events that occurred in the history of the family. I address this problem by treating a multidomain family as a set of co-evolving domains, each with its own history. If the family is evolving by vertical descent from a conserved set of ancestral domains, then all constituent domains will have the same phylogenetic history. Disagreement between domain tree topologies is evidence that the family evolved through processes other than speciation and gene duplication. My algorithms exploit this information to reconstruct the history of domain shuffling in the family, as well as the timing of these events and the ancestral domain composition. I have implemented these algorithms in software that outputs the most parsimonious history of events for each domain family. The software also reconstructs a composite family history, including duplications, insertions and losses of all constituent domains and ancestral domain composition.
My approach is capable of more detailed and accurate reconstructions than the widely used domain architecture model, which ignores sequence variation between domain instances. In contrast, my approach is based on an explicit model of events and captures sequence variation between domain instances. I demonstrate the utility of this method through case studies of notch-related proteins, protein tyrosine kinases, and membrane-associated guanylate kinases. I further present a largescale analysis of domain shuffling processes through comparison of all pairs of domain families that co-occur in a protein in the human genome. These analyses suggest that (1) a remarkably greater amount of domain shuffling may have occurred than previously thought and (2) that it is not uncommon for the same domain architecture to arise more than once through independent events. This stands in contrast to earlier reports that convergent evolution of domain architecture is rare and suggests that incorporating sequence variation in evolutionary analyses of multidomain families is a crucial requirement for accurate inference.
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A Fluorescence Based Method for Studying the Membrane Topology of the Anti-Apoptotic Protein BCL-XLAtkinson, Helen A. 10 1900 (has links)
Bcl-XL is a membrane-associated protein that inhibits programmed cell death
(apoptosis) in mammalian cells. Very little is known about the membrane topology of
Bel-XL or how its association with membranes contributes to its function. It was the aim
of this thesis to use fluorescence spectroscopy to investigate the location of a specific
amino acid ofBcl-XL relative to the membrane.
Bel-XL purified from E. coli could bind both to large unilamellar vesicles and
endoplasmic reticulum (ER) microsomes isolated from canine pancreas. A cysteine
residue at position 151 in Bcl-XL could be covalently labelled with the environmentally
sensitive fluorescent molecule NBD. Emission intensity measurements in the presence
and absence of membranes, combined with aqueous and lipophilic quenching
experiments, indicate that Cys 151 is inserted into the interior of the membrane bilayer
when Bcl-XL is bound to membranes. The methods outlined in this thesis form the basis
for an experimental system that can be used to determine the membrane topology ofBclXL
under a variety of conditions. / Thesis / Master of Science (MSc)
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Protein Quality Control at the Inner Nuclear Membrane – The Asi complex in Saccharomyces cerevisiaePantazopoulou, Marina January 2016 (has links)
The nuclear envelope is a barrier comprised of outer and inner membranes that separate the cytoplasm from the nucleoplasm. The outer (ONM) and inner (INM) membranes have different physical characteristics and protein compositions. In contrast to the extensive data available on the protein quality control processes operating in the cytoplasm, endoplasmic reticulum and the nucleoplasm, the mechanisms controlling protein turnover at the INM are poorly documented. The work presented in this thesis focuses on Asi1, Asi2 and Asi3, three bona-fide integral INM proteins of the yeast Saccharomyces cerevisiae. By contrast to mammalian cells, yeast progress through the cell cycle with a closed mitosis, that is cells divide in the absence of the cyclical fragmentation/reassembly of the nuclear membrane. Consequently, examining the processes affecting the stability of the Asi proteins in yeast may provide useful paradigms for understanding the turnover of INM components in non-dividing, terminally differentiated and post-mitotic cells of metazoan origin.The results have contributed to the elucidation of the biological function of Asi1 and Asi3, which are homologous proteins with C-terminal RING domains. Asi1 and Asi3 function together as a dimeric E3 ubiquitin ligase complex that operates with ubiquitin conjugating enzymes Ubc6 and Ubc7. The Asi1/3 complex ubiquitylates transcription factors Stp1 and Stp2 when they gain inappropriate access to the nucleus in the absence of SPS-sensor activation. Intriguingly, the Asi1/3 complex also mediates the turnover of multiple membrane proteins that primarily localize to other cell membranes. This latter finding indicates that the barrier function of nuclear pore complexes is not as tight as previously thought. Consistently, asi1 null mutations are synthetic lethal when introduced into hrd1Δ ire2Δ cells with compromised ER-associated degradation (ERAD) and unfolded protein response (UPR) pathways. Together the results define Asi1/3 as components of a novel quality control pathway operating in association with the INM that acts to safeguard the identity and maintain the function of the nuclear compartment. Asi1 and Asi2 exhibit rapid turnover and their turnover is ubiquitin-dependent, exhibiting a clear requirement for Ubc7. The ubiquitylated forms of Asi1 and Asi2 are degraded by nuclear-localized proteasomes; the ubiquitylated forms exhibit enhanced stability in sts1-2 mutants. Asi1 turnover requires Cue1, the AAA ATPase Cdc48 and co-factor Ubx1. Asi1 turnover occurs unimpeded in cells lacking a functional Asi1/3 complex and in cells lacking Doa10, an E3 ligase complex also known to function at the INM. Consequently, Asi1 is subject to a quality control pathway associated with INM but that is distinct from the Asi1/3 and Doa10 INM- associated degradative (INMAD) pathways. This thesis documents work that clearly demonstrates that the INM is a highly dynamic structure that possesses multiple and active quality control pathways. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>
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Susceptibilidade genética a periodontite apical crônica em indivíduos com fissura labiopalatina: estudo caso-controle / Genetic susceptibility to chronic apical periodontitis in individuals with cleft lip and/or palateDextre, Tulio Lorenzo Olano 03 August 2016 (has links)
A periodontite apical é um processo inflamatório que ocorre na região do periápice, em decorrência de contaminação microbiana do sistema de canais que se origina a partir da necrose pulpar ou canais radiculares tratados inadequadamente. Estudos recentes avaliando os fatores genéticos envolvidos no desenvolvimento da periodontite apical crônica (PAC) relatam forte relação com uma predisposição genética do indivíduo e determinam que variantes polimórficas em certos genes envolvidos na remodelação da matriz óssea poderiam contribuir na persistência desta. As metaloproteinases da matriz (MMPs) são enzimas que estão fortemente associadas com os níveis de inflamação e desempenham um papel importante na remodelação e reabsorção óssea, e níveis elevados de MMPs têm relação com a deficiência de reparo da lesão induzindo a PAC. Além disso, as MMPs também participam do complexo desenvolvimento raniofacial o que faz com que também sejam consideradas para a ocorrência de malformações faciais. O objetivo deste trabalho foi determinar se variantes polimórficas nos genes MMP-2 e MMP-3, envolvidos na resposta inflamatória estão associados a permanência de periodontite apical persistente após o tratamento endodôntico em indivíduos com fissura labiopalatina. Foram selecionados para este estudo 180 indivíduos divididos em 3 grupos: GI: 34 indivíduos com fissura labiopalatina, não sindrômicos, com história de PAC relacionada ao tratamento endodôntico; GII: 45 indivíduos sem fissura labiopalatina, não sindrômicos com história de PAC e GIII: grupo controle composto por 101 indivíduos sem fissura e sem relato de PAC. Como critério de inclusão para o diagnóstico de PAC foram considerados os escores 4 e 5 do índice PAI (PeriApical Index) analisado em radiografias periapicais de controle de um ano ou mais após o tratamento dos dentes envolvidos. Foram selecionadas para a genotipagem 3 variantes polimórficas no gene MMP-2 (rs243865, rs2285053 e rs2287074) e 2 no gene MMP-3 (rs679620 e rs522616). Os resultados foram analisados usando o software SDS 1.7 (Applied Biosystems) e os dados foram tabulados no programa Excel 8.0. As comparações entre as frequências dos genótipos e alelos foram realizadas através do teste do qui-quadrado (2) e Odds Ratio com intervalo de confiança de 95% (IC). Valores de p<0,05 foram considerados estatisticamente significativos. Das variantes polimórficas pesquisadas neste grupo populacional brasileiro, foi encontrada associação positiva do rs679620 no gene MMP-3 com a fissura labiopalatina e PAC somente quando comparado com o grupo com PAC e sem fissura. Sendo encontrada associação positiva também do rs522616 no gene MMP-3 com PAC e sem fissura, somente no comparativo com o grupo controle. Não foi encontrada nenhuma associação positiva das variantes no gene MMP-2 (rs243865, rs2285053 e rs2287074) nem com PAC nem com fissura labiopalatina. Dessa forma, são necessários estudos semelhantes analisando outras variantes polimórficas em outros genes envolvidos na cascata de sinalização da inflamação e na embriologia craniofacial, com amostras maiores, para esclarecer o papel do fator genético tanto para a periodontite apical crônica quanto para a fissura / Apical Periodontitis is an inflammatory process on periapical tissues, due to a microbial contamination of the canals system, arising from pulp necrosis or failed root canal treatment. Recent studies evaluating genetic factors involved on chronic apical periodontitis (CAP) show real relation with a genetic predisposition in individuals and determine whether polymorphic variability in some genes involved on bone matrix remodeling could contribute on the disease persistence. Matrix metalloproteinases (MMPs) are enzymes really associated with inflammation levels and take an important part of bone remodeling and resorption and high levels of MMPs have a relation with the lesion repair deficiency inducing CAP. Besides that, MMPs also participate on craniofacial development; being considered to the occurrence of some facial anomalies. The aim of this study was to determine whether polymorphic variability on genes MMP-2 e MMP-3 involved on inflammatory response is associated with the permanency of persistant apical periodontitis after endodontic therapy on individuals with cleft lip and/or palate. One hundred and eighty individuals were selected to this study, which was divided in 3 groups: GI: 34 individuals with cleft lip and/or palate, nonsyndromic, with CAP related to endodontic treatment; GII: 45 individuals without cleft lip and/or palate, nonsyndromic, with CAP, GIII: control group composed by 101 individuals without cleft and without report of chronic apical periodontitis. As inclusion criteria to CAP diagnosis were considered 4 and 5 of PAI score (PeriApical Index) analyzed on control periapical radiographies after one year or more after treatment of the teeth involved. Were selected for genotyping 3 polymorphic variabilities on gene MMP-2 (rs243865, rs2285053 e rs2287074) and 2 on gene MMP-3 (rs679620 e rs522616) were selected to genotyping. Results were analyzed by software SDS 1.7 (Applied Biosystems) and data were tabulated on 8.0 Excel program. Comparison among genotype frequencies and alleles were performed through qui square test (2) and Odds Ratio with 95% confidence. Values of p<0,05 were considered statistically significant. From polymorphic variability evaluated on this Brazilian group, it was only observed a positive association between rs679620 on MMP-3 gene with cleft lip and palate and chronic apical periodontitis only when compared with chronic apical periodontitis group and without cleft lip and palate. A positive association was also found on rs522616 gene MMP-3 with chronic apical periodontitis and without cleft lip and palate, only when compared to control group. It was not found any positive association of the variability of MMP-2 (rs243865, rs2285053 e rs2287074) gene neither with chronic apical periodontitis nor with cleft lip and palate. Therefore, other studies are required to analyze other polymorphic variability on other genes involved on inflammation cascade of signaling and craniofacial embryology with more samples, to a better understanding of genetic influence not only on chronic apical periodontitis but also on cleft lip and/or palate
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Susceptibilidade genética a periodontite apical crônica em indivíduos com fissura labiopalatina: estudo caso-controle / Genetic susceptibility to chronic apical periodontitis in individuals with cleft lip and/or palateTulio Lorenzo Olano Dextre 03 August 2016 (has links)
A periodontite apical é um processo inflamatório que ocorre na região do periápice, em decorrência de contaminação microbiana do sistema de canais que se origina a partir da necrose pulpar ou canais radiculares tratados inadequadamente. Estudos recentes avaliando os fatores genéticos envolvidos no desenvolvimento da periodontite apical crônica (PAC) relatam forte relação com uma predisposição genética do indivíduo e determinam que variantes polimórficas em certos genes envolvidos na remodelação da matriz óssea poderiam contribuir na persistência desta. As metaloproteinases da matriz (MMPs) são enzimas que estão fortemente associadas com os níveis de inflamação e desempenham um papel importante na remodelação e reabsorção óssea, e níveis elevados de MMPs têm relação com a deficiência de reparo da lesão induzindo a PAC. Além disso, as MMPs também participam do complexo desenvolvimento raniofacial o que faz com que também sejam consideradas para a ocorrência de malformações faciais. O objetivo deste trabalho foi determinar se variantes polimórficas nos genes MMP-2 e MMP-3, envolvidos na resposta inflamatória estão associados a permanência de periodontite apical persistente após o tratamento endodôntico em indivíduos com fissura labiopalatina. Foram selecionados para este estudo 180 indivíduos divididos em 3 grupos: GI: 34 indivíduos com fissura labiopalatina, não sindrômicos, com história de PAC relacionada ao tratamento endodôntico; GII: 45 indivíduos sem fissura labiopalatina, não sindrômicos com história de PAC e GIII: grupo controle composto por 101 indivíduos sem fissura e sem relato de PAC. Como critério de inclusão para o diagnóstico de PAC foram considerados os escores 4 e 5 do índice PAI (PeriApical Index) analisado em radiografias periapicais de controle de um ano ou mais após o tratamento dos dentes envolvidos. Foram selecionadas para a genotipagem 3 variantes polimórficas no gene MMP-2 (rs243865, rs2285053 e rs2287074) e 2 no gene MMP-3 (rs679620 e rs522616). Os resultados foram analisados usando o software SDS 1.7 (Applied Biosystems) e os dados foram tabulados no programa Excel 8.0. As comparações entre as frequências dos genótipos e alelos foram realizadas através do teste do qui-quadrado (2) e Odds Ratio com intervalo de confiança de 95% (IC). Valores de p<0,05 foram considerados estatisticamente significativos. Das variantes polimórficas pesquisadas neste grupo populacional brasileiro, foi encontrada associação positiva do rs679620 no gene MMP-3 com a fissura labiopalatina e PAC somente quando comparado com o grupo com PAC e sem fissura. Sendo encontrada associação positiva também do rs522616 no gene MMP-3 com PAC e sem fissura, somente no comparativo com o grupo controle. Não foi encontrada nenhuma associação positiva das variantes no gene MMP-2 (rs243865, rs2285053 e rs2287074) nem com PAC nem com fissura labiopalatina. Dessa forma, são necessários estudos semelhantes analisando outras variantes polimórficas em outros genes envolvidos na cascata de sinalização da inflamação e na embriologia craniofacial, com amostras maiores, para esclarecer o papel do fator genético tanto para a periodontite apical crônica quanto para a fissura / Apical Periodontitis is an inflammatory process on periapical tissues, due to a microbial contamination of the canals system, arising from pulp necrosis or failed root canal treatment. Recent studies evaluating genetic factors involved on chronic apical periodontitis (CAP) show real relation with a genetic predisposition in individuals and determine whether polymorphic variability in some genes involved on bone matrix remodeling could contribute on the disease persistence. Matrix metalloproteinases (MMPs) are enzymes really associated with inflammation levels and take an important part of bone remodeling and resorption and high levels of MMPs have a relation with the lesion repair deficiency inducing CAP. Besides that, MMPs also participate on craniofacial development; being considered to the occurrence of some facial anomalies. The aim of this study was to determine whether polymorphic variability on genes MMP-2 e MMP-3 involved on inflammatory response is associated with the permanency of persistant apical periodontitis after endodontic therapy on individuals with cleft lip and/or palate. One hundred and eighty individuals were selected to this study, which was divided in 3 groups: GI: 34 individuals with cleft lip and/or palate, nonsyndromic, with CAP related to endodontic treatment; GII: 45 individuals without cleft lip and/or palate, nonsyndromic, with CAP, GIII: control group composed by 101 individuals without cleft and without report of chronic apical periodontitis. As inclusion criteria to CAP diagnosis were considered 4 and 5 of PAI score (PeriApical Index) analyzed on control periapical radiographies after one year or more after treatment of the teeth involved. Were selected for genotyping 3 polymorphic variabilities on gene MMP-2 (rs243865, rs2285053 e rs2287074) and 2 on gene MMP-3 (rs679620 e rs522616) were selected to genotyping. Results were analyzed by software SDS 1.7 (Applied Biosystems) and data were tabulated on 8.0 Excel program. Comparison among genotype frequencies and alleles were performed through qui square test (2) and Odds Ratio with 95% confidence. Values of p<0,05 were considered statistically significant. From polymorphic variability evaluated on this Brazilian group, it was only observed a positive association between rs679620 on MMP-3 gene with cleft lip and palate and chronic apical periodontitis only when compared with chronic apical periodontitis group and without cleft lip and palate. A positive association was also found on rs522616 gene MMP-3 with chronic apical periodontitis and without cleft lip and palate, only when compared to control group. It was not found any positive association of the variability of MMP-2 (rs243865, rs2285053 e rs2287074) gene neither with chronic apical periodontitis nor with cleft lip and palate. Therefore, other studies are required to analyze other polymorphic variability on other genes involved on inflammation cascade of signaling and craniofacial embryology with more samples, to a better understanding of genetic influence not only on chronic apical periodontitis but also on cleft lip and/or palate
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Varicose/ Senz'Aria, A MAGUK Required for Junctional Assembly During Epithelial Morphogenesis in DrosophilaMoyer, Katherine Ellen 10 1900 (has links)
Scaffolding proteins belonging to the Membrane Associated GUanylate Kinase (MAGUK) superfamily function as adaptors linking cytoplasmic and cell surface proteins to the cytoskeleton to regulate cell-cell adhesion, cell-cell communication and signal
transduction. We have identified a novel Drosophila MAGUK member, Varicose (Vari), the homologue of vertebrate scaffolding protein PALS2. Similar to its vertebrate counterpart, Varicose localizes to pleated Septate Junctions (pSJs) of all embryonic,
ectodermally derived epithelia and peripheral glia. In vari mutants, essential SJ proteins NeurexinIV and FasciclinIII are mislocalized basally and the cells develop a leaky paracellular seal. Localization of SJ protein Discs Large is not affected, indicating Vari is not involved in cell polarization. In addition, vari mutants display irregular tracheal tube diameters and have reduced lumenal protein accumulation suggesting involvement in tracheal morphogenesis. We found that Vari is distributed in the cytoplasm of optic lobe neuroepithelium and is required for proper ommatidial patterning. As well, Vari is expressed in a subset of neuroblasts and differentiated neurons of the nervous system. We also present a novel MAGUK function in wing hair alignment during adult
morphogenesis. We conclude that Varicose is involved in scaffold assembly at the SJ and has a role in patterning adult epithelia and in nervous system development. / Thesis / Doctor of Philosophy (PhD)
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Biosíntesi de glicolípids de membrana en Mycoplasma genitalium: expressió, purificació i caracterització d'una glicosiltransferasa processiva en la formació de glicosildiacilglicerolsMartínez Mas, Núria 11 November 2011 (has links)
Aquesta tesi se centra en l’estudi de les possibles glicosiltransferases de Micoplasma genitalium involucrades en la síntesi de glicolípids. Aquests compostos formen part de la membrana plasmàtica del microorganisme, únic envolcall que el protegeix del seu entorn ja que no disposa de paret cel•lular. La hipòtesi sobre la qual s’ha desenvolupat el treball és la possibilitat que aquests glicolípids i els enzims encarregats de la seva producció, les glicosiltransferases, siguin essencials per a la viabilitat del micoplasma i per tant, la seva inhibició sigui una forma d’eradicar les infeccions causades pel patògen.
De les tres seqüències classificades com a glicosiltrasferases en el genoma de Mycoplasma genitalium, mg025, mg060 i mg517, s’ha determinat que mg025 és l’únic dels tres gens que no és essencial per al bacteri, tot i que s’ha observat que els tres s’expressen tant a la fase exponencial com a la fase estacionària del seu creixement. La funció de mg025 i mg060 és encara desconeguda, mentre que mg517 és la glicosiltransferasa encarregada de la síntesi dels dos principals glicolípids del micoplasma, el monoglicosildiacilglicerol (MGlcDG) i el diglicosildiacilglicerol (DGlcDG).
En aquesta tesi s’ha desenvolupat un protocol d’expressió per a la glicosiltransferasa codificada per mg517, anomenada GT-MG517, el qual fa ús d’una coexpressió amb xaperones i solubilitza la proteïna amb detergents, glicerol i una elevada força iònica. Aquesta metodologia ha estat necessària ja que GT-MG517 és una proteïna associada a membrana i la seva expressió recombinant en E.coli presenta dificultats. La proteïna s’ha purificat mitjançant cromatografia d’afinitat per Ni, tot i que el grau de puresa assolit no ha estat suficient per a intentar la seva cristal•lització. Les diverses proves realitzades usant cromatografia d’exclusió molecular han permès determinar que GT-MG517 forma oligòmers d’alt pes molecular.
A partir de la proteïna purificada s’ha realitzat un estudi cinètic de la seva doble activitat glicosildiacilglicerolsintasa. D’aquest estudi s’extreu que GT-MG517 pot transferir un sucre, Glc o Gal, a una molècula principalment hidrofòbica, com és el DOG, i a una molècula molt més hidrofílica, com el MGlcDG. Ambdós compostos però posseeixen un alcohol primari sobre el qual té lloc la transferència creant l’enllaç (16). Amb qualsevol dels substrats acceptors provats, l’enzim presenta activitats específiques superiors si el substrat donador és UDP-Gal. L’acceptor preferit de GT-MG517 és el lípid DOG, però la glicosiltransferasa és capaç d’elongar, ja sigui amb una Glc o amb una Gal, glicolípids com el MGlcDG i el MGDEG, preferint en aquest cas el compost amb una Gal a l’extrem no reductor. Tot i això, l’afinitat de l’enzim és superior per a donadors amb Glc (KM inferiors a les dels donadors amb Gal). D’altra banda, s’ha demostrat que el lípid aniònic DOPG es comporta com a activador de l’activitat enzimática.
GT-MG517 posseeix un teòric domini d’unió d’UDP-Glc a l’extrem N-terminal. Aquesta zona presenta similitud de seqüència amb altres glicosiltransferases i permet la classificació de GT-MG517 dins de la família GT-2. En canvi, el seu extrem C-terminal presenta una seqüència particular que no s’alinea amb altres proteïnes. En aquesta tesi es formula la hipòtesi que aquesta zona podria ser d’interacció amb la membrana i, a més, que aquesta interacció podria regular l’activitat enzimàtica. Per això es preparen diverses formes truncades de GT-MG517, en les quals s’eliminen alguns aminoàcids de l’extrem C-terminal, i una forma en la qual només es conserva l’hipotètic domini d’unió d’UDP-Glc. Aquestes noves proteïnes s’expressen, solubilitzen i purifiquen aplicant el protocol establert per a la forma completa. Amb l’anàlisi dels glicolípids sintetitzats s’estableix que els deu últims aminoàcids de GT-MG517 són prescindibles per a la seva activitat, ja que les formes truncades corresponents continuen tenint la capacitat de sintetitzar glicolípids. No obstant, l’eliminació d’un nombre superior de residus, inactiva la proteïna. La purificació de l’hipotètic domini d’unió d’UDP-Glc posa de manifest que forma oligòmers, de la mateixa forma que ho fa la proteïna completa. / Esta tesis se centra en el estudio de las posibles glicosiltransferasas de Mycoplasma genitalium involucradas en la síntesis de glicolípidos. Estas moléculas constituyen parte de la membrana plasmática del miroorganismo, única estructura de protección frente al entorno ya que los micoplasmas carecen de pared celular. La hipótesis sobre la cual se ha desarrollado el trabajo es la posibilidad que los mencionados glicolípidos y las enzimas encargadas de su producción, las glicosiltransferasas, sean esenciales para la viabilidad del micoplasma y, por tanto, su inhibición sea una forma de erradicar las infecciones causadas por el patógeno.
De las tres secuencias clasificadas como glicosiltransferasas en el genoma de Mycoplasma genitalium, mg025, mg060 y mg517, se determinó que mg025 es el único de los tres genes que no es esencial para la bacteria, aunque se observó que los tres se expresan tanto en la fase exponencial como en la fase estacionaria de su crecimiento. Se desconoce todavía la función de mg025 y mg060. En cambio, se conoce que mg517 es la glicosiltransferasa responsable de la síntesis de los dos principales glicolípidos del micoplasma, el monoglicosildiacilglicerol (MGlcDG) y el diglicosildiacilglicerol (DGlcDG).
En esta tesis se desarrolló un protocolo de expresión para la glicosiltransferasa codificada por mg517, llamada GT-MG517, el cual emplea una coexpresión con chaperonas y solubiliza la proteína con detergentes, glicerol y una fuerza iónica elevada. Dicha metodología fue necesaria ya que GT-MG517 es una proteína asociada a membrana y su expresión recombinante en E.coli presenta dificultades. La proteína se purificó mediante cromatografía de afinidad por Ni pero el grado de pureza obtenido no fue suficiente para intentar su cristalización. Distintas pruebas realizadas usando cromatografía d’exclusión molecular permitieron determinar que GT-MG517 forma oligómeros de alto peso molecular.
Con la proteína purificada se realizó un estudio cinético de su doble actividad glicosildiacilglicerolsintasa. De este estudio se extrae que GT-MG517 puede transferir un azúcar, Glc o Gal, a una molécula principalmente hidrofóbica, como es el DOG, y a una molécula mucho más hidrofílica, como el MGlcDG. Pese a su diferencia, los dos compuestos poseen un alcohol primario sobre el que tiene lugar la transferencia creándose el enlace (16). Con cualquiera de los sustratos aceptores probados, la enzima presenta actividades específicas superiores si el sustrato dador es UDP-Gal. El aceptor preferido de GT-MG517 es el lípido DOG, aunque la glicosiltransferasa es capaz de elongar, ya sea con una Glc o con una Gal, glicolípidos como el MGlcDG y el MGDEG, prefiriendo en este caso el compuesto con una Gal en el extremo no reductor. Aún así, la afinidad de la enzima es superior para dadores con Glc (KM inferiores a las de los dadores con Gal). Por otro lado, se demostró que el lípido aniónico DOPG se comporta como activador de la actividad enzimática.
GT-MG517 posee un dominio teórico de unión de UDP-Glc en su extremo N-terminal. Esta zona presenta similitud de secuencia con otras glicosiltransferasas y permite la clasificación de GT-MG517 dentro de la familia GT-2. En cambio, su extremo C-terminal presenta una secuencia particular que no se alinea con otras proteínas. En esta tesis se formula la hipótesis que dicha zona podría ser de interacción con la membrana y, además, que dicha interacción podría regular la actividad enzimática. Por eso se prepararon distintas formas trucadas de GT-MG517, en las cuales se eliminaron algunos aminoácidos del extremo C-terminal, y una forma en la cual sólo se conservó el hipotético dominio de unión de UDP-Glc. Las nuevas proteínas se expresaron, solubilizaron y purificaron aplicando el protocolo establecido para la forma completa. Con el análisis de los glicolípidos sintetizados se determinó que los diez últimos aminoácidos de GT-MG517 eran prescindibles para su actividad, ya que las correspondientes formas truncadas conservaban su capacidad de sintetizar glicolípidos. No obstante, la eliminación de un número superior de residuos inactiva la proteína. La purificación del hipotético dominio de unión de UDP puso de manifiesto que forma oligómeros, de la misma forma en que lo hace la proteína completa. / This thesis is focused on the glycolipid producing glycosyltransferases from Mycoplasma genitalium. Glycolipids are part of the microorganism plasma membrane, which is the external covering of mycoplasmas since they lack a cell wall. Our working hypothesis is that glycolipids and the enzymes responsible for their production are essential to mycoplasma. Thus, glycosyltransferase inhibition could be a way to eradicate this pathogen caused infections.
There are three genes described as putative glycosyltransferases in Mycoplasma genitalium genome, mg025, mg060 and mg517. We determined that mg025 is the only one essential to the bacteria, although the three of them are expressed both during the exponential and stationary growth phases. The function of mg025 and mg060 still remains unknown, whereas mg517 codifies for the glycosyltransferase responsible for monoglycosyldiacylglycerol (MGlcDG) and diglycosyldiacylglycerol (DGlcDG) synthesis, main mycoplasma glycolipids.
In this work an expression protocol for mg517 codified glycosyltransferase was designed. The protocol included chaperone coexpression and protein solubilization with detergents, glycerol and high ionic strength. This methodology was necessary since GT-MG517 is a membrane associated protein and its recombinant expression in E.coli presents some difficulties. GT-MG517 was purified by Ni affinity chromatography. However, a suitable purity degree to attempt protein crystallization was not achieved. Some experiments using size exclusion chromatography revealed that GT-MG517 forms high molecular weight oligomers.
A kinetic study of the protein double glycosyldiacylglycerol synthase activity was performed. From this study we learned that GT-MG517 is able to transfer a sugar moiety, Glc or Gal, both to a hydrophobic molecule such as DOG or to a more hydrophilic compound such as MGlcDG. These acceptor substrates share a primary alcohol where the sugar transfer takes place forming a (16) bond. The enzyme presents higher specific activities when UDP-Gal acts as reaction donor, regardless of the acceptor tested. DOG is the preferred acceptor although the enzyme is able to transfer Glc or Gal moieties to glycolipids such as MGlcDG and MGDEG. In this case, the reaction is faster with acceptors with Gal in the non-reducing end. As for donor substrate, enzyme’s affinity is higher for Glc containing molecules, with lower KM values than for Gal donors. In addition, our study proved the anionic lipid DOPG to act as an enzymatic activity enhancer.
GT-MG517 has a putative binding domain for UDP-Glc in the N-terminal end. This sequence is similar to other glycosyltransferases and allows its classification in Cazy’s GT-2 family. On the contrary, the C-terminal end has a particular sequence which does not match up with any other protein. Our hypothesis was that this C-terminal end of GT-MG517 could contain a membrane interaction sequence, which could at the same time modulate enzymatic activity. To test this hypothesis some truncated forms of GT-MG517, where C-terminal aminoacids had been removed, were prepared. Moreover, a form where only the putative UDP-Glc binding domain was conserved was also expressed. All these proteins were expressed, solubilized and purified with the same protocol used for the full-length form of GT-MG517. Glycolipids produced by truncated forms were analysed and results implied that the last ten aminoacids were not involved in the enzyme’s activity. Elimination of a higher number of aminoacids caused protein inactivation. When the putative UDP-binding domain was purified, it showed high molecular weight oligomers such as those developed by the complete form of the protein.
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MicroRNAs cause micro changes: Regulation of expression of membrane-associated complement inhibitors and its effect on Neisseria gonorrhoeaeSavin, Avital 18 May 2021 (has links)
No description available.
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Self-Organization of β-Peptide Nucleic Acid Helices for Membrane ScaffoldingHöger, Geralin 14 February 2019 (has links)
No description available.
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Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variationGraf, Justin T. January 2008 (has links)
This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation.
In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation.
The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity.
Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes.
This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.
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