GORAB is a protein that localises to the trans-Golgi network (TGN) and is known to interact with Rab6. The loss of GORAB leads to gerodermia osteodysplastica (GO), an autosomal recessive disorder which results in lax skin, precocious skin aging, osteoporosis, susceptibility to fractures and joint hyperelasticity. Both the function of GORAB and the mechanism of pathogenesis in GO patients are poorly defined. In this study, the cellular functions of GORAB have been investigated. Using a variety of approaches (yeast two-hybrid assays, pull-downs with recombinant proteins, proximity biotinylation assays combined with mass spectrometry and co-immunoprecipitations) it was possible to establish a network of interactions with Golgi-localised proteins, including Arf GTPases and the COPI-associated protein Scyl1, that suggests a possible role of GORAB in COPI-mediated trafficking. Consistent with this hypothesis, ultrastructural changes of the Golgi apparatus were observed as were abnormal protein glycosylation in primary skin fibroblasts derived from GO patients, detected using both lectin binding assays and mass spectrometric glycan profiling. Moreover, immuno-electron microscopy studies revealed unequal distribution of GORAB within the TGN and fluorescence recovery after photobleaching experiments show GORAB being very stably associated with Golgi membranes. These properties of GORAB seem to result from its ability to oligomerise and to interact with vimentin filaments. Based on these data, a model of GORAB acting as a scaffolding protein that organises sites of COPI budding at the TGN has been proposed. Additionally, analysis of both published and newly identified GORAB mutations found in GO patients revealed that they affect various properties of GORAB including its interaction with small GTPases and GORAB ability to oligomerise, which suggests that these features are important for GORAB cellular functions. Together, these data suggest that the underlying cause of the skin and bone defects observed in GO patients is impaired COPI trafficking at the Golgi apparatus resulting in abnormal glycosylation of extracellular matrix proteins.

Orientadores: Carlos Eduardo Steiner, Edi Lucia Sartorato / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T07:50:14Z (GMT). No. of bitstreams: 1 Scherrer_DanielZanetti_M.pdf: 2073780 bytes, checksum: 5690d67c200b80bd6e6b14beca0d0f07 (MD5) Previous issue date: 2007 / Resumo: Cutis laxa é um distúrbio raro do tecido conectivo em que a pele perde sua firmeza, cedendo excessivamente e conferindo ao indivíduo um aspecto envelhecido. Dentre as formas geneticamente determinadas, há uma de herança autossômica dominante, uma de herança ligada ao X e três de herança autossômica recessiva. Sugere-se que cutis laxa autossômica recessiva tipo II ou cutis laxa com déficit de crescimento e de desenvolvimento seja a mesma entidade descrita com as denominações de ¿síndrome da pele enrugada¿ e gerodermia osteodisplástica. Tal confusão quanto à nomenclatura é, em parte, causada pelo desconhecimento de suas bases biológicas. Recentemente se definiu que a cutis laxa autossômica recessiva tipo I é causada por mutações nos genes FBLN4 e FBLN5, localizados nos cromossomos 11q13.1 e 14q32.1, respectivamente. Por outro lado, dois indivíduos com cutis laxa descritos na literatura apresentaram alterações da lisil oxidase, codificada pelo gene LOX localizado em 5q23. Deste modo, o presente estudo visou analisar três famílias não aparentadas com quadro típico de cutis laxa autossômica recessiva tipo II, ¿síndrome da pele enrugada¿ ou gerodermia osteodisplástica para mutações nos genes LOX, FBLN4 e FBLN5. Para tanto, foram obtidas amostras de DNA genômico a partir de linfócitos do sangue periférico, utilizando métodos descritos na literatura pertinente. Todos os genes candidatos tiveram suas regiões codificantes amplificadas e submetidas à análise de seqüenciamento direto. Nenhuma mutação que caracterizasse o fenótipo foi encontrada nesses genes. Assim, o presente estudo excluiu os genes LOX, FBLN4 e FBLN5 como responsáveis para essa forma de cutis laxa nessas famílias / Abstract: Cutis Laxa is a rare disorder of connective tissue in which the skin sags excessively, giving to the individual an aged aspect. Concerning the genetic forms, there is an autosomal dominant, an X-linked recessive, and three autosomal recessive conditions. It has been suggested that type II autosomal recessive cutis laxa or cutis laxa with growth and developmental delay is the same entity described with the denominations of wrinkly skin syndrome and gerodermia osteodysplastica. Such confusion is caused, in part, by the fact that its biological basis remains unknown. Recently, type I autosomal recessive cutis laxa has been associated with mutations in FBLN4 and FBLN5 genes located on 11q13 and 14q32, respectively. On the other hand, some patients with cutis laxa show abnormalities in lysyl oxidase, coded by the LOX gene located on 5q23. In this way, the present study analyzed three unrelated families with typical picture of type II autosomal recessive cutis laxa, ¿wrinkly skin syndrome¿ or gerodermia osteodysplastica for mutations in LOX, FBLN4, and FBLN5 genes. Molecular studies were performed in genomic DNA samples from peripheral blood. All codifier regions of the three candidate genes were amplified and submitted to direct sequence analysis. No mutation that characterized the phenotype was found in the genes. Thus, the present study excluded LOX, FBLN4 and FBLN5 as responsible for this form of cutis laxa in these families / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Orientadores: Carlos Eduardo Steiner, Claudia Vianna Maurer Morelli / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T22:10:07Z (GMT). No. of bitstreams: 1 Scherrer_DanielZanetti_D.pdf: 3629453 bytes, checksum: 18c59f0316e08a663d1352236ede8f6e (MD5) Previous issue date: 2012 / Resumo: Cutis laxa autossômica tipo II (CLAR tipo II) é um distúrbio raro do tecido conectivo em que a pele perde sua firmeza, cedendo excessivamente e conferindo ao indivíduo um aspecto envelhecido. Sugere-se que CLAR tipo II seja a mesma entidade descrita como "síndrome da pele enrugada" e gerodermia osteodisplástica. Tal confusão quanto à nomenclatura é, em parte, causada pelo conhecimento limitado de suas bases biológicas. O presente estudo visou analisar três famílias não aparentadas com quadro típico de CLAR tipo II para mutações nos genes ATP6V0A2, SCYL1BP1 e PYCR1. Nenhuma mutação foi identificada nos genes ATP6V0A2 e SCYL1BP1. Por outro lado, a triagem de mutações por sequenciamento direto do DNA no gene PYCR1, que desempenha um papel crítico na biossíntese de prolina, revelou duas novas variantes (p.Q10X e p.A241V), com efeito patogênico predito por análises de bioinformática. Em complementação, foi determinado o perfil de expressão gênica em tecido de pele e fibroblasto a partir de dois indivíduos com CLAR tipo II pela investigação de microarranjos, identificando os genes e as vias metabólicas possivelmente alteradas em tal condição, com a finalidade de entender os mecanismos subjacentes a este tipo de cutis laxa. Foi utilizado o Human Genome U133 Plus 2.0 array (Affymetrix¿), e analisados por meio dos pacotes Affy e RankProd do BioConductor. O perfil transcricional apresentado por pele fresca pela análise de microarranjos nos indivíduos com CLAR tipo II detectou 542 genes diferencialmente expressos em comparação com controles saudáveis pareados por sexo, idade e topografia anatômica. A análise de enriquecimento das categorias de ontologia gênica, utilizando o programa DAVID, incluiu a diferenciação e desenvolvimento epidérmico, ectodérmico e de queratinócitos. As vias de sinalização mais ativadas analisadas por software Ingenuity foram relacionadas a doenças e condições dermatológicas, além de "câncer, desenvolvimento e função do tecido conjuntivo, esquelético e muscular", bem como o metabolismo lipídico. Este é o primeiro estudo utilizando investigação de microarranjos na doença CLAR tipo II. Os resultados incluíram duas novas alterações genéticas (p.Q10X e p.A241V), além de fornecer uma visão completa das vias celulares diferencialmente expressas em tal condição. Desta forma, estes resultados contribuem para uma melhor compreensão da CLAR tipo II quanto aos mecanismos moleculares e nosologia / Abstract: Autosomal recessive cutis laxa, type 2 (ARCL2), is a rare disorder of connective tissue in which the skin sags excessively, giving to the individual an aged aspect. It has been suggested that ARCL2 is the same entity described under the denominations of wrinkly skin syndrome and gerodermia osteodysplastica. Such confusion is caused, in part, due to limited knowledge of its molecular basis. In the present study three unrelated families with ARCL2 were analyzed for mutations in ATP6V0A2, SCYL1BP1, and PYCR1. No causative mutations were identified in ATP6V0A2 and SCYL1BP1. However, screening for mutations in PYCR1 revealed two new variant (p.Q10X and p.A241V) by direct DNA sequencing, with predicted pathogenic effect on bioinformatic analysis. In complementation, in order to shed some light into the molecular mechanisms underlying this type of cutis laxa, gene expression profile in skin tissue and fibroblast were studied in two patients with ARCL2 using microarray investigation. This study was performed using the Human Genome U133 Plus 2.0 array (Affymetrix¿), and analyzed using Affy and RankProd packages from Bioconductor. Transcriptional profiling in skin tissue by microarray analysis from ARCL2 patients detected 542 differentially expressed genes compared to healthy controls matched by gender, age, and anatomic region. The top enriched gene ontology categories analyzed by DAVID included the differentiation and development of keratinocytes, epidermis and ectoderm. Among the most activated signaling pathways analyzed by Ingenuity software were related diseases and dermatological conditions and "cancer, connective tissue development and function, skeletal and muscular systems" as well as lipid metabolism. This is the first study using microarray investigation in the ARCL2 disease. Our results include two new genetic alterations (p.Q10X and p.A241V) and also provide a complete view of cellular pathways differentially expressed in such condition. The present study contributed to a better understanding of ARCL2 molecular mechanisms and nosology of this group / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas