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Doctoral Thesis
DOI
https://doi.org/10.11606/T.17.2020.tde-11022020-134336
Document
Author
Full name
Ana Carla Medeiros
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2019
Supervisor
Committee
Gomes, Marcelo Damario (President)
Ferro, Emer Suavinho
Lucena, Malson Neilson de
Sebollela, Adriano Silva
Title in Portuguese
Caracterização subcelular e bioquímica da proteína SAMHD1
Keywords in Portuguese
DSBs
Início alternativo de tradução
SAMHD1
Abstract in Portuguese
SAMHD1 é uma proteína de 626 aminoácidos com um motivo N-terminal SAM e um domínio contendo histidina e ácido aspártico (HD). O domínio SAM é envolvido em interações do tipo proteína-proteína e proteína-DNA/RNA. Proteínas com o domínio HD fazem parte de uma superfamília de fosfohidrolases comumente envolvidas no metabolismo de ácidos nucléicos. SAMHD1 tem a função de diminuir o pool de dNTPs intracelular, e mutações no gene de SAMHD1 está associada a Síndrome de Aicardi-Goutières (AGS). Nesse trabalho, avaliamos a especificidade de anticorpos comerciais anti-SAMHD1 em diferentes linhagens celulares, determinamos sua localização subcelular em resposta ao dano no DNA induzido por camptotecina (CPT) que induz duplas quebras na fita de DNA (DSBs - DoubleStrand Breaks). Por último, investigamos a possibilidade de existência de uma forma alternativa truncada endógena de SAMHD1. Vimos que o anticorpo anti-SAMHD1 monoclonal possui reatividade cruzada com uma proteína de aproximadamente 50 kDa, de função ainda desconhecida, enquanto que o anticorpo policlonal reconhece especificamente uma banda de massa molecular correspondente a SAMHD1. Além disso, identificamos que a hiperexpressão de SAMHD1 produz uma forma truncada SAMHD1(ATG2) sem os 114 primeiros aminoácidos, incluindo o domínio SAM. Esta forma foi produzida graças a um códon de início alternativo localizado a 345 nucleotídeos upstream do ATG1, do gene SAMHD1. Por fim, concluímos que SAMHD1 ativa a resposta ao dano, porém não é recrutada para o foci nuclear, diferente do que foi recentemente publicado na literatura.
Title in English
Biochemical and subcellular characterization of SAMHD1 protein
Keywords in English
Alternative translation start
DSBs
SAMHD1
Abstract in English
SAMHD1 is a 626 amino acid protein in an N-terminal SAM motif and a domain containing histidine and aspartic acid (HD). The SAM domain is involved in protein-protein and proteinDNA / RNA interactions. Proteins with the HD domain are part of a superfamily of phosphohydrolases commonly involved in the metabolism of nucleic acids. SAMHD1 has the function of decreasing the pool of intracellular dNTPs, and mutations in the SAMHD1 gene are associated with Aicardi-Goutières Syndrome (AGS). In this work, we evaluated the specificity of commercial anti-SAMHD1 antibodies in different cell lines, determined their subcellular location in response to DNA damage induced by camptothecin (CPT) that induces double strand breaks (DSBs). Finally, we investigated the possibility of an endogenous truncated alternative form of SAMHD1. We have seen that the anti-SAMHD1 monoclonal antibody has cross-reactivity with a protein of approximately 50 kDa, of yet unknown function, whereas the polyclonal antibody specifically recognizes a band of molecular mass corresponding to SAMHD1. In addition, we have identified that the overexpression of SAMHD1 produces a truncated form SAMHD1 (ATG2) without the first 114 amino acids, including the SAM domain. This form was produced thanks to an alternative start codon located at 345 nucleotides upstream of the ATG1, SAMHD1 gene. Finally, we conclude that SAMHD1 activates the response to the damage, but it is not recruited to the nuclear foci, different from what has recently been published in the literature.
 
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ANACARLAMEDEIROS.pdf (1.83 Mbytes)
Publishing Date
2020-04-29
 
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