Análise dos efeitos da superexpressão do componente RNA da telomerase de Leishmania major (LeishTER)
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Data
2018-10-27
Autores
Vassilievitch, Alessandro Cabral
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Universidade Estadual Paulista (Unesp)
Resumo
Parasitos do gênero Leishmania pertencem à família Trypanosomatidae, os quais causam a leishmaniose, doença tropical negligenciada, que pode se apresentar em três formas clínicas: cutânea, mucocutânea e visceral. O Brasil é um dos países mais afetados pela doença, devido principalmente às condições socioeconômicas, às mudanças climáticas e ambientais. Pesquisas relacionadas à biologia da Leishmania contribuem para o entendimento dos mecanismos fisiológicos do parasito, e assim fornecem a possibilidade de encontrar novos alvos terapêuticos. O estudo dos telômeros de Leishmania se mostram promissores, já que estão relacionados com a estabilidade do genoma. Os telômeros estão localizados nas extremidades dos cromossomos e são responsáveis por proteger os cromossomos assegurando que a informação genética seja corretamente copiada durante a duplicação celular. Os telômeros são elongados por uma transcritase reversa especializada denominada telomerase. A telomerase é uma ribonucleoproteína, constituída por duas subunidades, uma proteína com função de transcriptase reversa denominada TERT, e um componente RNA (TER) que contém a sequência do molde da repetição telomérica copiado pela TERT. Estudos recentes mostram que o TER possui outras funções além de conter apenas um molde para elongamento dos telômeros. Sua estrutura secundária possui domínios com funções de controle da inserção de nucleotídeos pelo TERT, reconhecimento da sequência e ligação de proteínas acessórias. Recentemente foi identificado e parcialmente caracterizado o componente TER da telomerase de Leishmania spp. (LeishTER), mas sua biogênese e funções ainda permanecem desconhecidas. No presente trabalho foi verificado que a superexpressão de LeishTER tem um efeito dominante negativo sobre o desenvolvimento, morfologia e crescimento do parasito. Os resultados mostram que a superexpressão do TER possui efeito drástico na proliferação dos parasitos, fazendo com que a densidade populacional apresente um declínio considerável durante o crescimento exponencial, culminando na morte celular sem que a população atinja a fase estacionária de crescimento. Além disso, pode-se observar uma discreta diminuição no tamanho dos telômeros desses parasitos. Concluímos que a superexpressão de LeishTER em L. major afeta o ciclo celular e o crescimento do parasito, provavelmente devido a impossibilidade dos parasitos em manterem seus telômeros adequadamente, embora mais estudos sejam necessários para elucidar como o excesso de LeishTER afeta mecanismos e processos celulares importantes para manter a homeostase do parasito.
Parasites of the Leishmania genus belong to the Trypanosomatide family, which present peculiar and particular characteristics. Among them are the species that cause leishmaniasis, a neglected tropical disease that can be expressed in three different clinical forms: cutaneous, mucocutaneous and visceral. Brazil is one of the most affected countries, due mainly to socioeconomic conditions, climate change and environmental alterations. Research related to the biology of Leishmania contributes to the understanding of the important physiological mechanisms of the parasite, and thus provide new therapeutic targets against the disease. The study of Leishmania telomeres appears promising since they related are to the genome stability. Telomeres are nucleoprotein structures located at the ends of the chromosomes and are responsible for protecting the chromosomes ensuring that the genetic information copied is correctly during cell duplication. DNA polymerase does not elongate telomeres as the rest of the genetic material, and thus maintained are by the action of a specialized reverse transcriptase named telomerase. Telomerase is a ribonucleoprotein minimally composed by two subunits, a protein with reverse transcriptase function TERT, and an RNA component (TER) that contains the telomeric repeat template sequence copied by TERT. Recent studies shown that TER has other functions besides being just a template for telomeres elongation. Its secondary structure has domains with control functions of nucleotide insertion by TERT, sequence recognition and attachment of accessory proteins. The TER component of Leishmania telomerase (LeishTER) has recently been identified and partially characterized, but known is nothing about its biogenesis and its functions. The present work aims to verify the effects of the overexpression of LeishTER on parasite development, morphology and telomere maintenance. Preliminary results shown here demonstrated that we were able to clone the gene in a cloning vector and to subclone the gene in a Leishmania expression vector. The results also show that the overexpression of the TER has a dominant negative effect over parasite’s proliferation causing a rapid decline on population doubling inducing parasite death before they reach the stationary phase of growth. In addition, during the exponential growth there is a slight decrease in parasite telomere length. We conclude that the overexpression of LeishTER in L. major strongly affects parasite cell cycle probably by the impairment of telomere maintenance, although more studies are needed to elucidate the mechanisms and processes affected by the excess of this lncRNAs and why they are important to maintain parasite homeostasis.
Parasites of the Leishmania genus belong to the Trypanosomatide family, which present peculiar and particular characteristics. Among them are the species that cause leishmaniasis, a neglected tropical disease that can be expressed in three different clinical forms: cutaneous, mucocutaneous and visceral. Brazil is one of the most affected countries, due mainly to socioeconomic conditions, climate change and environmental alterations. Research related to the biology of Leishmania contributes to the understanding of the important physiological mechanisms of the parasite, and thus provide new therapeutic targets against the disease. The study of Leishmania telomeres appears promising since they related are to the genome stability. Telomeres are nucleoprotein structures located at the ends of the chromosomes and are responsible for protecting the chromosomes ensuring that the genetic information copied is correctly during cell duplication. DNA polymerase does not elongate telomeres as the rest of the genetic material, and thus maintained are by the action of a specialized reverse transcriptase named telomerase. Telomerase is a ribonucleoprotein minimally composed by two subunits, a protein with reverse transcriptase function TERT, and an RNA component (TER) that contains the telomeric repeat template sequence copied by TERT. Recent studies shown that TER has other functions besides being just a template for telomeres elongation. Its secondary structure has domains with control functions of nucleotide insertion by TERT, sequence recognition and attachment of accessory proteins. The TER component of Leishmania telomerase (LeishTER) has recently been identified and partially characterized, but known is nothing about its biogenesis and its functions. The present work aims to verify the effects of the overexpression of LeishTER on parasite development, morphology and telomere maintenance. Preliminary results shown here demonstrated that we were able to clone the gene in a cloning vector and to subclone the gene in a Leishmania expression vector. The results also show that the overexpression of the TER has a dominant negative effect over parasite’s proliferation causing a rapid decline on population doubling inducing parasite death before they reach the stationary phase of growth. In addition, during the exponential growth there is a slight decrease in parasite telomere length. We conclude that the overexpression of LeishTER in L. major strongly affects parasite cell cycle probably by the impairment of telomere maintenance, although more studies are needed to elucidate the mechanisms and processes affected by the excess of this lncRNAs and why they are important to maintain parasite homeostasis.
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Palavras-chave
Leishmania major, Leishmaniose, RNA telomerase (LeishTER), Superexpressão, Leishmaniasis, Telomerase RNA, Super expression