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Estudo da Interação do Exão-1 da Huntingtina com Membranas Lipídicas
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Resumo(s)
A doença de Huntington (DH) é uma desordem neurodegenerativa rara associada a uma expansão
anormal da repetição de poliglutaminas (poliQ) no exão-1 da proteína huntingtina (Htt). Expansões
acima de um limite crítico de 37 resíduos de glutamina (37Q) resultam na formação de fibras do tipo
amilóide ou de corpos de inclusão intranucleares. Quando ocorre o splicing aberrante do gene HTT
mutado ou a clivagem proteolítica das proteínas poliQ expandidas são gerados fragmentos altamente
tóxicos do exão-1 da Htt (Httex1), capazes de reproduzir a patologia de DH. Há vários estudos que
indicam que as membranas biológicas podem desencadear o misfolding/agregação do fragmento Httex1
e que as regiões adjacentes do poliQ – segmento N17 e a região rica em prolinas (PRR) – regulam a sua
ligação à membrana.
Este trabalho focou-se na avaliação da importância relativa das interações hidrofóbicas e
eletrostáticas na ligação do fragmento Httex1-23Q a lipossomas preparadas com uma composição
lipídica variável através da realização de medidas de fluorescência em estado estacionário e resolvidas
no tempo. A interação do fragmento Httex1-23Q com membranas lipídicas aniónicas foi estudada
mediante a sua marcação específica na posição A2C (no segmento N17) ou A82C (no C-terminal da
região PRR) com acrilodano, uma sonda de polaridade fluorescente. Os resultados obtidos confirmaram
que a ligação do fragmento Httex1-23Q é mediada através do seu segmento N17, que fica ancorado na
superfície da membrana, enquanto a região PRR permanece exposta à solução aquosa. A ligação às
membranas do fragmento Httex1-23Q-A2C-acrilodano tem uma componente eletrostática moderada
pois a sua partição para os lipossomas (i) aumentou apenas ligeiramente com o aumento do seu conteúdo
em fosfolípidos aniónicos, e (ii) era essencialmente independente da força iónica do tampão. A alteração
da curvatura das membranas através da variação do diâmetro das vesículas também teve um efeito
negligenciável nas propriedades de partição do fragmento Httex1-23Q-A2C-acrilodano.
Huntington's disease (HD) is a rare neurodegenerative disorder associated to the abnormal expansion of the polyglutamine (polyQ) repeat in the exon-1 of the huntingtin (Htt) protein. Expansions above a critical threshold of about 37 glutamine residues (37Q) induce the formation of “amyloid-like” fibrils or intranuclear inclusion bodies. When the aberrant splicing of the mutant HTT gene or the proteolytic cleavage of expanded polyQ proteins occur, highly toxic fragments of Htt exon-1 (Httex1) are generated, capable of reproducing the pathology of HD. Several studies support that biological membranes can trigger Httex1 misfolding/aggregation and suggest that the flanking polyQ regions - N17 segment and proline-rich region (PRR) - regulate its membrane binding. This work focused on using steady-state and time-resolved fluorescence measurements to evaluate the relative contribution of hydrophobic and electrostatic interactions to the binding of Httex1-23Q fragment towards liposomes prepared with a variable lipid composition. The interaction of Httex1-23Q fragment with anionic lipid membranes was first studied by its site-specific fluorescently-labeling at position A2C (at the N17 segment) or A82C (at the C-terminal of the PRR region) with acrylodan, a fluorescent polarity probe. Our fluorescence data confirms that membrane binding of Httex1-23Q is mediated via its N17 segment, which is anchored at the lipid surface, while the PRR region remains highly exposed to the aqueous solution. The membrane binding of Httex1-23Q-A2C-acrylodan fragment was found to have a moderate electrostatic component, since its partition towards the liposomes (i) only slightly increased upon raising the anionic phospholipid content of the vesicles, and (ii) was essentially independent of the ionic strength of the buffer. Finally, changing the membrane curvature by varying the vesicle diameter from 50 to 100 nm had a negligible effect on the partition properties of Httex1- 23Q-A2C-acrylodan.
Huntington's disease (HD) is a rare neurodegenerative disorder associated to the abnormal expansion of the polyglutamine (polyQ) repeat in the exon-1 of the huntingtin (Htt) protein. Expansions above a critical threshold of about 37 glutamine residues (37Q) induce the formation of “amyloid-like” fibrils or intranuclear inclusion bodies. When the aberrant splicing of the mutant HTT gene or the proteolytic cleavage of expanded polyQ proteins occur, highly toxic fragments of Htt exon-1 (Httex1) are generated, capable of reproducing the pathology of HD. Several studies support that biological membranes can trigger Httex1 misfolding/aggregation and suggest that the flanking polyQ regions - N17 segment and proline-rich region (PRR) - regulate its membrane binding. This work focused on using steady-state and time-resolved fluorescence measurements to evaluate the relative contribution of hydrophobic and electrostatic interactions to the binding of Httex1-23Q fragment towards liposomes prepared with a variable lipid composition. The interaction of Httex1-23Q fragment with anionic lipid membranes was first studied by its site-specific fluorescently-labeling at position A2C (at the N17 segment) or A82C (at the C-terminal of the PRR region) with acrylodan, a fluorescent polarity probe. Our fluorescence data confirms that membrane binding of Httex1-23Q is mediated via its N17 segment, which is anchored at the lipid surface, while the PRR region remains highly exposed to the aqueous solution. The membrane binding of Httex1-23Q-A2C-acrylodan fragment was found to have a moderate electrostatic component, since its partition towards the liposomes (i) only slightly increased upon raising the anionic phospholipid content of the vesicles, and (ii) was essentially independent of the ionic strength of the buffer. Finally, changing the membrane curvature by varying the vesicle diameter from 50 to 100 nm had a negligible effect on the partition properties of Httex1- 23Q-A2C-acrylodan.
Descrição
Tese de mestrado, Bioquímica (Bioquímica), 2022, Universidade de Lisboa, Faculdade de Ciências
Palavras-chave
Doença de Huntington huntingtina membranas biológicas interação proteína-lípido espectroscopia de fluorescência Teses de mestrado - 2023