Dissecting the intracellular cholesterol transport in Tetrahymena thermophila.

COSTA J; GABRIELLI M; UTTARO AD; HERNÁNDEZ J

Salta

Congreso; LV Reunión anual de la SAIB y XIV congreso PABMB; 2019

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB) y Panamerican Association of Biochemistry and Molecular Biology (PABMB)

The ciliated protozoa Tetrahymena thermophila has not sterol requirements, but when available, they are incorporated and converted to 7,22-bisdehydrocholesterol, replacing the endogenous sterol surrogate, tetrahymanol, in the cell membranes. In this process, genes involved in sterolbioconversion, such as sterol C22 desaturase (Des22), are induced whereas tetrahymanol synthesis genes, like squalene synthase (SqS), arerepressed. Although phagocytosis through the oral apparatus is the main route for sterol uptake, we have previously found that there is at least asecond internalization pathway, and furthermore, some genes responded differently depending on the entry mechanism. However, the details ofthe intracellular traffic of sterols in T. thermophila and the associated signaling pathways involved in the regulation of gene expression have notyet been established. Using radiolabeled cholesterol and following its incorporation and esterification by thin-layer chromatography, we havepreviously observed that U18666A, an inhibitor of the export of sterols from lysosomes to the endoplasmic reticulum, impaired cholesterolesterification. This effect was not due to inhibition of ACAT activity since no differences in cholesteryl esters levels were detected in assays withcell-free extracts, therefore indicating that sterol transport to endoplasmic reticulum was indeed affected. In addition, the time-course analysis ofgene expression upon treatment with U18666A revealed a delay in the cholesterol-induced upregulation of Des22 and an absence of SqS repression.When cells were treated with the Golgi disrupting agent Brefeldin A neither cholesterol uptake nor its esterification were affected, but, interestingly,RT-qPCR data showed higher levels of Des22 mRNA and a further downregulation of SqS. Together, these results suggest that cholesterol reachesthe endoplasmic reticulum where it is modified and from where it triggers signaling pathways leading to changes in gene expression.