Stard4 and stard5new players in intracellular cholesterol metabolism

  1. Calderón Domínguez, María
Dirigida per:
  1. Daniel Rodríguez Agudo Director/a
  2. Miguel Ángel Medina Torres Director/a

Universitat de defensa: Universidad de Málaga

Fecha de defensa: 08 de de març de 2013

Tribunal:
  1. Ramón Muñoz Chápuli President/a
  2. Raúl Montañez Martínez Secretari/ària
  3. Laura Herrero Rodríguez Vocal
  4. Luca Galluzzi Vocal
  5. Francisca Sánchez Jiménez Vocal

Tipus: Tesi

Teseo: 339504 DIALNET

Resum

Cholesterol is a structural component of all mammalian cell membranes, as well as a precursor of bile acid and steroid hormones. The regulation of the levels of this key molecule is vital for the proper function of cells and organs. Although cholesterol metabolism is well known in many aspects, the mechanisms of intracellular cholesterol transport and the responsible protein carriers remain poorly understood. Among these protein carriers are the members of the START domain superfamily of proteins, which are characterized by a similar lipid binding domain. START domain proteins bind different ligands or cholesterol derivatives based on modifications of that binding domain, which also appears in a wide range of proteins involved in other cellular functions, including lipid transport and metabolism, signal transduction and transcriptional regulation. Some of the START domain proteins have been shown to participate in several pathways of intracellular traffic of lipids, including cholesterol. This is the case of StarD4 and StarD5, members of the StarD4 subfamily of START domain proteins, with a characteristic lipid binding pocket specific for cholesterol, similar to StarD1, the first START domain protein identified and the best studied of this family of proteins until now. However, the function and regulation of StarD4 and StarD5 proteins remain still unclear. Therefore, the objective of this thesis was to define StarD4 and StarD5 regulation and subcellular localization in order to pursue their functions. First, StarD4 was studied by immunoblot, showing that it is highly expressed in the mouse fibroblast cell line 3T3-L1, in human THP-1 macrophages, Kupffer cells (liver macrophages), hepatocytes and in various other tissues. StarD4 protein was found to be highly regulated by sterol levels, and appeared localized to the cytoplasm and the endoplasmic reticulum (ER) of cells. More specifically, in THP-1 macrophages StarD4 co-localized to areas of the ER enriched in Acetyl-CoA acetyltransferase 1 (ACAT-1), and was closely associated with lipid droplets. In a follow up study, StarD4¿s ability to transfer cholesterol to ACAT-1 was determined in vitro with purified StarD4 recombinant protein, leading to a 2-fold increase in ACAT activity, indicating that StarD4 could serve as a cholesterol carrier to ACAT-1. The initial studies on StarD5 focused on the regulation of the gene. StarD5 was found to be highly regulated/induced in conditions of ER stress, which also led to an increase in intracellular free cholesterol correlated with an increase in HMG-CoA reductase expression. Despite this, the molecular mechanisms of this induction were not known, and therefore mechanistic studies were performed using ER stress signal transducers. These studies demonstrated that the activation of StarD5 expression was mediated by the transcriptional ER stress factor XBP-1, and also revealed that StarD5 mRNA was stabilized during ER stress. The immunocytochemistry studies performed revealed that StarD5 protein was mainly localized to the nuclear region in 3T3-L1 cells but, upon induction of ER stress, it redistributed to the cytosol and membranes of the cells. In summary, the results here described provide strong evidence for StarD4 and StarD5 as highly regulated, non-vesicular, directional, intracellular cholesterol transporters. More specifically, StarD4 could play a role in the distribution of cholesterol to the ER and the synthesis of cholesteryl esters. In the case of StarD5, the results point toward a role for StarD5 in the redistribution of cholesterol during the cell protective phase of the ER stress response.