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Knock-Out of ACBD3 Leads to Dispersed Golgi Structure, but Unaffected Mitochondrial Functions in HEK293 and HeLa Cells.

ABSTRACT: The Acyl-CoA-binding domain-containing protein (ACBD3) plays multiple roles across the cell. Although generally associated with the Golgi apparatus, it operates also in mitochondria. In steroidogenic cells, ACBD3 is an important part of a multiprotein complex transporting cholesterol into mitochondria. Balance in mitochondrial cholesterol is essential for proper mitochondrial protein biosynthesis, among others. We generated ACBD3 knock-out (ACBD3-KO) HEK293 and HeLa cells and characterized the impact of protein absence on mitochondria, Golgi, and lipid profile. In ACBD3-KO cells, cholesterol level and mitochondrial structure and functions are not altered, demonstrating that an alternative pathway of cholesterol transport into mitochondria exists. However, ACBD3-KO cells exhibit enlarged Golgi area with absence of stacks and ribbon-like formation, confirming the importance of ACBD3 in Golgi stacking. The glycosylation of the LAMP2 glycoprotein was not affected by the altered Golgi structure. Moreover, decreased sphingomyelins together with normal ceramides and sphingomyelin synthase activity reveal the importance of ACBD3 in ceramide transport from ER to Golgi.

SUBMITTER: Danhelovska T

PROVIDER: S-EPMC8303370 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:FIP200 (focal adhesion kinase family interacting protein of 200 kDa) has been shown to interact with other proteins to regulate several intracellular signaling pathways. To study a potential role of FIP200 in tumorigenesis and possibly other disease processes in vivo, we created and analyzed murine mammary tumor virus-Cre-mediated FIP200 conditional knock-out (CKO) mice. We found that deletion of FIP200 in mammary epithelial cells did not result in spontaneous development of breast cancer. Moreover, deletion of FIP200 did not further accelerate or inhibit lymphomagenesis induced by inactivation of p53 in mice. Interestingly, however, FIP200 and p53 double conditional knock-out (dCKO) mice exhibited significant hyperplasia of epidermis (acanthosis), thickening of the cornified layer (hyperkeratosis), and increased vascularity in the dermis. FIP200 CKO mice also showed similar, although less severe, skin defects as dCKO mice. Analyses of primary keratinocytes isolated from dCKO mice did not detect increased proliferation of these cells in vitro, suggesting that epidermis hyperproliferation is not epidermal cell-autonomous but may be a consequence of increased inflammation triggered by immune cells in vivo. Consistent with this possibility, we found infiltration of leukocytes including T cells, macrophages, and granulocytes into the dermis and epidermis, associated with activation of NF-kappaB and increased expression of several proinflammatory cytokines and chemokines in skin of the dCKO mice. We further found that cultured FIP200 KO keratinocytes showed reduced NF-kappaB phosphorylation in response to tumor necrosis factor alpha stimulation, suggesting a paracrine regulation of aberrant NF-kappaB activation in the skin microenviroment of dCKO and FIP200 CKO mice. Together, these results demonstrate that ablation of FIP200, although not promoting tumorigenesis, can lead to skin inflammatory disorders, suggesting a novel function of FIP200 in the maintenance of normal skin homeostasis in vivo.

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Knock-Out of ACBD3 Leads to Dispersed Golgi Structure, but Unaffected Mitochondrial Functions in HEK293 and HeLa Cells.

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