Characterization of a novel CREBH-induced protein FACI and its involvement in clathrin-mediated endocytosis

Abstract

Cyclic AMP-responsive element-binding protein H (CREBH) is a transcriptional factor enriched in liver and small intestine, where it plays a crucial role in glucose and lipid metabolism. FLJ22675 or C11orf86, an uncharacterized novel target gene of CREBH, was identified via a comparative analysis of differentially expressed genes in the liver of wild-type and Crebh-/- mice. Previous studies have shown the expression of this novel CREBH target is highly induced by fasting and CREBH. Therefore, the gene was renamed as fasting- and CREBH-induced protein (FACI). However, details of its biochemical properties, regulation and functions are still poorly understood. In this study, FACI was systematically analyzed and characterized in different aspects. FACI is a small protein with 115 amino acids which can be divided into two parts, an N-terminal intrinsically disordered region and a C-terminal α-helix region, according to the structure. By multiple alignments among FACI homologs, 5 regions were identified evolutionarily conserved and named as Motifs A to E. FACI is specifically enriched in the small intestine and the liver. Subcellularly, FACI is mainly localized on the plasma membrane and in the recycling endosome compartments. FACI transcription is regulated by nutrient-responsive transcription factors CREB-H, SREBP, HNF4α, CREB and PGC1α. The motif E of FACI is composed of an amphipathic helix and a hydrophobic region, which contributes to FACI’s high affinity to membrane PI(4,5)P2 and its plasma membrane localization. Interactome analysis revealed that FACI had high proximity to a group of proteins in the clathrin-mediated endocytosis (CME) pathway. TIRFM image showed FACI’s colocalization with clathrin-coated pits on the plasma membrane, indicating the participation of FACI in the CME process. Immunoprecipitation results demonstrated the direct interaction between FACI and AP2 complex through the DxxxLI motif (Motif D) of FACI. Mass spectrometry result showed two phosphorylation sites on FACI, S9p on Motif A and S37p on Motif C. By kinase assay screening, PKCα was found to be responsible for this phosphorylation. Activation of PKC by PMA treatment dissociated FACI from clathrin-coated pits. And mutated form of motif A or C blocked PMA-induced FACI dissociation from CCPs. FACI was found to colocalize with LDL receptor both on the plasma membrane and in the cytoplasm. In vitro FACI knockout and overexpression experiments in cultured cells showed altered cellular LDL uptake comparing to WT cells, indicating that FACI can promote LDLR endocytosis. Importantly, diet-induced hypercholesterolemia was exacerbated in Faci-/- mice and alleviated in AAV-induced liver-specific FACI overexpressed mice. Taken together, my work identified and characterized a novel protein involved in CME and cellular LDL uptake. The elucidation of its exact function in LDL uptake may reveal novel targets for the prevention and treatment of dyslipidemia.