The ChREBP/Mondo-Mlx transcription factors are activated by sugars and are essential for sugar tolerance. They promote the conversion of sugars to lipids, but beyond this, their physiological roles are insufficiently understood. Here, we demonstrate that in an organism-wide setting in Drosophila, Mondo-Mlx controls the majority of sugar-regulated genes involved in nutrient digestion and transport as well as carbohydrate, amino acid, and lipid metabolism. Furthermore, human orthologs of the Mondo-Mlx targets display enrichment among gene variants associated with high circulating triglycerides. In addition to direct regulation of metabolic genes, Mondo-Mlx maintains metabolic homeostasis through downstream effectors, including the Activin ligand Dawdle and the Gli-similar transcription factor Sugarbabe. Sugarbabe controls a subset of Mondo-Mlx-dependent processes, including de novo lipogenesis and fatty acid desaturation. In sum, Mondo-Mlx is a master regulator of other sugar-responsive pathways essential for adaptation to a high-sugar diet.

OSBP (oxysterol-binding protein) homologues, ORPs (OSBP-related proteins), constitute a 12-member family in mammals. We employed an in vitro [3H]25OH (25-hydroxycholesterol)-binding assay with purified recombinant proteins as well as live cell photo-cross-linking with [3H]photo-25OH and [3H]photoCH (photo-cholesterol), to investigate sterol binding by the mammalian ORPs. ORP1 and ORP2 [a short ORP consisting of an ORD (OSBP-related ligand-binding domain) only] were in vitro shown to bind 25OH. GST (glutathione S-transferase) fusions of the ORP1L [long variant with an N-terminal extension that carries ankyrin repeats and a PH domain (pleckstrin homology domain)] and ORP1S (short variant consisting of an ORD only) variants bound 25OH with similar affinity (ORP1L, K(d)=9.7x10(-8) M; ORP1S, K(d)=8.4 x10(-8) M), while the affinity of GST-ORP2 for 25OH was lower (K(d)=3.9x10(-6) M). Molecular modelling suggested that ORP2 has a sterol-binding pocket similar to that of Saccharomyces cerevisiae Osh4p. This was confirmed by site-directed mutagenesis of residues in proximity of the bound sterol in the structural model. Substitution of Ile249 by tryptophan or Lys150 by alanine markedly inhibited 25OH binding by ORP2. In agreement with the in vitro data, ORP1L, ORP1S, and ORP2 were cross-linked with photo-25OH in live COS7 cells. Furthermore, in experiments with either truncated cDNAs encoding the OSBP-related ligand-binding domains of the ORPs or the full-length proteins, photo-25OH was bound to OSBP, ORP3, ORP4, ORP5, ORP6, ORP7, ORP8, ORP10 and ORP11. In addition, the ORP1L variant and ORP3, ORP5, and ORP8 were cross-linked with photoCH. The present study identifies ORP1 and ORP2 as OSBPs and suggests that most of the mammalian ORPs are able to bind sterols.

Oxysterol binding protein-related protein 2 (ORP2) is a member of the oxysterol binding protein family, previously shown to bind 25-hydroxycholesterol and implicated in cellular cholesterol metabolism. We show here that ORP2 also binds 22(R)-hydroxycholesterol [22(R)OHC], 7-ketocholesterol, and cholesterol, with 22(R)OHC being the highest affinity ligand of ORP2 (K d 1.4 3 10 28 M). We report the localization of ORP2 on cytoplasmic lipid droplets (LDs) and its function in neutral lipid metabolism using the human A431 cell line as a model. The ORP2 LD association depends on sterol binding: Treatment with 5 mM 22(R)OHC inhibits the LD association, while a mutant defective in sterol binding is constitutively LD bound. Silencing of ORP2 using RNA interference slows down cellular triglyceride hydrolysis. Furthermore, ORP2 silencing increases the amount of (5), and inhibit the processing of sterol regulatory element binding proteins (SREBPs) via binding to the Insig proteins, which retain SREBP/SCAP complexes in the endoplasmic reticulum (ER) (6). The cytosolic oxysterol receptor, oxysterol binding protein (OSBP), was identified in the 1980s (7). Families of OSBP-related proteins (ORPs) have recently been identified in practically all eukaryotic organisms studied (8). Most of the information on the ORP proteins has been obtained using yeast (Saccharomyces cerevisiae) or mammalian cells. The yeast ORPs (Osh proteins) are suggested to play major roles in the intracellular transport of sterols (9), in vesicle budding from the Golgi apparatus (10, 11), and in the establishment of cell polarity (12, 13), while mammalian ORPs have been suggested to participate in the regulation of lipid metabolism, vesicle transport, and cellular signaling (8).All ORPs contain in their C-terminal part a structure designated OSBP-related domain (ORD), which is homologous to the oxysterol binding domain of OSBP (8). In addition to the ORD, most ORPs contain an N-terminal region involved in their subcellular targeting. The N-terminal extensions containing a pleckstrin homology domain target ORP1L Abbreviations: 22(R)OHC, 22(R)-hydroxycholesterol; 25OHC, 25-hydroxycholesterol; CE, cholesteryl ester; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; FCS, fetal calf serum; FFAT, two phenylalanines in an acidic tract; GST, glutathione S-transferase; LD, lipid droplet; mab, monoclonal mouse antibody; mbCD, methyl-b-cyclodextrin; ORD, oxysterol binding protein-related domain; ORP, oxysterol binding protein-related protein; OSBP, oxysterol binding protein; siRNA, short interfering RNA; SREBP, sterol regulatory element binding protein; TG, triglyceride.