Protein-protein interactions are the key to organizing cellular processes in space and time. The only direct way to identify such interactions in their cellular environment is by photo-crosslinking. Here we present a new strategy for photo-cross-linking proteins in living cells. We designed two new photoactivatable amino acids that we termed photo-methionine and photo-leucine based on their structures and properties closely resembling the natural amino acids methionine and leucine, respectively. This similarity allows them to escape the stringent identity control mechanisms during protein synthesis and be incorporated into proteins by the unmodified mammalian translation machinery. Activation by ultraviolet light induces covalent cross-linking of the interacting proteins, which can be detected with high specificity by simple western blotting. Applying this technology to membrane protein complexes, we discovered a previously unknown direct interaction of the progesterone-binding membrane protein PGRMC1 with Insig-1, a key regulator of cholesterol homeostasis.

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.

Protein families related to OSBP (oxysterol-binding protein) are present in eukaryotes from yeast to human. The functions of the ORPs (OSBP-related proteins) have remained largely enigmatic. Even though they have been implicated in the function of ERJs (endoplasmic reticulum junctions), it is evident that any single model for their mechanism of action is insufficient. The existing evidence points in many different directions, such as integration of sterol and sphingomyelin metabolism, regulation of neutral lipid metabolism, control of signalling cascades, regulation of secretory vesicle generation, and function in the microtubule-based motility of endo/lysosomes. Some of these functions could involve ERJ and non-vesicular transport of lipids, but this is unlikely to be the unifying feature. We believe, rather, that the common denominator for ORP function is acting as sterol sensors that relay information to a spectrum of cellular processes.

BackgroundBilharzia is one of the major parasitic infections affecting the public health and socioeconomic circumstances in (sub) tropical areas. Its causative agents are schistosomes. Since these worms remain in their host for decades, they have developed mechanisms to evade or resist the immune system. Like several other parasites, their surface membranes are coated with a protective layer of glycoproteins that are anchored by a lipid modification.Methods and FindingsWe studied the release of glycosyl-phosphatidylinositol (GPI)-anchored proteins of S. mansoni and found them in the circulation associated with host lipoprotein particles. Host cells endocytosed schistosomal GPI-anchored proteins via their lipoprotein receptor pathway, resulting in disturbed lysosome morphology. In patients suffering from chronic schistosomiasis, antibodies attacked the parasite GPI-anchored glycoproteins that were associated with the patients' own lipoprotein particles. These immunocomplexes were endocytosed by cells carrying an immunoglobulin-Fc receptor, leading to clearance of lipoproteins by the immune system. As a consequence, neutral lipids accumulated in neutrophils of infected hamsters and in human neutrophils incubated with patient serum, and this accumulation was associated with apoptosis and reduced neutrophil viability. Also, Trypanosoma brucei, the parasite that causes sleeping sickness, released its major GPI-anchored glycoprotein VSG221 on lipoprotein particles, demonstrating that this process is generalizable to other pathogens/parasites.ConclusionsTransfer of parasite antigens to host cells via host lipoproteins disrupts lipid homeostasis in immune cells, promotes neutrophil apoptosis, may result in aberrant antigen presentation in host cells, and thus cause an inefficient immune response against the pathogen.