Identification And Functional Studies Of OSBP-related Proteins Family Interactome

Protein families characterized by a ligand binding domain related to that of oxysterol binding protein (OSBP) have been identified in eukaryotic species from yeast to humans. These proteins, designated OSBP-related (ORPs) or OSBP-likeproteins (OSBPL), have been implicated in various cellular functions. Data from our and other laboratories suggest that binding of sterol ligands may be a unifying theme. Work with Saccharomyces cerevisiae ORPs suggests a function of these proteins in the nonvesicular intracellular transport of sterols, in secretory vesicle transport from the Golgi complex, and in the establishment of cell polarity. Mammals have more ORP genes, and differential splicing substantially increases the complexity of the encoded protein family. Functional studies on mammalian ORPs point in different directions:integration of sterol and sphingomyelin metabolism, sterol transport, regulation of neutral lipid metabolism, control of the microtubule-dependent motility of endosomes/ lysosomes, and regulation of signaling cascades. However, the detailed mechanisms of their action have remained elusive. An important way of learning protein function is to analyze their interacting-protein partners. In this study, the interacting proteins of ORPs family have be screened by Yeast Two-Hybrid using Human Fetal Kidney cDNA library and Mate & Plate Library-Universal Human (Normalized) which used cDNA from a wide spectrum of human tissues. Results showed ORPs interacted with proteins involved in various cellular functions, including signaling transduction, lipid metabolism, vesicle transport, nuclear transport, ion transport, blood coagulation, carbohydrate metabolism, protein synthesis and protein degradation etc. Interesting interactions have been chosen and studied further.The interaction of ORP8 and NUP62 has been confirmed by GST-Pull down and Co-IP. Moderate adenovirus-mediated overexpression of human ORP8 in mouse liver induced a decrease of cholesterol, phospholipids, and triglycerides in serum(-34%,-26%,-37%, respectively) and liver tissue (-40%,-12%,-24%, respectively), coinciding with reduction of hepatic nuclear (n)SREBP-1 and-2. Consistently, excess ORP8 reduced nSREBPs in HuH7 cells, and ORP8 overexpression or silencing suppressed or induced the expression of SREBP-2 target genes, respectively. The impact of overexpressed ORP8 on nSREBPs was shown to require a normal level of Nup62. Together, these experiments showed a new mechanism that ORP8 regulates cellular cholesterol homeostasis.Further more, we showed that ORP9 and ORP11 formed dimer with Co-IP experiment. We carry out characterization of ORP11, a previously unexplored ORP family member which is devoid of a FFAT motif or other known ER targeting signal. Using an affinity-purified antiserum generated during this study, we report the tissue and cell-type specific expression patterns of this new protein and demonstrate that it distributes in cells between Golgi and late endosome membranes. Overexpressed ORP9 recruited EGFP-ORP11 to endoplasmic reticulum and Golgi, and ORP9 silencing inhibited ORP11 Golgi association. The results identify ORP11 as an OSBP homologue distributing at the Golgi-LE interface and define the ORP9-ORP11 dimer as a functional unit that may act as an intracellular lipid sensor or transporter. We also confirmed the interaction of ORP4 and IP3 receptor(ITPR1) by Co-IP and Bimolecular Fluorescence Complementation (BiFC) experiment. ITPR1 is a Ca2+ channel localized in the membrane of Endoplasmic reticulum. Our primary data showed that the silencing of ORP4 in HeLa cell reduced Histamine-induced calcium release and enhanced apoptosis after treatment of thapsigargin.