Phosphatidylinositol transfer proteins are highly conserved, soluble eukaryotic proteins that binds reversibly one molecule of phosphatidylinositol (PtdIns), phosphatidylcholine (PtdCho), or sphingomyelin (CerPCho). An in vitro activity of transferring the structurally diverse glycerophospholipids PtdIns and PtdCho between a variety of membranes was described over 30 years ago (University of Utrecht, Utrecht NL) and served as a means of quantitating "catalytic" activity and monitoring purification of proteins from bovine brain and liver.
Typically, the rate at which a "tagged" phospholipid (radiolabelled, fluorescent, spin-labeled) moves through the aqueous phase from a donor membrane to an acceptor membrane is markedly enhanced in the presence of PtdIns transfer proteins These proteins are, therefore, capable of extracting phospholipids from and inserting them into a membrane surface. Evidence from the laboratories of Shamshad Cockcroft (University College, London UK) and Thomas Martin (University of Wisconsin) supports the participation of PtdIns transfer proteins in signal transduction processes that proceed through the phosphorylation and hydrolysis of phosphoinositides.
In the past few years my laboratory has been investigating structure-function relationships of the recombinant rat and human PtdIns transfer protein isoforms PITPα and PITPβ. Much of my current research is being performed in collaboration with the laboratories of Lynwood Yarbrough (University of Kansas Medical Center) and Marilyn Yoder (University of Missouri - Kansas City). Our physical characterization studies are being carried out on recombinant and crystallized PtdIns transfer protein isoforms and a variety of structural mutants.
Recent experimental results from a number of laboratories suggest that PtdIns transfer protein participates in several important cellular pathways. In both yeasts and higher eukaryotic cells the trafficking of vesicles among intracellular organelles and secretion (exocytosis) at the plasma membrane appear to require PtdIns transfer protein. In this process, the protein may function as a regulatory molecule or may be needed to modify the lipid composition of specific membrane surfaces. PtdIns transfer protein similarly plays a part in those signal transduction pathways linked to the metabolism of phosphoinositides, most likely in directing substrate PtdIns to membrane-bound kinases.
Cellular activities of PtdIns transfer protein are being evaluated by regulating the expression of the protein in cultured cells. Conditions to increase or decrease the basal level of protein have been developed for hepatoma and fibroblast cell lines. Tools used are transient and stable transfection with constituitive and regulated expression vectors encoding either human or rat PtdIns transfer protein and subcellular localization of the protein by biochemical and immunological techniques.
Phenotypic changes being monitored include the secretion of lipoproteins, the integrity of lipid-mediated signal transduction pathways, and progression through the cell cycle and differentiation. While an overexpression of the protein appears to have little effect on cell function, a significant decrease in the cellular level of PtdIns transfer protein is accompanied by a loss of cell viability.
A particularly fascinating aspect of my research deals with the association of PtdIns transfer protein with its monomeric phospholipid. We have demonstrated that PtdIns transfer protein still retains a remarkable interaction with phospholipid in the presence of denaturants such as guanidinium chloride and can only be "stripped" of this lipid by chromatography on lipophilic matrices. With both apo- and holo-species, refolding and renaturation to catalytically active proteins occurs rapidly and efficiently.
While productive refolding does take place in the absence of phospholipid, achievement of the native conformation requires association with a transferable phospholipid molecule. Measurements that monitor the fluorescent and circular dichroic spectral properties suggest a critical role of the phospholipid in the generation and maintenance of the structural integrity of PtdIns transfer protein.
Our research took a rather surprising direction following efforts to subject PtdIns transfer protein to proteolytic digestion. The protein alone is remarkably resistant to attack by trypsin, chymotrypsin, and other proteases. Similarly, if phospholipid vesicles to which the protein binds only weakly are mixed with PtdIns transfer protein and a proteolytic enzyme, little digestion is seen. But when phospholipid vesicles to which the protein binds strongly, as shown by chromatographic analysis and transfer inhibition studies, are added, a region of the C-terminus is exposed to rapid proteolysis.
If trypsin is used, two large truncated products are generated, catalytic activity is lost, and the protein becomes irreversibly "fixed" to the membrane surface. These observations suggested that the transient interactions with membranes, which must occur as the protein donates and accepts phospholipids, may be modulated by a specific region of PtdIns transfer protein. A bacterial expression system permitted the efficient production of native and truncated proteins whose functional and structural properties were studied. Coupled with subsequent x-ray crystallographic data, this specific region of the protein is now known to consist of about thirty amino acid residues, folded into the long α-helix and a short unstructured segment at the C-terminus.
Structure-function relationships of PtdIns transfer proteins have been investigated by comparison of primary sequences and secondary structural motifs and the identification of specific amino acid residues in the binding domain for phospholipid ligands and in the domain for protein-protein or protein-lipid surface interactions. Comparision of the lipid binding domains of the rat PtdIns transfer isoforms revealed several differences that were then exploited by point mutation. One locus, Phe225α/Leu224β, is common to rodents and appears to be critical for the modest isoform differences toward PtdIns, PtdCho, and CerPCho. Interestingly, although other vertebrate PtdIns transfer proteins exhibit only Phe at this locus, they still have some significant transfer differences.
In all metazoan PtdIns transfer proteins there is a short region of α-helix: 7 amino acids, one of which is Arg, Lys, or Gln, that are bracketed by the absolutely conserved Pro70 and Pro78 residues. We hypothesize that this helix, found near the 'opening' of the lipid binding domain, may insert into the acceptor membrane to promote an exchange of protein-bound and membrane-bound lipid molecules. These approaches have provided information about the mechanism by which PtdIns transfer protein functions in vitro as well as in the cellular environment.
The three-dimensional structure of the alpha isoform of rat phosphatidylinositol transfer protein complexed with one molecule of phospholipid has been determined by x-ray diffraction techniques. Multiple anomolous dispersion of seleno-methionine-substituted protein crystals yielded data to a resolution of 2.2 angstroms.
A seven-strand beta-sheet and several long alpha-helices define an enclosed internal cavity, in which a single molecule of sn-1,2-dioleoyl-phosphatidylcholine, one of its physiological ligands, is accommodated with its polar head group in the center of the protein and fatty acyl chains projected toward the surface. Other structural features suggest mechanisms by which cytosolic phosphatidylinositol transfer protein interacts with membranes for lipid exchange and associates with a variety of lipid and protein kinases.
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