ABSTRACT
Phosphatidylinositol transfer protein (PITP) is a ubiquitous eukaryotic protein that preferentially binds either phosphatidylinositol or phosphatidylcholine and catalyzes the exchange of these lipids between membranes. Mammalian cytosolic PITPs include the ubiquitously expressed PITPalpha and PITPbeta isoforms (269-270 residues). The crystal structure of rat PITPbeta complexed to dioleoylphosphatidylcholine was determined to 2.18 A resolution with molecular replacement using rat PITPalpha (77% sequence identify) as the phasing model. A structure comparison of the alpha and beta isoforms reveals minimal differences in protein conformation, differences in acyl conformation in the two isoforms, and remarkable conservation of solvent structure around the bound lipid. A comparison of transfer activity by human and rat PITPs, using small unilamellar vesicles with carefully controlled phospholipid composition, indicates that the beta isoforms have minimal differences in transfer preference between PtdIns and PtdCho when donor vesicles contain predominantly PtdCho. When PtdCho and PtdIns are present in equivalent concentrations in donor vesicles, PtdIns transfer occurs at approximately 3-fold the rate of PtdCho. The rat PITPbeta isoform clearly has the most diminished transfer rate of the four proteins studied. With the two rat isoforms, site-directed mutations of two locations within the lipid binding cavity that possess differing biochemical properties were characterized: I84alpha/F83beta and F225alpha/L224beta. The 225/224 locus is more critical in determining substrate specificity. Following the mutation of this locus to the other amino acid, the PtdCho transfer specific activity became PITPalpha (F225L) approximately PITPbeta and PITPbeta (L224F) approximately PITPalpha. The 225alpha/224beta locus plays a modest role in the specificity of both isoforms toward CerPCho.
Subject(s)
Phosphatidylcholines/chemistry , Phospholipid Transfer Proteins/chemistry , Phospholipids/metabolism , Amino Acid Sequence , Animals , Conserved Sequence , Crystallography, X-Ray , Humans , Models, Molecular , Mutagenesis, Site-Directed , Phosphatidylcholines/metabolism , Phospholipid Transfer Proteins/genetics , Phospholipid Transfer Proteins/metabolism , Phospholipids/chemistry , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , RatsABSTRACT
Pectate lyase A (PelA) is a pectate-degrading enzyme secreted by plant pathogens. PelA from Erwinia chrysanthemi has 61% amino-acid identity and a conserved structural similarity to pectate lyase E (PelE). Although similar in structure and sequence, the enzymatic characteristics of PelA differ from those for PelE. A structural alignment of PelA and PelE reveals differences in the T1.5 loop. The sequence of the T1.5 loop in PelA was mutated to the homologous sequence in PelE. The crystal structure of the PelA T1.5 mutant has been solved to 1.6 and 2.9 A resolution. The enzymatic and structural properties of the T1.5 mutant are discussed.
Subject(s)
Dickeya chrysanthemi/enzymology , Mutation , Polysaccharide-Lyases/chemistry , Bacterial Proteins/chemistry , Binding Sites , Cloning, Molecular , Crystallization , Hydrogen-Ion Concentration , Polysaccharide-Lyases/genetics , Polysaccharide-Lyases/isolation & purification , Protein Conformation , Sequence Alignment , X-Ray DiffractionABSTRACT
How stem cells are recruited to and maintained in their niches is crucial to understanding their regulation and use in regenerative medicine. Here, we demonstrate that DE-cadherin-mediated cell adhesion is required for anchoring germline stem cells (GSCs) in their niches in the Drosophila ovary. Two major components of this adhesion process, DE-cadherin and Armadillo/beta-catenin, accumulate at high levels in the junctions between GSCs and cap cells, one of the niche components. Removal of these proteins from GSCs results in stem cell loss. Furthermore, DE-cadherin is required for recruiting GSCs to their niche. Our study demonstrates that anchorage of GSCs in their niche by DE-cadherin-mediated adhesion is important for stem cell maintenance and function.
Subject(s)
Adherens Junctions/physiology , Cadherins/physiology , Drosophila Proteins , Drosophila/physiology , Oocytes/cytology , Stem Cells/physiology , Trans-Activators , Alleles , Animals , Armadillo Domain Proteins , Cadherins/genetics , Cell Adhesion , Cell Differentiation , Drosophila/cytology , Drosophila/genetics , Drosophila/growth & development , Female , Insect Proteins/genetics , Insect Proteins/physiology , Larva/physiology , Mutation , Oocytes/physiology , Oocytes/ultrastructure , Ovary/cytology , Ovary/growth & development , Ovary/physiology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/physiology , Signal Transduction , Stem Cells/cytology , Stem Cells/ultrastructure , Transcription Factors , Wnt1 ProteinABSTRACT
Pectate lyase A is a virulence factor secreted by the plant-pathogenic bacteria Erwinia chrysanthemi. The enzyme cleaves the glycosidic bond of pectate polymers by a calcium-dependent beta-elimination mechanism. The crystal structure of pectate lyase A from E. chrysanthemi EC16 has been determined in two crystal forms, monoclinic C2 to 1.8 A and rhombohedral R3 to 2.1 A. The protein structure is compared with two other pectate lyase isoforms from E. chrysanthemi EC16, pectate lyase C and pectate lyase E. Pectate lyase A is unique as it is the only acidic pectate lyase and has end products that are significantly more varied in length in comparison to those of the other four major pectate lyase isozymes. Differences and similarities in polypeptide trace, size and volume of the active-site groove and surface electrostatics are discussed.
