Point mutational analysis of the liganding site in human glycolipid transfer protein. Functionality of the complex

Mammalian glycolipid transfer proteins (GLTPs) facilitate the selective transfer of glycolipids between lipid vesicles in vitro. Recent structural determinations of the apo- and glycolipid-liganded forms of human GLTP have provided the first insights into the molecular architecture of the protein and its glycolipid binding site (Malinina, L., Malakhova, M. L., Brown, R. E., and Patel, D. J. (2004) Nature 430, 1048-1053). In the present study, we have evaluated the functional consequences of point mutation of the glycolipid liganding site of human GLTP within the context of a carrier-based mechanism of glycolipid intermembrane transfer. Different approaches were developed to rapidly and efficiently assess the uptake and release of glycolipid by GLTP. They included the use of glass-immobilized, glycolipid films to load GLTP with glycolipid and separation of GLTP/glycolipid complexes from vesicles containing glycolipid (galactosylceramide or lactosylceramide) or from monosialoganglioside dispersions by employing nickel-nitrilotriacetic acid-based affinity or gel filtration strategies. Point mutants of the sugar headgroup recognition center (Trp-96, Asp-48, Asn-52) and of the ceramide-accommodating hydrophobic tunnel (Phe-148, Phe-183, Leu-136) were analyzed for their ability to acquire and release glycolipid ligand. Two manifestations of point mutation within the liganding site were apparent: (i) impaired formation of the GLTP/glycolipid complex; (ii) impaired acquisition and release of bound glycolipid by GLTP. The results are consistent with a carrier-based mode of GLTP action to accomplish the intermembrane transfer of glycolipid. Also noteworthy was the inefficient release of glycolipid by wtGLTP into phosphatidylcholine acceptor vesicles, raising the possibility of a function other than intermembrane glycolipid transfer in vivo.     

Pulmonary surfactant protein A inhibits the lipid peroxidation stimulated by linoleic acid hydroperoxide of rat lung mitochondria and microsomes

Reactive oxygen species play an important role in several acute lung injuries. The lung tissue contains polyunsaturated fatty acids (PUFAs) that are substrates of lipid peroxidation that may lead to loss of the functional integrity of the cell membranes. In this study, we compare the in vitro protective effect of pulmonary surfactant protein A (SP-A), purified from porcine surfactant, against ascorbate-Fe(2+) lipid peroxidation stimulated by linoleic acid hydroperoxide (LHP) of the mitochondria and microsomes isolated from rat lung; deprived organelles of ascorbate and LHP were utilized as control. The process was measured simultaneously by chemiluminescence as well as by PUFA degradation of the total lipids isolated from these organelles. The addition of LHP to rat lung mitochondria or microsomes produces a marked increase in light emission; the highest value of activation was produced in microsomes (total chemiluminescence: 20.015+/-1.735 x 10(5) cpm). The inhibition of lipid peroxidation (decrease of chemiluminescence) was observed with the addition of increasing amounts (2.5 to 5.0 microg) of SP-A in rat lung mitochondria and 2.5 to 7.5 microg of SP-A in rat lung microsomes. The inhibitory effect reaches the highest values in the mitochondria, thus, 5.0 microg of SP-A produces a 100% inhibition in this membranes whereas 7.5 microg of SP-A produces a 51.25+/-3.48% inhibition in microsomes. The major difference in the fatty acid composition of total lipids isolated from native and peroxidized membranes was found in the arachidonic acid content; this decreased from 9.68+/-1.60% in the native group to 5.72+/-1.64% in peroxidized mitochondria and from 7.39+/-1.14% to 3.21+/-0.77% in microsomes. These changes were less pronounced in SP-A treated membranes; as an example, in the presence of 5.0 microg of SP-A, we observed a total protection of 20:4 n-6 (9.41+/-3.29%) in mitochondria, whereas 7.5 microg of SP-A produced a 65% protection in microsomes (5.95+/-0.73%). Under these experimental conditions, SP-A produces a smaller inhibitory effect in microsomes than in mitochondria. Additional studies of lipid peroxidation of rat lung mitochondria or microsomes using equal amounts of albumin and even higher compared to SPA were carried out. Our results indicate that under our experimental conditions, BSA was unable to inhibit lipid peroxidation stimulated by linoleic acid hydroperoxide of rat lung mitochondria or microsomes, thus indicating that this effect is specific to SP-A.     

The adaptor protein 3BP2 binds human CD244 and links this receptor to Vav signaling, ERK activation, and NK cell killing

Adaptor proteins, molecules that mediate intermolecular interactions, are crucial for cellular activation. The adaptor 3BP2 has been shown to positively regulate NK cell-mediated cytotoxicity. In this study we present evidence for a physical interaction between 3BP2 and the CD244 receptor. CD244, a member of the CD150 family, is a cell surface protein expressed on NK, CD8+ T, and myeloid cells. CD244 interacts via its Src homology 2 domain with the X-linked lymphoproliferative disease gene product signaling lymphocytic activation molecule-associated protein (SAP)/SH2 domain protein 1A. 3BP2 interacts with human but not murine CD244. CD244-3BP2 interaction was direct and regulated by phosphorylation, as shown by a three-hybrid analysis in yeast and NK cells. Tyr337 on CD244, part of a consensus motif for SAP/SH2 domain protein 1A binding, was critical for the 3BP2 interaction. Although mutation of Tyr337 to phenylalanine abrogated human 3BP2 binding, we still observed SAP association, indicating that this motif is not essential for SAP recruitment. CD244 ligation induced 3BP2 phosphorylation and Vav-1 recruitment. Overexpression of 3BP2 led to an increase in the magnitude and duration of ERK activation, after CD244 triggering. This enhancement was concomitant with an increase in cytotoxicity due to CD244 ligation. However, no differences in IFN-gamma secretion were found when normal and 3BP2-transfected cells were compared. These results indicate that CD244-3BP2 association regulates cytolytic function but not IFN-gamma release, reinforcing the hypothesis that, in humans, CD244-mediated cytotoxicity and IFN-gamma release involve distinct NK pathways.     

Characterization of liposomal tacrolimus in lung surfactant-like phospholipids and evaluation of its immunosuppressive activity

Tacrolimus (FK506) is a hydrophobic immunosuppressive agent that rapidly penetrates the plasmatic membrane and inhibits the signal transduction cascade of T lymphocytes. The objective of this study was the characterization of liposomal FK506 with surfactant-like phospholipids to be administered intratracheally after lung transplantation or in inflammatory lung diseases. We evaluated the optimal incorporation of FK506 in dipalmitoylphosphatidylcholine (DPPC) and DPPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) monolayers and bilayers and the effects of FK506 on the physical properties of DPPC and DPPC/POPG (8:2 w/w) vesicles. In addition, we assessed the immunosuppressive effects of surfactant-like phospholipid vesicles containing different amounts of FK506 on T-cell proliferation and interleukin 2 production. From surface pressure measurements of FK506/DPPC and FK506/DPPC/POPG mixed monolayers, we determined that FK506 was embedded into these monolayers up to an FK506 concentration of about 0.4 mol %. Beyond this concentration, FK506 was not quantitatively incorporated into the monolayer, suggesting possible concentration-dependent aggregation of tacrolimus. The incorporation of FK506 into DPPC monolayers, at concentrations 相似文献   

Two positively charged residues of phi29 DNA polymerase, conserved in protein-primed DNA polymerases, are involved in stabilisation of the incoming nucleotide

In DNA polymerases from families A and B in the closed conformation, several positively charged residues, located in pre-motif B and motif B, have been shown to interact with the phosphate groups of the incoming nucleotide at the polymerisation active site: the invariant Lys of motif B and the nearly invariant Lys of pre-motif B (family B) correspond to a His in family A DNA polymerases. In phi29 DNA polymerase, belonging to the family B DNA polymerases able to start replication by protein-priming, the corresponding residues, Lys383 and Lys371, have been shown to be dNTP-ligands. Since in several DNA polymerases a third residue has been involved in dNTP binding, we have addressed here the question if in the DNA polymerases of the protein-primed subfamily, and especially in phi29 DNA polymerase, there are more than these two residues involved in nucleotide binding. By site-directed mutagenesis in phi29 DNA polymerase the functional role of the remaining two conserved positively charged amino acid residues of pre-motif B and motif B (besides Lys371 and Lys383) has been studied. The results indicate that residue Lys379 of motif B is also involved in dNTP binding, possibly through interaction with the triphosphate moiety of the incoming nucleotide, since the affinity for nucleotides of mutant DNA polymerase K379T was reduced in DNA and TP-primed reactions. On the other hand, we propose that, when the terminal protein (TP) is present at the polymerisation active site, residue Lys366 of pre-motif B is involved in stabilising the incoming nucleotide in an appropriate position for efficient TP-deoxynucleotidylation. Although mutant DNA polymerase K366T showed a wild-type like phenotype in DNA-primed polymerisation in the presence of DNA as template, in TP-primed reactions as initiation and transition it was impaired, especially in the presence of the phi29 DBP, protein p6.     

A deleterious mutation in SAMD9 causes normophosphatemic familial tumoral calcinosis

Familial tumoral calcinosis (FTC) is a rare autosomal recessive disorder characterized by the progressive deposition of calcified masses in cutaneous and subcutaneous tissues, which results in painful ulcerative lesions and severe skin and bone infections. Two major types of FTC have been recognized: hyperphosphatemic FTC (HFTC) and normophosphatemic FTC (NFTC). HFTC was recently shown to result from mutations in two different genes: GALNT3, which codes for a glycosyltransferase, and FGF23, which codes for a potent phosphaturic protein. To determine the molecular cause of NFTC, we performed homozygosity mapping in five affected families of Jewish Yemenite origin and mapped NFTC to 7q21-7q21.3. Mutation analysis revealed a homozygous mutation in the SAMD9 gene (K1495E), which was found to segregate with the disease in all families and to interfere with the protein expression. Our data suggest that SAMD9 is involved in the regulation of extraosseous calcification, a process of considerable importance in a wide range of diseases as common as atherosclerosis and autoimmune disorders.