BBA - Molecular and Cell Biology of Lipids (v.1811, #5)

Exploring the inhibitory activity of short-chain phospholipids against amyloid fibrillogenesis of hen egg-white lysozyme by Steven S.-S. Wang; Ying-Tz Hung; Wen-Sing Wen; Keng-Chi Lin; Geng-Yuan Chen (301-313).
Amyloid fibrillogenesis is an important pathological feature of a group of degenerative human diseases. The 129-residue enzyme hen egg-white lysozyme has been shown to form fibrils in vitro at pH 2.0 and 55 °C. In this research, using various spectroscopic techniques, light scattering, and transmission electron microscopy, we first examined the influence of short-chain phospholipids on the amyloid fibrillogenesis and the structural changes derived from hen lysozyme in vitro. Both model short-chain phospholipids were observed to mitigate the fibrillogenesis of hen lysozyme. Also, urea-induced unfolding results suggested that the susceptibility of hen lysozyme to conformational changes elicited by the denaturant was observed to decrease upon addition of short-chain phospholipids. Moreover, our molecular dynamics simulations results demonstrated that the observed inhibitory action of short-chain phosoholipids against hen lysozyme fibrillogenesis might be attributable to the interference of β-strand extension by the binding of phospholipids to lysozyme's β-sheet-rich region. We believe that the outcome from this study may contribute to a better understanding the molecular factors affecting amyloid fibrillogenesis and the molecular mechanism(s) of the interactions between phospholipids/lipids and amyloid-forming proteins.Display Omitted► Short-chain phospholipids (DHPC) suppress the fibrillogenesis of hen lysozyme. ► Addition of DHPC triggers the structural changes of amyloid protein. ► DHPC inhibits fibrillogenesis by interference of β-strand extension. ► Addition of DHPC leads to formation of amorphous aggregates.
Keywords: Phospholipid; Amyloid; Fibril; Inhibition; Lysozyme;

Novel sterol glucosyltransferase in the animal tissue and cultured cells: Evidence that glucosylceramide as glucose donor by Hisako Akiyama; Narie Sasaki; Shuwa Hanazawa; Mari Gotoh; Susumu Kobayashi; Yoshio Hirabayashi; Kimiko Murakami-Murofushi (314-322).
Cholesteryl glucoside (CG), a membrane glycolipid, regulates heat shock response. CG is rapidly induced by heat shock before the activation of heat shock transcription factor 1 (HSF1) and production of heat shock protein 70 (HSP70), and the addition of CG in turn induces HSF1 activation and HSP70 production in human fibroblasts; thus, a reasonable correlation is that CG functions as a crucial lipid mediator in stress responses in the animal. In this study, we focused on a CG-synthesizing enzyme, animal sterol glucosyltransferase, which has not yet been identified. In this study, we describe a novel type of animal sterol glucosyltransferase in hog stomach and human fibroblasts (TIG-3) detected by a sensitive assay with a fluorescence-labeled substrate. The cationic requirement, inhibitor resistance, and substrate specificity of animal sterol glucosyltransferase were studied. Interestingly, animal sterol glucosyltransferase did not use uridine diphosphate glucose (UDP-glucose) as an immediate glucose donor, as has been shown in plants and fungi. Among the glycolipids tested in vitro, glucosylceramide (GlcCer) was the most effective substrate for CG formation in animal tissues and cultured cells. Using chemically synthesized [U- 13 C]Glc-β-Cer as a glucose donor, we confirmed by mass spectrometry that [U- 13 C]CG was synthesized in hog stomach homogenate. These results suggest that animal sterol glucosyltransferase transfers glucose moiety from GlcCer to cholesterol. Additionally, using GM-95, a mutant B16 melanoma cell line that does not express ceramide glucosyltransferase, we showed that GlcCer is an essential substrate for animal sterol glucosyltransferase in the cell. Display Omitted► Animal sterol glucosyltransferase does not use UDP-glucose as glucose donor. ► Glucosylceramide is essential substrate for animal sterol glucosyltransferase. ► Animal sterol glucosyltransferase is novel-type enzyme.
Keywords: Sterol glucosyltransferase; Cholesteryl glucoside; Cholesterol; Glucosylceramide; Heat shock response;

Purification, molecular cloning, and application of a novel sphingomyelin-binding protein (clamlysin) from the brackishwater clam, Corbicula japonica by Taketoshi Takara; Tetsuto Nakagawa; Masami Isobe; Nozomu Okino; Sachiyo Ichinose; Akira Omori; Makoto Ito (323-332).
A novel sphingomyelin-binding protein (clamlysin) was purified from the foot muscle of a brackishwater clam, Corbicula japonica. The purified 24.8-kDa protein lysed sheep, horse and rabbit erythrocytes and the hemolytic activity was inhibited by sphingomyelin, but not other phospholipids or glycosphingolipids. The open reading frame of the clamlysin gene encoded a putative 26.9-kDa protein (clamlysin B) which showed high sequence similarity with the actinoporin family. A surface plasmon resonance assay confirmed that clamlysin B specifically bound to sphingomyelin. Furthermore, two cDNA variants of clamlysin, encoding putative 31.4 kDa (clamlysin A) and 11 kDa (clamlysin C) proteins, were isolated. Only the 31.4-kDa variant was found to exhibit sphingomyelin-binding activity. Clamlysin A and B, but not C, shared a sequence (domain II) conserved in all known sphingomyelin-binding proteins. Domain II fused with a glutathione S-transferase bound to sphingomyelin. Horse erythrocytes, mouse melanoma B16 and GM95 cells, and Chinese hamster ovary CHO-K1 cells, but not the same cells treated with bacterial sphingomyelinase, were immunostained with clamlysin B. These results indicate that clamlysin B binds to the sphingomyelin of living cells and thus would be useful as a molecular probe to detect sphingomyelin.► A novel sphingomyelin-binding protein (clamlysin) was purified from the foot muscle of a brackishwater clam, Corbicula japonica, and its cDNA was cloned. ► Domain II of the recombinant clamlysin fused with a glutathione S-transferase was found to bind to sphingomyelin. ► Horse erythrocytes, mouse melanoma B16 and GM95 cells, and Chinese hamster ovary CHO-K1 cells, but not the same cells treated with bacterial sphingomyelinase, were immunostained with clamlysin.
Keywords: Sphingomyelin; Hemolytic protein; Actinoporin family; Clamlysin;

A physiologically-based kinetic model for the prediction of plasma cholesterol concentrations in the mouse by Niek C.A. van de Pas; Ruud A. Woutersen; Ben van Ommen; Ivonne M.C.M. Rietjens; Albert A. de Graaf (333-342).
The LDL cholesterol (LDL-C) and HDL cholesterol (HDL-C) concentrations are determined by the activity of a complex network of reactions in several organs. Physiologically-based kinetic (PBK) computational models can be used to describe these different reactions in an integrated, quantitative manner. A PBK model to predict plasma cholesterol levels in the mouse was developed, validated, and analyzed. Kinetic parameters required for defining the model were obtained using data from published experiments. To construct the model, a set of appropriate submodels was selected from a set of 65,536 submodels differing in the kinetic expressions of the reactions. A submodel was considered appropriate if it had the ability to correctly predict an increased or decreased plasma cholesterol level for a training set of 5 knockout mouse strains. The model thus defined consisted of 8 appropriate submodels and was validated using data from an independent set of 9 knockout mouse strains. The model prediction is the average prediction of 8 appropriate submodels. Remarkably, these submodels had in common that the rate of cholesterol transport from the liver to HDL was not dependent on hepatic cholesterol concentrations. The model appeared able to accurately predict in a quantitative way the plasma cholesterol concentrations of all 14 knockout strains considered, including the frequently used Ldlr−/− and Apoe−/− mouse strains. The model presented is a useful tool to predict the effect of knocking out genes that act in important steps in cholesterol metabolism on total plasma cholesterol, HDL-C and LDL-C in the mouse.► We model the intake, synthesis, transport, and degradation of cholesterol in mice. ► The model constructed can predict plasma cholesterol concentrations in knockout mice. ► A good prediction was given for 14 knockout strains, including Ldlr−/− and Apoe−/−. ► The model hints cholesterol transport to HDL to be independent on liver cholesterol.
Keywords: Plasma cholesterol; PBK modeling; Knockout mouse strains; LDL cholesterol; HDL cholesterol;

Phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids among lipoproteins. Over half of the PLTP in human plasma has been found to have little phospholipid transfer activity (inactive PLTP). We recently observed that plasma PLTP specific activity is inversely correlated with high-density lipoprotein (HDL) level and particle size in healthy adults. The purpose of this study was to evaluate the factors that contribute to the variation in plasma PLTP specific activity. Analysis of the specific activity of PLTP complexes in nine plasma samples from healthy adults revealed two clusters of inactive PLTP complexes with mean molecular weights (MW) of 342 kDa and 146 kDa. The large and small inactive PLTP complexes represented 52 ± 8% (range 39–63%) and 8 ± 8% (range 1–28%) of the plasma PLTP, respectively. Active PLTP complexes had a mean MW of 207 kDa and constituted 40 ± 6% (range 33–50%) of the plasma PLTP. The specific activity of active PLTP varied from 16 to 32 μmol/μg/h. These data demonstrate for the first time the existence of small inactive plasma PLTP complexes. Variation in the amount of the two clusters of inactive PLTP complexes and the specific activity of the active PLTP contribute to the variation in plasma PLTP specific activity.► Inactive PLTP resides in large (mean 342 kDa) and small (mean 146 kDa) complexes. ► Large and small inactive PLTP complexes constitute 52 ± 8% and 8 ± 8% of plasma PLTP, respectively. ► Active PLTP complexes (mean 207 kDa) constitute 33–50% of plasma PLTP. ► The specific activity (SA) of active PLTP complexes varies among plasma samples. ► Variation in plasma active PLTP SA and inactive PLTP contribute to variation in SA.
Keywords: High-density lipoproteins; Phospholipid transfer protein; PLTP specific activity; Active PLTP; Inactive PLTP;

Expression of the human atypical kinase ADCK3 rescues coenzyme Q biosynthesis and phosphorylation of Coq polypeptides in yeast coq8 mutants by Letian X. Xie; Edward J. Hsieh; Shota Watanabe; Christopher M. Allan; Jia Y. Chen; UyenPhuong C. Tran; Catherine F. Clarke (348-360).
Coenzyme Q (ubiquinone or Q) is a lipid electron and proton carrier in the electron transport chain. In yeast Saccharomyces cerevisiae eleven genes, designated COQ1 through COQ9, YAH1 and ARH1, have been identified as being required for Q biosynthesis. One of these genes, COQ8 (ABC1), encodes an atypical protein kinase, containing six (I, II, III, VIB, VII, and VIII) of the twelve motifs characteristically present in canonical protein kinases. Here we characterize seven distinct Q-less coq8 yeast mutants and show that unlike the coq8 null mutant, each maintained normal steady-state levels of the Coq8 polypeptide. The phosphorylation states of Coq polypeptides were determined with two-dimensional gel analyses. Coq3p, Coq5p, and Coq7p were phosphorylated in a Coq8p-dependent manner. Expression of a human homolog of Coq8p, ADCK3(CABC1) bearing an amino-terminal yeast mitochondrial leader sequence, rescued growth of yeast coq8 mutants on medium containing a nonfermentable carbon source and partially restored biosynthesis of Q6. The phosphorylation state of several of the yeast Coq polypeptides was also rescued, indicating a profound conservation of yeast Coq8p and human ADCK3 protein kinase function in Q biosynthesis.► Yeast coq8 mutants were identified that maintain normal steady-state levels of Coq8p. ► S. cerevisiae Coq8 polypeptide is required for phosphorylation of Coq3, Coq5 and Coq7. ► The human Coq8 homolog ADCK3 expressed in yeast coq8 mutants rescues Q biosynthesis. ► Human ADCK3 rescues phosphorylation of yeast Coq polypeptides in yeast coq8 mutants. ► The results indicate a profound conservation of Coq8/ADCK3 function in Q synthesis.
Keywords: Coenzyme Q; Ubiquinone; Saccharomyces cerevisiae; Mitochondria; Lipid metabolism; Protein kinase;