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The uptake of cholesterol carried by low-density lipoprotein (LDL) is tightly controlled in the body. Macrophages are not well suited to counteract the cellular consequences of excess cholesterol leading to their transformation into "foam cells," an early step in vascular plaque formation. We have uncovered and characterized a novel mechanism involving phospholipase D (PLD) in foam cell formation. Utilizing bone marrow-derived macrophages from genetically PLD deficient mice, we demonstrate that PLD2 (but not PLD1)-null macrophages cannot fully phagocytose aggregated oxidized LDL (Agg-Ox-LDL), which was phenocopied with a PLD2-selective inhibitor. We also report a role for PLD2 in coupling Agg-oxLDL phagocytosis with WASP, Grb2, and Actin. Further, the clearance of LDL particles is mediated by both CD36 and PLD2, via mutual dependence on each other. In the absence of PLD2, CD36 does not engage in Agg-Ox-LDL removal and when CD36 is blocked, PLD2 cannot form protein-protein heterocomplexes with WASP or Actin. These results translated into humans using a GEO database of microarray expression data from atheroma plaques versus normal adjacent carotid tissue and observed higher values for NFkB, PLD2 (but not PLD1), WASP, and Grb2 in the atheroma plaques. Human atherectomy specimens confirmed high presence of PLD2 (mRNA and protein) as well as phospho-WASP in diseased arteries. Thus, PLD2 interacts in macrophages with Actin, Grb2, and WASP during phagocytosis of Agg-Ox-LDL in the presence of CD36 during their transformation into "foam cells." Thus, this study provides new molecular targets to counteract vascular plaque formation and atherogenesis., (©2018 Society for Leukocyte Biology.)

Rab small GTPases, well-known regulators of membrane trafficking pathways in eukaryotic cells, comprise approximately 60 different members in mammals. During the past decade, our understanding of the functions of mammalian Rab32 subfamily members (Rab32 and Rab38) have deepened, especially on the biogenesis of lysosome-related organelles, such as melanosomes, and the protection mechanisms against several pathogenic microbial infections. Endosome-mediated membrane trafficking by Rab32 subfamily members plays pivotal roles in these events. In this review, we provide an overview of the regulatory mechanisms of mammalian Rab32-family members in endosomal trafficking, especially focusing on their GEF, GAP and effector molecules, and describe the latest findings on physiological and pathological functions regulated by these molecules., (© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

The lipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5K) produces a versatile signaling phospholipid, phosphatidylinositol 4,5-bisphosphate. Three PIP5K isozymes, PIP5K1A, PIP5K1B, and PIP5K1C, have been identified in mammals so far. Although the functions of these three PIP5K isozymes have been extensively studied in vitro, the in vivo physiological roles of these PIP5K isozymes remain largely unknown. In this study, we examined the functions of PIP5K1A and PIP5K1B in spermatogenesis, using Pip5k1a-knockout (KO), Pip5k1b-KO, and Pip5k1a/Pip5k1b double (D)-KO mice. Pip5k1a-KO and D-KO males were subfertile and completely sterile, respectively. F-actin in the seminiferous epithelium was disorganized in the D-KO mice, although F-actin bundles at the apical ectoplasmic specialization was not affected. D-KO seminiferous tubules contained a greatly decreased number of elongated spermatids. Flagella of sperm from Pip5k1a-KO and D-KO mice remarkably underwent morphological change, whereas Pip5k1b-KO sperm were morphologically normal. Notably, the flagellar shape of D-KO sperm was more severely impaired than that of Pip5k1a-KO sperm. These results suggest that PIP5K1A and PIP5K1B may coordinately and/or redundantly function in the maintenance of sperm number and morphology during spermatogenesis.

Phosphoinositide kinases comprise a unique family of enzymes that catalyze the phosphorylation of phosphatidylinositol and its phosphorylated metabolites to produce seven phosphoinositides. Recent advances have revealed that these phosphoinositides have specific physiological functions, such as in actin cytoskeletal reorganization, membrane transport, cell proliferation and survival, in eukaryotic cells and that each phosphoinositide kinase is differently and precisely regulated. Here we describe the diverse regulation and physiological functions of phosphoinositide kinases involving their products.

The crystal structures of the GTP- and GDP-bound alpha subunits of heterotrimeric GTP-binding proteins were recently determined, and a conserved Thr residue in the G2 (linker 2) region of the alpha subunits, which corresponds to Thr182 in Gi2 alpha, was deduced to interact with the gamma-phosphate of GTP and Mg2+. To investigate biochemically the significance of the Thr residue, we produced a mutant Gi2 alpha, in which Thr182 was substituted for Ala (T182A), in Escherichia coli. The rate of guanosine 5'-(gamma-thio)tri-phosphate (GTP gamma S) binding to T182A was higher than that to the wild-type Gi2 alpha, especially with a high concentration (10 mM) of Mg2+. The rate of dissociation of bound GDP from T182A was also much faster than that from the wild-type with the high Mg2+ concentration. Moreover, T182A had much lower GTPase activity than the wild-type, like the gip mutant (R179C) of Gi2 alpha found in human endocrine tumors. The ability of T182A to interact with beta gamma subunits and membrane-bound receptors was the same as that of the wild-type alpha subunit. T182A could take on a GTP-bound active conformation, as judged from its sensitivity to tryptic digestion. These results indicated that Thr182 plays an important role not only in the Mg(2+)-sensitive GDP-GTP exchange reaction but also in the GTPase activity of Gi2 alpha. The T182A mutant of Gi2 alpha, characterized by the faster GDP release and the slower GTP hydrolysis, would be a novel mutant that retains the ability to interact with receptors and beta gamma subunits.

An ecto-enzyme of NAD glycohydrolase (NADase) induced by retinoic acid in HL-60 cells is attributed to the molecule of CD38 antigen [Kontani, K., Nishina, H., Ohoka, Y., Takahashi, K., and Katada, T. (1993) J. Biol. Chem. 268, 16895-16898]. CD38 antigen has an amino acid sequence homologous to Aplysia ADP-ribosyl cyclase which generates cyclic adenosine diphosphoribose (cADPR) and nicotinamide (NA) from beta-NAD+. On the basis of this sequence homology, we compared enzyme properties between CD38 NADase expressed as a fusion protein in Escherichia coli and ADP-ribosyl cyclase purified from the ovotestis of Aplysia kurodai. 1) beta-NAD+ analogs, nicotinamide 1, N6-ethenoadenine dinucleotide, and nicotinamide hypoxanthine dinucleotide, did not serve as good substrates for the ADP-ribosyl cyclase, suggesting that the intact adenine ring of beta-NAD+ was required for the cyclase-catalyzed reaction. On the other hand, CD38 NADase utilized the NAD analogs to form ADP-ribose and NA. 2) Kinetic analyses of the ADP-ribosyl cyclase reaction revealed that NA was first released from the substrate (beta-NAD+)-enzyme complex, followed by the release of another product, cADPR, which was capable of interacting with the free enzyme. 3) The enzyme reaction catalyzed by the ADP-ribosyl cyclase was fully reversible; beta-NAD+ could be formed from cADPR and NA with a velocity similar to that observed in the degradation of beta-NAD+. However, CD38 NADase did not catalyze the reverse reaction to form beta-NAD+ from ADP-ribopase and NA. 4) The CD38 NADase activity was, but the ADP-ribosyl cyclase activity was not, inhibited by dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear protein transport was inhibited in permeabilized HeLa cells which had been prepared after culture with pertussis toxin, suggesting that the pertussis toxin-sensitive protein(s) might be involved in the nuclear protein transport. To investigate the mechanism whereby pertussis toxin inhibited the nuclear protein transport, an accumulation of proteins containing a nuclear localization signal sequence (NLS) into isolated rat liver nuclei was investigated. The NLS-containing protein accumulation required ATP and cytosolic proteins, and was temperature- and wheat germ agglutinin-sensitive as had been observed in permeabilized cells. Non-hydrolyzable GTP analogues, such as guanosine 5'-(gamma-thio)triphosphate and guanosine 5'-(beta, gamma-methylene)triphosphate, but not ATP analogues, inhibited the NLS-containing protein accumulation in the isolated nuclei. The NLS-containing protein accumulation was also inhibited by prior treatment of the nuclei with pertussis toxin plus NAD, and the effect of pertussis toxin was blocked when guanosine 5'-(gamma-thio)triphosphate was simultaneously added during the pretreatment with pertussis toxin. The inhibition induced by pertussis toxin and the blockage by guanosine 5'-(gamma-thio)triphosphate were well correlated to ADP-ribosylation of 40-kDa protein in nuclear fraction. These results suggested that the nuclear pertussis toxin-sensitive GTP-binding protein is involved in a pathway of nuclear protein transport.

Stimulation by fMet-Leu-Phe analogs of GTPase activity in plasma membranes from rabbit neutrophils was compared with the stimulation of degranulation in intact neutrophils. All four formyl peptides examined (fMet-Leu-Phe-Phe, fMet-Leu-Phe, fNle-Leu-Phe, and fVal-Leu-Phe) were full agonists for both responses. Their ED50 values for the two responses correlated well, although those for GTPase stimulation were uniformly about tenfold greater. The specific antagonist tBoc-Phe-Leu-Phe-Leu-Phe competitively inhibited both GTPase activity and degranulation stimulated by fMet-Leu-Phe; its Ki values were similar for the two responses. Pertussis toxin treatment, in contrast, inhibited the maximal stimulation of both responses by fMet-Leu-Phe with minimal shift in ED50. The inhibitory actions of tBoc-Phe-Leu-Phe-Leu-Phe and pertussis toxin on GTPase activity thus paralleled the effects on degranulation. These observations substantiate the hypothesis that a guanine nucleotide-binding protein that is a pertussis toxin substrate couples the formyl peptide receptors to physiological function in the neutrophil.