Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid synthesis. In yeast and mammals, ACP exists as a separate domain within a large multifunctional fatty acid synthase polyprotein (type I FAS), whereas it is a small monomeric protein in bacteria and plastids (type II FAS). Bacterial ACPs are also acyl donors for synthesis of a variety of products, including endotoxin and acylated homoserine lactones involved in quorum sensing; the distinct and essential nature of these processes in growth and pathogenesis make ACP-dependent enzymes attractive antimicrobial drug targets. Additionally, ACP homologues are key components in the production of secondary metabolites such as polyketides and nonribosomal peptides. Many ACPs exhibit characteristic structural features of natively unfolded proteins in vitro, with a dynamic and flexible conformation dominated by 3 parallel alpha helices that enclose the thioester-linked acyl group attached to a phosphopantetheine prosthetic group. ACP conformation may also be influenced by divalent cations and interaction with partner enzymes through its "recognition" helix II, properties that are key to its ability to alternately sequester acyl groups and deliver them to the active sites of ACP-dependent enzymes. This review highlights recent progress in defining how the structural features of ACP are related to its multiple carrier roles in fatty acid metabolism.

Oxysterol-binding protein (OSBP) is a high-affinity receptor for a variety of oxysterols, such as 25-hydroxycholesterol, that down-regulate cholesterol synthesis and stimulate cholesterol esterification. To examine a potential role for OSBP in regulating cholesterol metabolism, we stably overexpressed this protein in Chinese-hamster ovary (CHO)-K1 cells. Compared with mock-transfected controls, several cell lines overexpressing wild-type OSBP (CHO-OSBP) displayed a 50% decrease in cholesteryl ester synthesis when cultured in medium with delipidated serum, 25-hydroxycholesterol or low-density lipoprotein (LDL). CHO-OSBP cells showed a 40-60% decrease in acyl-CoA:cholesterol acyltransferase activity and mRNA, a 50% elevation in mRNA for three sterol-regulated genes [LDL receptor, 3-hydroxy-3-methylgluraryl (HMG)-CoA reductase and HMG-CoA synthase], and an 80% increase in [14C]acetate incorporation into cholesterol. CHO-K1 cells overexpressing two OSBP mutants with a complete or N-terminal deletion of the pleckstrin homology (PH) domain had cholesterol esterification and synthesis rates that were similar to those shown by mock-transfected controls. Unlike wild-type OSBP, both PH domain mutants displayed diffuse cytoplasmic immunofluorescence staining and did not translocate to the Golgi apparatus in the presence of 25-hydroxycholesterol. CHO-K1 cells overexpressing OSBP have pronounced alterations in cholesterol esterification and synthesis, indicating a potential role for this receptor in cholesterol homoeostasis. The phenotype observed in cells overexpressing OSBP is dependent on the PH domain, which appears to be necessary for ligand-dependent localization of OSBP to the Golgi apparatus.

Human newborns are more susceptible than adults to infection by gram-negative bacteria. We hypothesized that this susceptibility may be associated with a decreased response by leukocytes to lipopolysaccharide (LPS). In this study, we compared LPS-induced secretion of tumor necrosis factor alpha (TNF-␣) by mononuclear cells (MNC) from adult peripheral blood and newborn umbilical cord blood in vitro and attempted to determine the mechanisms involved in its regulation. At a high concentration of LPS (10 ng/ml) and in the presence of autologous plasma, MNC from adults and newborns secreted similar amounts of TNF-␣. However, in the absence of plasma, MNC from newborns secreted significantly less TNF-␣ compared to MNC from adults. Moreover, at a low concentration of LPS (0.1 ng/ml) and in the presence of plasma, TNF-␣ secretion was significantly lower for newborn MNC compared to adult MNC. Adults and newborns had similar numbers of CD14 and Toll-like receptor 4 (TLR-4)-positive cells as measured by flow cytometry. However, the intensity of the CD14 marker was greater for adult than for newborn cells. Incubation of cells with LPS led to an increase in CD14 and TLR-4 intensity for adult cells but not for newborn cells. The effect of LPS stimulation of adult or newborn cells was similar for ERK, p38, and IB␣ phosphorylation, as well as IB␣ degradation. Finally, we assessed levels of the TLR-4 adapter protein, the myeloid differentiation antigen 88 (MyD88). We found a direct relation between adult and newborn TNF-␣ secretion and MyD88, which was significantly decreased in newborn monocytes. Since TLR-4 signals intracellularly through the adapter protein, MyD88, we hypothesize that MyD88-dependent factors are responsible for delayed and decreased TNF-␣ secretion in newborn monocytes.

Niemann-Pick type II disease is an autosomal recessive disorder characterized by a defect in intracellular trafficking of sterols. We have determined the intron/exon boundaries of eight exons from the conserved 3' portion of NPC1, the gene associated with most cases of the disease. SSCP analyses were designed for these exons and were used to identify the majority of mutations in 13 apparently unrelated families. Thirteen mutations were found, accounting for 19 of the 26 alleles. These mutations included eight different missense mutations (including one reported by Greer et al. [1998]), one 4-bp and two 2-bp deletions that generate premature stop codons, and two intronic mutations that are predicted to alter splicing. Two of the missense mutations were present in predicted transmembrane (TM) domains. Clustering of these and other reported NPC1 mutations in the carboxy-terminal third of the protein indicates that screening of these exons, by means of the SSCP analyses reported here, will detect most mutations. The carboxy-terminal half of the Npc1 protein shares amino acid similarity with the TM domains of the morphogen receptor Patched, with the largest stretch of unrelated sequence lying between two putative TM spans. Alignment of this portion of the human Npc1 protein sequence with Npc1-related sequences from mouse, yeast, nematode, and a plant, Arabidopsis, revealed conserved cysteine residues that may coordinate the structure of this domain. That 7 of a total of 13 NPC1 missense mutations are concentrated in this single Npc1-specific domain suggests that integrity of this region is particularly critical for normal functioning of the protein.

Niemann-Pick type D (NPD) disease is a progressive neurodegenerative disorder characterized by the accumulation of tissue cholesterol and sphingomyelin. This disorder is relatively common in southwestern Nova Scotia, because of a founder effect. Our previous studies, using classic linkage analysis of this large extended kindred, defined the critical gene region to a 13-cM chromosome segment between D18S40 and D18S66. A recently isolated gene from this region, NPC1, is mutated in the majority of patients with Niemann-Pick type C disease. We have identified a point mutation within this gene (G3097-->T; Gly992-->Trp) that shows complete linkage disequilibrium with NPD, confirming that NPD is an allelic variant of NPC1.