Differentiation of human adipose stromal cells in vitro into insulin-sensitive adipocytes.

Adipose tissue-related diseases such as obesity and type 2 diabetes are worldwide epidemics. In order to develop adipose tissue cultures in vitro that mimic more faithfully the in vivo physiology, new well-characterized and publicly accepted differentiation methods of human adipose stem cells are needed. The aims of this study are (1) to improve the existing natural adipose tissue extract (ATE)-based induction method and (2) to study the effects of a differentiation method on insulin responsiveness of the resulting adipocytes. Different induction media were applied on human adipose stromal cell (hASC) monocultures to study the differentiation capacity of the induction media and the functionality of the differentiated adipocytes. Cells were differentiated for 14 days to assess triglyceride accumulation per cell and adipocyte-specific gene expression (PPARγ, adiponectin, AP2, leptin, Glut4, Prdm16, CIDEA, PGC1-α, RIP140, UCP and ADCY5). Insulin response was studied by measuring glucose uptake and inhibition of lipolysis after incubation with 100 or 500 nM insulin. The selected differentiation method included a 3-day induction with ATE, 6 days in serum-free medium supplemented with 1.15 µM insulin and 9.06 µM Troglitazone, followed by 4 days in a defined serum- and insulin-free stimulation medium. This protocol induced prominent general adipocyte gene expression, including markers for both brown and white adipocytes and triglyceride accumulation. Moreover, the cells were sensitive to insulin as observed from increased glucose uptake and inhibition of lipolysis. This differentiation protocol provides a promising approach for the induction of hASC adipogenesis to obtain functional and mature human adipocytes.

Adipose tissue is inflamed in NAFLD due to obesity but not in NAFLD due to genetic variation in PNPLA3.

AIMS/HYPOTHESIS: The rs738409 C>G single-nucleotide polymorphism in PNPLA3 leads to a missense mutation (I148M) which increases liver fat but does not cause insulin resistance. We hypothesised that patients with non-alcoholic fatty liver disease (NAFLD) due to the PNPLA3 variant ('PNPLA3 NAFLD' = PNPLA3-148MM) do not have adipose tissue (AT) inflammation in contrast with those with NAFLD due to obesity ('obese NAFLD'). METHODS: Biopsy specimens of AT were taken, and PNPLA3 genotype and liver fat ((1)H-magnetic resonance spectroscopy) were determined in 82 volunteers, who were divided into groups based on either median BMI (obese 36.2 ± 0.7 kg/m(2); non-obese 26.0 ± 0.4 kg/m(2)) or PNPLA3 genotype. All groups were similar with respect to age and sex. The PNPLA3 subgroups were equally obese (PNPLA3-148MM, 31.1 ± 1.3 kg/m(2); PNPLA3-148II, 31.2 ± 0.8 kg/m(2)), while the obese and non-obese subgroups had similar PNPLA3 genotype distribution. Gene expression of proinflammatory (MCP-1, CD68) and anti-inflammatory (Twist1, ADIPOQ) markers was measured using quantitative real-time RT-PCR. RESULTS: Liver fat was similarly increased in obese NAFLD (9.5 ± 1.3% vs 5.1 ± 0.9%, obese vs non-obese, p = 0.007) and PNPLA3 NAFLD (11.4 ± 1.7% vs 5.3 ± 0.8%, PNPLA3-148MM vs PNPLA3-148II, p < 0.001). Fasting serum insulin was higher in the obese than the non-obese group (76 ± 6 vs 47 ± 6 pmol/l, p < 0.001), but similar in PNPLA3-148MM and PNPLA3-148II (60 ± 8 vs 62 ± 5 pmol/l, NS). In obese vs non-obese, MCP-1 and CD68 mRNAs were upregulated, whereas those of Twist1 and ADIPOQ were significantly downregulated. AT gene expression of MCP-1, CD68, Twist1 and ADIPOQ was similar in PNPLA3-148MM and PNPLA3-148II groups. CONCLUSIONS/INTERPRETATION: PNPLA3 NAFLD is characterised by an increase in liver fat but no insulin resistance or AT inflammation, while obese NAFLD has all three of these features.

The OSBP-related proteins (ORPs): global sterol sensors for co-ordination of cellular lipid metabolism, membrane trafficking and signalling processes?

Protein families related to OSBP (oxysterol-binding protein) are present in eukaryotes from yeast to human. The functions of the ORPs (OSBP-related proteins) have remained largely enigmatic. Even though they have been implicated in the function of ERJs (endoplasmic reticulum junctions), it is evident that any single model for their mechanism of action is insufficient. The existing evidence points in many different directions, such as integration of sterol and sphingomyelin metabolism, regulation of neutral lipid metabolism, control of signalling cascades, regulation of secretory vesicle generation, and function in the microtubule-based motility of endo/lysosomes. Some of these functions could involve ERJ and non-vesicular transport of lipids, but this is unlikely to be the unifying feature. We believe, rather, that the common denominator for ORP function is acting as sterol sensors that relay information to a spectrum of cellular processes.

The OSBP-related protein family in humans.

Oxysterols are oxygenated derivatives of cholesterol that have a number of biological effects and play a key role in the maintenance of the body cholesterol balance. In this study, we describe the cDNA sequences and genomic structures of the recently identified human oxysterol-binding protein (OSBP)-related protein (ORP) family (Laitinen, S. et al. 1999. J. Lipid Res. 40: 2204-2211). The family now includes 12 genes/proteins, which can be divided into six distinct subfamilies. The ORP have two major structural features: a highly conserved OSBP-type sterol-binding domain in the C-terminal half and a pleckstrin homology domain present in the N-terminal region of most family members. Several ORP genes are present in S. cerevisiae, D. melanogaster, and C. elegans, suggesting that the protein family has functions of fundamental importance in the eukaryotic kingdom. Analysis of ORP mRNA levels in unloaded or acetylated LDL-loaded human macrophages revealed that the expression of ORP genes was not significantly affected by the loading, with the exception of ORP6, which was up-regulated 2-fold. The present study summarizes the basic characteristics of the OSBP-related gene/protein family in humans, and provides tools for functional analysis of the encoded proteins.

The Rab GTPase family.

SUMMARY: The Rab family is part of the Ras superfamily of small GTPases. There are at least 60 Rab genes in the human genome, and a number of Rab GTPases are conserved from yeast to humans. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane traffic pathways. In the GTP-bound form, the Rab GTPases recruit specific sets of effector proteins onto membranes. Through their effectors, Rab GTPases regulate vesicle formation, actin- and tubulin-dependent vesicle movement, and membrane fusion.

Dynamic changes in mouse lipoproteins induced by transiently expressed human phospholipid transfer protein (PLTP): importance of PLTP in prebeta-HDL generation.

The plasma phospholipid transfer protein (PLTP) plays an important role in the regulation of plasma high density lipoprotein (HDL) levels and governs the distribution of HDL sub-populations. In the present study, adenovirus mediated overexpression of human PLTP in mice was employed to investigate the distribution of PLTP in serum and its effect on plasma lipoproteins. Gel filtration experiments showed that the distributions of PLTP activity and mass in serum are different, suggesting that human PLTP circulated in mouse plasma as two distinct forms, one with high and the other with low specific activity. Our study further demonstrates that overexpression of PLTP leads to depletion of HDL and that, as PLTP activity declines, replenishment of the HDL fraction occurs. During this process, the lipoprotein profile displays transient particle populations, including apoA-IV and apoE-rich particles in the LDL size range and small particles containing apoA-II only. The possible role of these particles in HDL reassembly is discussed. The increased PLTP activity enhanced the ability of mouse sera to produce pre(beta)-HDL. The present results provide novel evidence that PLTP is an important regulator of HDL metabolism and plays a central role in the reverse cholesterol transport (RCT) process.

The impact of phospholipid transfer protein (PLTP) on HDL metabolism.

High-density lipoproteins (HDL) play a major protective role against the development of coronary artery disease. Phospholipid transfer protein (PLTP) is a main factor regulating the size and composition of HDL in the circulation and plays an important role in controlling plasma HDL levels. This is achieved via both the phospholipid transfer activity of PLTP and its capability to cause HDL conversion. The present review focuses on the impact of PLTP on HDL metabolism. The basic characteristics and structure of the PLTP protein are described. The two main functions of PLTP, PLTP-mediated phospholipid transfer and HDL conversion are reviewed, and the mechanisms and control, as well as the physiological significance of these processes are discussed. The relationship between PLTP and the related cholesteryl ester transfer protein (CETP) is reviewed. Thereafter other functions of PLTP are recapitulated: the ability of PLTP to transfer cholesterol, alpha-tocopherol and lipopolysaccharide (LPS), and the suggested involvement of PLTP in cellular cholesterol traffic. The discussion on PLTP activity and mass in (patho)physiological settings includes new data on the presence of two forms of PLTP in the circulation, one catalytically active and the other inactive. Finally, future directions for PLTP research are outlined.

Phospholipid transfer is a prerequisite for PLTP-mediated HDL conversion.

Phospholipid transfer protein (PLTP) is an important regulator of high-density lipoprotein (HDL) metabolism. The two main functions of PLTP are transfer of phospholipids between lipoprotein particles and modulation of HDL size and composition in a process called HDL conversion. These PLTP-mediated processes are physiologically important in the transfer of surface remnants from lipolyzed triglyceride-rich lipoproteins to nascent HDL particles and in the generation of prebeta-HDL, the initial acceptor of excess peripheral cell cholesterol. The aim of the study presented here was to investigate the interrelationship between the two functions of PLTP. Plasma PLTP was chemically modified using diethylpyrocarbonate or ethylmercurithiosalicylate. The modified proteins displayed a dose-dependent decrease in phospholipid transfer activity and a parallel decrease in the ability to cause HDL conversion. Two recombinant PLTP mutant proteins, defective in phospholipid transfer activity due to a mutation in the N-terminal lipid-binding pocket, were produced, isolated, and incubated together with radioactively labeled HDL(3). HDL conversion was analyzed using three methods: native gradient gel electrophoresis, ultracentrifugation, and crossed immunoelectrophoresis. The results demonstrate that the mutant proteins (i) are able to induce only a modest increase in HDL particle size compared to the wild-type protein, (ii) are unable to release apoA-I from HDL(3), and (iii) do not generate prebeta-mobile particles following incubation with HDL(3). These data suggest that phospholipid transfer is a prerequisite for HDL conversion and demonstrate the close interrelationship between the two main activities of PLTP.

Distribution of phospholipid transfer protein in human plasma: presence of two forms of phospholipid transfer protein, one catalytically active and the other inactive.

Plasma phospholipid transfer protein (PLTP) plays an important role in the maintenance of plasma high-density lipoprotein (HDL) content and remodeling of HDL in the circulation. In the present study we have used different fractionation methods to investigate the distribution of PLTP in human plasma. A novel enzyme-linked immunosorbent assay developed during the study allowed for simultaneous assessment of both PLTP mass and activity in the fractions obtained. Size-exclusion chromatography and plasma fractionation by nondenaturing polyacrylamide gel electrophoresis (PAGE) yielded similar results demonstrating that PLTP associates in native plasma with two distinct particle populations, while ultracentrifugation with high salt leads to detachment of PLTP from lipoprotein particles and loss of a majority of its phospholipid transfer activity. Interestingly, analysis of the size-exclusion chromatography fractions demonstrated that PLTP exists in the circulation as an active population that elutes in the position of HDL corresponding to an average molecular mass of 160+/-40 kDa and an inactive form with an average mass of 520+/-120 kDa. The inactive fraction containing approximately 70% of the total PLTP protein eluted between HDL and low density lipoprotein (LDL). Thus, the two PLTP pools are associated with different types of lipoprotein particles, suggesting that the PLTP activity in circulation is modulated by the plasma lipoprotein profile and lipid composition.

Selective targeting of avidin/mannose 6-phosphate receptor chimeras to early or late endosomes.

In this study we have used the Semliki forest virus expression system to transiently express chimeric proteins that contain transmembrane and cytoplasmic domains of the cation-independent mannose 6-phosphate receptor (CI-MPR) fused to chicken avidin. Immunofluorescence and electron microscopy studies showed that the chimeric protein with the entire cytoplasmic domain of CI-MPR was transported to late endosomes, where it accumulated. We made use of the biotin-binding capacity of lumenal avidin, and found that, in agreement with this distribution, the chimeric protein could be labelled with biotinylated HRP endocytosed for a long, but not a brief, period of time. However, truncation of the C-terminal tail distal to the rapid endocytosis motif (YKYSKV), caused the truncated chimera to be transported to, and accumulated within, early endosomes. This truncated chimera did not reach recycling early endosomes labelled with internalised transferrin, to any significant extent, but was accessible to biotinylated HRP internalised for 5 min (or for longer periods at 19 degrees C). Coinfection of these chimeras showed that they follow the same route from the TGN to the early endosomes. We conclude that the sequence distal to the endocytosis motif contains the signals which are required for efficient transport to late endosomes. Our results also suggest that the YKYSKV sequence close to the CI-MPR transmembrane segment is sufficient for targeting to sorting early endosomes.

Quantification of human plasma phospholipid transfer protein (PLTP): relationship between PLTP mass and phospholipid transfer activity.

A sensitive sandwich-type enzyme-linked immunosorbent assay (ELISA) for human plasma phospholipid transfer protein (PLTP) has been developed using a monoclonal capture antibody and a polyclonal detection antibody. The ELISA allows for the accurate quantification of PLTP in the range of 25-250 ng PLTP/assay. Using the ELISA, the mean plasma PLTP concentration in a Finnish population sample (n = 159) was determined to be 15.6 +/- 5.1 mg/l, the values ranging from 2.30 to 33.4 mg/l. PLTP mass correlated positively with HDL-cholesterol (r = 0.36, P < 0.001), apoA-I (r = 0.37, P < 0.001), apoA-II (r = 0.20, P < 0.05), Lp(A-I) (r=0.26, P=0.001) and Lp(A-I/A-II) particles (r=0.34, P<0.001), and negatively with body mass index (BMI) (r = -0.28, P < 0.001) and serum triacylglycerol (TG) concentration (r = -0.34, P < 0.001). PLTP mass did not correlate with phospholipid transfer activity as measured with a radiometric assay. The specific activity of PLTP, i.e. phospholipid transfer activity divided by PLTP mass, correlated positively with plasma TG concentration (r=0.568, P<0.001), BMI (r=0.45, P<0.001), apoB (r = 0.45, P < 0.001). total cholesterol (r=0.42, P < 0.001), LDL-cholesterol (r = 0.34, P < 0.001) and age (r = 0.36, P < 0.001), and negatively with HDL-cholesterol (r= -0.33, P < 0.001), Lp(A-I) (r= -0.21, P < 0.01) as well as Lp(A-I/A-II) particles (r = -0.32, P < 0.001). When both PLTP mass and phospholipid transfer activity were adjusted for plasma TG concentration, a significant positive correlation was revealed (partial correlation, r = 0.31, P < 0.001). The results suggest that PLTP mass and phospholipid transfer activity are strongly modulated by plasma lipoprotein composition: PLTP mass correlates positively with parameters reflecting plasma high density lipoprotein (HDL) levels, but the protein appears to be most active in subjects displaying high TG concentration.

Dissecting the role of the golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface.

In this study, we compared the transport of newly synthesized cholesterol with that of influenza virus hemagglutinin (HA) from the endoplasmic reticulum to the plasma membrane. The arrival of cholesterol on the cell surface was monitored by cyclodextrin removal, and HA transport was monitored by surface trypsinization and endoglycosidase H digestion. We found that disassembly of the Golgi complex by brefeldin A treatment resulted in partial inhibition of cholesterol transport while completely blocking HA transport. Further, microtubule depolymerization by nocodazole inhibited cholesterol and HA transport to a similar extent. When the partitioning of cholesterol into lipid rafts was analyzed, we found that newly synthesized cholesterol began to associate with low-density detergent-resistant membranes rapidly after synthesis, before it was detectable on the cell surface, and its raft association increased further upon chasing. When cholesterol transport was blocked by using 15 degrees C incubation, the association of newly synthesized cholesterol with low-density detergent-insoluble membranes was decreased and cholesterol accumulated in a fraction with intermediate density. Our results provide evidence for the partial contribution of the Golgi complex to the transport of newly synthesized cholesterol to the cell surface and suggest that detergent-resistant membranes are involved in the process.

In vivo interaction of the adapter protein CD2-associated protein with the type 2 polycystic kidney disease protein, polycystin-2.

We identified a developmentally regulated gene from mouse kidney whose expression is up-regulated in metanephrogenic mesenchyme cells when they are induced to differentiate to epithelial cells during kidney organogenesis. The deduced 70.5-kDa protein, originally named METS-1 (mesenchyme-to-epithelium transition protein with SH3 domains), has since been cloned as a CD2-associated protein (CD2AP). CD2AP is strongly expressed in glomerular podocytes, and the absence of CD2AP in mice results in congenital nephrotic syndrome. We have found that METS-1/CD2AP (hereafter referred to as CD2AP) is expressed at lower levels in renal tubular epithelial cells in the adult kidney, particularly in distal nephron segments. Independent yeast two-hybrid screens using the COOH-terminal region of either CD2AP or polycystin-2 as bait identified the COOH termini of polycystin-2 and CD2AP, respectively, as strong interacting partners. This interaction was confirmed in cultured cells by co-immunoprecipitation of endogenous polycystin-2 with endogenous CD2AP and vice versa. CD2AP shows a diffuse reticular cytoplasmic and perinuclear pattern of distribution, similar to polycystin-2, in cultured cells, and the two proteins co-localize by indirect double immunofluorescence microscopy. CD2AP is an adapter molecule that associates with a variety of membrane proteins to organize the cytoskeleton around a polarized site. Such a function fits well with that hypothesized for the polycystin proteins in renal tubular epithelial cells, and the present findings suggest that CD2AP has a role in polycystin-2 function.

Munc18-2, a functional partner of syntaxin 3, controls apical membrane trafficking in epithelial cells.

The Sec1-related proteins bind to syntaxin family t-SNAREs with high affinity, thus controlling the interaction of syntaxins with their cognate SNARE partners. Munc18-2 is a Sec1 homologue enriched in epithelial cells and forms a complex with syntaxin 3, a t-SNARE localized to the apical plasma membrane. We generated here a set of Munc18-2 point mutants with substitutions in conserved amino acid residues. The mutants displayed a spectrum of different syntaxin binding efficiencies. The in vitro and in vivo binding patterns were highly similar, and the association of the Munc18-2 variants with syntaxin 3 correlated well with their ability to displace SNAP-23 from syntaxin 3 complexes when overexpressed in Caco-2 cells. Even the Munc18-2 mutants that do not detectably bind syntaxin 3 were membrane associated in Caco-2 cells, suggesting that the syntaxin interaction is not the sole determinant of Sec1 protein membrane attachment. Overexpression of the wild-type Munc18-2 was shown to inhibit the apical delivery of influenza virus hemagglutinin (HA). Interestingly, mutants unable to bind syntaxin 3 behaved differently in the HA transport assay. While one of the mutants tested had no effect, one inhibited and one enhanced the apical transport of HA. This implies that Munc18-2 function in apical membrane trafficking involves aspects independent of the syntaxin 3 interaction.

Family of human oxysterol binding protein (OSBP) homologues. A novel member implicated in brain sterol metabolism.

Oxysterol binding protein (OSBP) is a cytosolic protein that undergoes ligand-induced binding to the Golgi apparatus and has been implicated in the regulation of cellular cholesterol metabolism. In the yeast Saccharomyces cerevisiae an OSBP homologue is involved in membrane trafficking through the Golgi complex. Prompted by the multitude of OSBP-related genes in the yeast genome, we carried out a search for human expressed sequence tags (ESTs) displaying homology to the sterol-binding domain of OSBP. This revealed a minimum of six novel OSBP-related proteins, designated ORP-1 to ORP-6. ORP cDNA probes were generated by reverse transcription-PCR from human liver mRNA, and used for Northern blot analysis of human tissue transcript panels. This verified that each of them represents a different gene product and showed that they display distinct tissue-specific expression patterns. The ORP-1 and -2 mRNA expression levels were similar to or higher than that of OSBP while the ORP-3 to -6 mRNAs were detected at lower levels in specific tissues. The most abundantly expressed new gene, ORP-1, was transcribed at strikingly high levels in the cortical areas of human brain and displayed sterol-regulated expression in a cultured human neuroblastoma cell line. This indicates that ORP-1 may play an important role in maintaining the sterol balance in cells of the central nervous system. Together with OSBP, the identified gene products constitute a novel human protein family that may provide a link between organellar sterol status and membrane dynamics.

Syntaxin 2 splice variants exhibit differential expression patterns, biochemical properties and subcellular localizations.

The syntaxins are a large protein family implicated in the targeting and fusion of intracellular transport vesicles. A subset of proteins of this family are the four syntaxin 2 splice variants, syntaxins 2A (2), 2B (2'), 2C (2") and 2D. Each syntaxin 2 variant contains an identical, or nearly identical, amino-terminal cytoplasmic domain followed by a distinct hydrophobic (syntaxins 2A and 2B) or hydrophilic (syntaxins 2C and 2D) carboxyl-terminal domain. To investigate whether the difference among the syntaxin 2 variants is functionally important, we have examined comparatively their RNA transcript and protein expression patterns, membrane associations, protein-protein interactions and intracellular localizations. Analysis of the RNA transcript and protein expression patterns demonstrated that syntaxins 2A, 2B and 2C are broadly, but not uniformly, expressed while syntaxin 2D expression is restricted to the brain. Subcellular fractionation studies showed that syntaxins 2A and 2B behave as integral membrane proteins while syntaxin 2C is only partially associated with membranes. In vitro biochemical assays demonstrated that the syntaxin 2 variants exhibit similar yet distinct interactions with other proteins implicated in vesicular trafficking, including SNAP-25, SNAP-23, VAMP-2 and n-sec1. In a variety of nonpolarized cell types, syntaxins 2A and 2B localized to both the plasma membrane and endosomal membranes. However, in two polarized epithelial cell lines, MDCK and Caco-2, syntaxin 2A localized predominantly to the apical plasma membrane while syntaxin 2B was associated with both the apical and the basolateral membranes. These observations indicate that the distinct carboxyl-terminal domains of the syntaxin 2 variants influence their biochemical and localization properties and may therefore confer upon these variants different functional roles in the regulation of intracellular membrane trafficking.

Vesicular transport and kidney development.

Vesicular transport processes play crucial roles in the biogenesis of cellular membranes and in the polarized transport functions of epithelial cells. During the 1990's we have witnessed major progress in elucidation of the machineries responsible for the intracellular membrane trafficking. The components of these machineries are abundant in tissues with a high content of epithelial cells, such as the kidney. However, the developmental role of the membrane trafficking apparatus in higher eukaryotes has been addressed hardly at all. We summarize here data on the presence and the functional role of vesicle transport proteins in the kidney, and describe work addressing the developmentally regulated expression and localization of three molecules suggested to be involved in polarized trafficking in kidney epithelia, Rab17, syntaxin 3, and Munc-18-2. The results show that specialized transport machinery is induced during differentiation of renal epithelia. However, the expression levels of the components under study are highest in the mature structures, indicating that the proteins are predominantly required for the function of mature epithelia and possibly for the maintenance of the polarized phenotype of specific epithelial cells. The proteins are, however, detected at low levels already in earlier, differentiating structures, and could thus also be involved in the differentiation of kidney epithelia.

Syntaxin 3 and Munc-18-2 in epithelial cells during kidney development.

BACKGROUND: Differentiation of epithelial cells involves the assembly of polarized membrane transport machineries necessary for the generation and maintenance of the apical and basolateral membrane domains characteristic of this cell type. We have analyzed the expression patterns of vesicle-docking proteins of the syntaxin family in mouse kidney, focusing on syntaxin 3 and its interaction partner, the Sec1-related Munc-18-2. METHODS: Expression patterns were studied by in situ hybridization and immunocytochemistry and the complex formation of syntaxin 3 and Munc-18-2 by coimmunoprecipitation and Western blotting. RESULTS: We have previously shown by in situ hybridization that Munc-18-2 is present in the proximal tubules and collecting ducts of embryonic day 17 mouse kidney. We compared this with the expression patterns of syntaxin 1A, 2, 3, 4, and 5, and found that syntaxin 3 was enriched in the same epithelial structures in which Munc-18-2 was abundant. By immunocytochemistry, the two proteins colocalized at the apical plasma membrane of proximal tubule and collecting duct epithelial cells, and they were shown to form a physical complex in the kidney. The expression of both proteins was up-regulated during kidney development. The most prominent changes in expression levels coincided with the differentiation of proximal tubules, suggesting a role in the generation of the highly active reabsorption machinery characterizing this segment of the nephron. CONCLUSION: The results show that Munc-18-2 and syntaxin 3 form a complex in vivo and suggest that they participate in epithelial cell differentiation and targeted vesicle transport processes in the developing kidney.

Structure and phospholipid transfer activity of human PLTP: analysis by molecular modeling and site-directed mutagenesis.

The plasma phospholipid transfer protein (PLTP) is an important regulator of high density lipoprotein (HDL) metabolism. We have here, based on sequence alignments of the plasma LPS-binding/lipid transfer protein family and the X-ray structure of the bactericidal/permeability increasing protein (BPI), modeled the structure of PLTP. The model predicts a two-domain architecture with conserved lipid-binding pockets consisting of apolar residues in each domain. By site-directed mutagenesis of selected amino acid residues and transient expression of the protein variants in HeLa cells, the pockets are shown to be essential for PLTP-mediated phospholipid transfer. A solid phase ligand binding assay was used to determine the HDL-binding ability of the mutants. The results suggest that the observed decreases in phospholipid transfer activity of the N-terminal pocket mutants cannot be attributed to altered HDL-binding, but the C-terminal lipid-binding pocket may be involved in the association of PLTP with HDL. Further, the essential structural role of a disulfide bridge between cysteine residues 146 and 185 is demonstrated. The structural model and the mutants characterized here provide powerful tools for the detailed analysis of the mechanisms of PLTP function.