The myelin protein PMP2 is regulated by SOX10 and drives melanoma cell invasion.

The transcription factor sex determining region Y-box 10 (SOX10) plays a key role in the development of melanocytes and glial cells from neural crest precursors. SOX10 is involved in melanoma initiation, proliferation, invasion, and survival. However, specific mediators which impart its oncogenic properties remain widely unknown. To identify target genes of SOX10, we performed RNA sequencing after ectopic expression of SOX10 in human melanoma cells. Among nine differentially regulated genes, peripheral myelin protein 2 (PMP2) was consistently upregulated in several cell lines. Direct regulation of PMP2 by SOX10 was shown by chromatin immunoprecipitation, electrophoretic mobility shift, and luciferase reporter assays. Moreover, a coregulation of PMP2 by SOX10 and early growth response 2 in melanoma cells was found. Phenotypical investigation demonstrated that PMP2 expression can increase melanoma cell invasion. As PMP2 protein was detected only in a subset of melanoma cell lines, it might contribute to melanoma heterogeneity.

Changes in gene expression in small bowel neuroendocrine tumors associated with progression to metastases.

BACKGROUND: Small bowel neuroendocrine tumors (SBNETs) present frequently with metastases, yet little is known about the molecular basis of this progression. This study sought to identify the serial differential expression of genes between normal small bowel, primary small bowel neuroendocrine tumors, and liver metastases. METHODS: RNA isolated from matched normal small bowel tissue, primary small bowel neuroendocrine tumors, and liver metastases in 12 patients was analyzed with whole transcriptome expression microarrays and RNA-Seq. Changes in gene expression between primary small bowel neuroendocrine tumors and normal small bowels, and liver metastases versus primary small bowel neuroendocrine tumors were calculated. Common genes that were differentially expressed serially (increasing or decreasing from normal small bowel to primary small bowel neuroendocrine tumors to liver metastases) were identified, and 10 were validated using qPCR. RESULTS: Use of 2 transcriptome platforms allowed for a robust discrimination of genes important in small bowel neuroendocrine tumors progression. Serial differential expression was validated in 7/10 genes, all of which had been described previously in abdominal cancers, and with several interacting with members of the AKT, MYC, or MAPK3 pathways. Liver metastases had consistent underexpression of PMP22, while high expression of SERPINA10 and SYT13 was characteristic of both pSBTs and liver metastases. CONCLUSION: Identification of the serial differential expression of genes from normal tissues to primary tumors to metastases lends insight into important pathways for SBNETs progression. Differential expression of various genes, including PMP22, SYT13 and SERPINA10, are associated with the progression of SBNETs and warrant further investigation.

B1-induced caspase-independent apoptosis in MCF-7 cells is mediated by down-regulation of Bcl-2 via p53 binding to P2 promoter TATA box.

The Bcl-2 family contains a panel of proteins which are conserved regulators of apoptosis in mammalian cells, like the anti-apoptotic protein Bcl-2. According to its significant role in altering susceptibility to apoptosis, the deciphering of the mechanism of Bcl-2 expression modulation may be crucial for identifying therapeutics strategies for cancer. Treatment with naphthalimide-based DNA intercalators, including M2-A and R16, generally leads to a decrease in Bcl-2 intracellular amounts. Whereas the interest for these chemotherapeutics is accompanied by advances in the fundamental understanding of their anticancer properties, the molecular mechanism underlying changes in Bcl-2 expression remains poorly understood. We report here that p53 contributes to Bcl-2 down-regulation induced by B1, a novel naphthalimide-based DNA intercalating agent. Indeed, the decrease in Bcl-2 protein levels observed during B1-induced apoptosis was correlated to the decrease in mRNA levels, as a result of the inhibition of Bcl-2 transcription and promoter activity. In this context, we evaluated p53 contribution in the Bcl-2 transcriptional down-regulation. We found a significant increase of p53 binding to P(2) promoter TATA box in MCF7 cells by chromatin immunoprecipitation. These data suggest that B1-induced caspase-independent apoptosis in MCF-7 cells is associated with the activation of p53 and the down-regulation of Bcl-2. Our study strengthens the links between p53 and Bcl-2 at a transcriptional level, upon naphthalimide-based DNA intercalator treatment.

Improved glucose and lipid metabolism in genetically obese mice lacking aP2.

Adipocyte fatty acid-binding protein, aP2, is a member of the intracellular fatty acid binding protein family. Previously, studies have shown increased insulin sensitivity in aP2-deficient mice with dietary obesity. Here, we asked whether aP2-related alterations in lipolytic response and insulin production are features of obesity-induced insulin resistance and investigated the effects of aP2-deficiency on glucose homeostasis and lipid metabolism in ob/ob mice, a model of extreme obesity. ob/ob mice homozygous for the aP2 null allele (ob/ ob-aP2-/-) became more obese than ob/ob mice as indicated by significantly increased body weight and fat pad size but unaltered body length. However, despite their extreme adiposity, ob/ob-aP2-/- animals were more insulin-sensitive compared with ob/ob controls, as demonstrated by significantly lower plasma glucose and insulin levels and better performance in both insulin and glucose tolerance tests. These animals also showed improvements in dyslipidemia and had lower plasma triglyceride and cholesterol levels. Lipolytic response to beta-adrenergic stimulation and lipolysis-associated insulin secretion was significantly reduced in ob/ob-aP2-/- mice. Interestingly, glucose-stimulated insulin secretion, while virtually abolished in ob/ob controls, was significantly improved in ob/ob-aP2-/- animals. There were no apparent morphological differences in the structure or size of the pancreatic islets between genotypes. Taken together, the data indicate that in obesity, aP2-deficiency not only improves peripheral insulin resistance but also preserves pancreatic beta cell function and has beneficial effects on lipid metabolism.

Plasma FFA utilization and fatty acid-binding protein content are diminished in type 2 diabetic muscle.

In this study, we investigated the hypothesis that impairments in forearm skeletal muscle free fatty acid (FFA) metabolism are present in patients with type 2 diabetes both in the overnight fasted state and during beta-adrenergic stimulation. Eight obese subjects with type 2 diabetes and eight nonobese controls (Con) were studied using the forearm balance technique and indirect calorimetry during infusion of the stable isotope tracer [U-(13)C]palmitate after an overnight fast and during infusion of the nonselective beta-agonist isoprenaline (Iso, 20 ng. kg lean body mass(-1) x min(-1)). Additionally, activities of mitochondrial enzymes and of cytoplasmatic fatty acid-binding protein (FABP) were determined in biopsies from the vastus lateralis muscle. Both during fasting and Iso infusion, the tracer balance data showed that forearm muscle FFA uptake (Con vs. type 2: fast 449+/-69 vs. 258 +/-42 and Iso 715+/-129 vs. 398+/-70 nmol. 100 ml tissue(-1) x min(-1), P<0.05) and FFA release were lower in type 2 diabetes compared with Con. Also, the oxidation of plasma FFA by skeletal muscle was blunted during Iso infusion in type 2 diabetes (Con vs. type 2: Iso 446 +/- 274 vs. 16+/-70 nmol. 100 ml tissue(-1) x min(-1), P<0.05). The net forearm glycerol release was increased in type 2 diabetic subjects (P< 0.05), which points to an increased forearm lipolysis. Additionally, skeletal muscle cytoplasmatic FABP content and the activity of muscle oxidative enzymes were lowered in type 2 diabetes. We conclude that the uptake and oxidation of plasma FFA are impaired in the forearm muscles of type 2 diabetic subjects in the overnight fasted state with and without Iso stimulation.

The mineralocorticoid receptor mediates aldosterone-induced differentiation of T37i cells into brown adipocytes.

By use of targeted oncogenesis, a brown adipocyte cell line was derived from a hibernoma of a transgenic mouse carrying the proximal promoter of the human mineralocorticoid receptor (MR) linked to the SV40 large T antigen. T37i cells remain capable of differentiating into brown adipocytes upon insulin and triiodothyronine treatment as judged by their ability to express uncoupling protein 1 and maintain MR expression. Aldosterone treatment of undifferentiated cells induced accumulation of intracytoplasmic lipid droplets and mitochondria. This effect was accompanied by a significant and dose-dependent increase in intracellular triglyceride content (half-maximally effective dose 10(-9) M) and involved MR, because it was unaffected by RU-38486 treatment but was totally abolished in the presence of aldosterone antagonists (spironolactone, RU-26752). The expression of early adipogenic gene markers, such as lipoprotein lipase, peroxisome proliferator-activated receptor-gamma, and adipocyte-specific fatty acid binding protein 2, was enhanced by aldosterone, confirming activation of the differentiation process. We demonstrate that, in the T37i cell line, aldosterone participates in the very early induction of brown adipocyte differentiation. Our findings may have a broader biological significance and suggest that MR is not only implicated in maintaining electrolyte homeostasis but could also play a role in metabolism and energy balance.

The fatty acid transport function of fatty acid-binding proteins.

The intracellular fatty acid-binding proteins (FABPs) comprise a family of 14-15 kDa proteins which bind long-chain fatty acids. A role for FABPs in fatty acid transport has been hypothesized for several decades, and the accumulated indirect and correlative evidence is largely supportive of this proposed function. In recent years, a number of experimental approaches which more directly examine the transport function of FABPs have been taken. These include molecular level in vitro modeling of fatty acid transfer mechanisms, whole cell studies of fatty acid uptake and intracellular transfer following genetic manipulation of FABP type and amount, and an examination of cells and tissues from animals engineered to lack expression of specific FABPs. Collectively, data from these studies have provided strong support for defining the FABPs as fatty acid transport proteins. Further studies are necessary to elucidate the fundamental mechanisms by which cellular fatty acid trafficking is modulated by the FABPs.

Fatty acid binding proteins from different tissues show distinct patterns of fatty acid interactions.

Fatty acid binding proteins (FABP) form a family of proteins displaying tissue-specific expression. These proteins are involved in fatty acid (FA) transport and metabolism by mechanisms that also appear to be tissue-specific. Cellular retinoid binding proteins are related proteins with unknown roles in FA transport and metabolism. To better understand the origin of these tissue-specific differences we report new measurements, using the acrylodated intestinal fatty acid binding protein (ADIFAB) method, of the binding of fatty acids (FA) to human fatty acid binding proteins (FABP) from brain, heart, intestine, liver, and myelin. We also measured binding of FA to a retinoic acid (CRABP-I) and a retinol (CRBP-II) binding protein and we have extended to 19 different FA our characterization of the FA-ADIFAB and FA-rat intestinal FABP interactions. These studies extend our previous analyses of human FABP from adipocyte and rat FABPs from heart, intestine, and liver. Binding affinities varied according to the order brain approximately myelin approximately heart > liver > intestine > CRABP > CRBP. In contrast to previous studies, no protein revealed a high degree of selectivity for particular FA. The results indicate that FA solubility (hydrophobicity) plays a major role in governing binding affinities; affinities tend to increase with increasing hydrophobicity (decreasing solubility) of the FA. However, our results also reveal that, with the exception of the intestinal protein, FABPs exhibit an additional attractive interaction for unsaturated FA that partially compensates for their trend toward lower affinities due to their higher aqueous solubilities. Thermodynamic potentials were determined for oleate and arachidonate binding to a subset of the FABP and retinoid binding proteins. FA binding to all FABPs was enthalpically driven. The DeltaH degrees values for paralogous FABPs, proteins from the same species but different tissues, reveal an exceptionally wide range of values, from -22 kcal/mol (myelin) to -7 kcal/mol (adipocyte). For orthologous FABPs from the same tissue but different species, DeltaH degrees values were similar. In contrast to the enthalpic dominance of FA binding to FABP, binding of FA to CRABP-I was entropically driven. This is consistent with the notion that FA specificity for FABP is determined by the enthalpy of binding. Proteins from different tissues also revealed considerable heterogeneity in heat capacity changes upon FA binding, DeltaC(p) values ranged between 0 and -1.3 kcal mol(-1) K(-1). The results demonstrate that thermodynamic parameters are quite different for paralogous but are quite similar for orthologous FABP, suggesting tissue-specific differences in FABP function that may be conserved across species.

Fenofibrate and rosiglitazone lower serum triglycerides with opposing effects on body weight.

Activators of peroxisome proliferator activated receptors (PPARs) are effective drugs to improve the metabolic abnormalities linking hypertriglyceridemia to diabetes, hyperglycemia, insulin-resistance, and atherosclerosis. We compared the pharmacological profile of a PPARalpha activator, fenofibrate, and a PPARgamma activator, rosiglitazone, on serum parameters, target gene expression, and body weight gain in (fa/fa) fatty Zucker rats and db/db mice as well as their association in db/db mice. Fenofibrate faithfully modified the expression of PPARalpha responsive genes. Rosiglitazone increased adipose tissue aP2 mRNA in both models while increasing liver acyl CoA oxidase mRNA in db/db mice but not in fatty Zucker rats. Both drugs lowered serum triglycerides yet rosiglitazone markedly increased body weight gain while fenofibrate decreased body weight gain in fatty Zucker rats. KRP 297, which has been reported to be a PPARalpha and gamma co-activator, also affected serum triglycerides and insulin in fatty Zucker rats although no change in body weight gain was noted. These results serve to clearly differentiate the metabolic finality of two distinct classes of drugs, as well as their corresponding nuclear receptors, having similar effects on serum triglycerides.

Isolation, characterization and binding properties of two rat liver fatty acid-binding protein isoforms.

Mammalian liver has only one fatty acid-binding protein (L-FABP) while the liver of non-mammalian vertebrates expresses a liver basic FABP (Lb-FABP) in addition to other members of the FABP family. We explore the possibility that L-FABP isoforms accomplish, in the liver of mammals, the metabolic functions corresponding to the different FABPs present in the liver of non-mammalian vertebrates. We have isolated isoforms I and II which have a different residue 105, Asn in the former and Asp in the latter. We made a conformational comparison of the apo-isoforms by intrinsic fluorescence emission and fourth-derivative spectroscopy, native-state proteolysis and unfolding curves. Ligand affinity was studied by measuring cis-parinaric acid displacement by different ligands. They have differences in their molecular conformation, including the environment of the binding site. Isoform II has probably a more open conformation than isoform I, thus allowing the binding of a greater variety of ligands. The affinity of isoform II for lysophospholipids, prostaglandins, retinoids, bilirubin and bile salts is greater than that of isoform I. These characteristics of rat L-FABP isoforms I and II suggest that they may accomplish different functions as happens with those of the different FABP types in non-mammalian species.

Biochemical alterations in rat Thiry-Vella fistulas.

In order to obtain baseline information on the secretory function of normal rat bowel for our work on intestinal graft ischemia, we studied several biochemical parameters in rat Thiry-Vella fistulas (TVF). TVFs were created in 200-g male Lewis rats (n = 11) using the 25-cm segment of jejunum normally used as a graft in our intestinal transplant model. The stomas were matured primarily and the animals were allowed to recover. The TVFs were flushed at 0, 6, and 24 h and then daily for up to 21 days with 12 mL normal saline solution. The effluent was collected and analyzed for total protein (TP), secretory phospholipase A2 (sPLA2), intestinal fatty acid binding protein (I-FABP), lactate dehydrogenase (LDH), and N-acetylglucosamine (NAGA). TP content was 0.12 +/- 0.01 mg/mL up to 48 h, then gradually increased and stabilized at 0.39 +/- 0.05 mg/mL at day 21. By sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), one major protein band was identified in the low-molecular-mass range (15 kD), consistent with I-FABP and sPLA2. Secretory PLA2 levels decreased over the first 4 days to a low of 115 +/- 24.8% hydrolysis/min/fraction, then gradually rose to a plateau at approximately 529.76 +/- 88.36% hydrolysis/min/fraction by day 18. I-FABP levels rose rapidly from 0 ng/mL at 2 h to 900 +/- 250.0 ng/mL at 6 h and approximately 3000 +/- 304.9 ng/mL by day 14. LDH levels at 2 h and 48 h did not differ, with 0.03 +/- 0.004 and 0.03 +/- 0.005 optical density units (OD)/min/mL, respectively. NAGA levels were 0.07 +/- 0.05 OD/h/mL at 2 h and rose to 0.14 +/- 0.04 OD/h/mL at 48 h. These data suggest that after an early equilibration period, biochemical secretion into the lumen of normal rat bowel reaches a state of equilibrium, and therefore appears to reflect the baseline biochemical status of the bowel. Some of these levels are not negligible as one would expect in "normal" bowel. This information should prove extremely helpful as a baseline study of abnormal conditions of the intestine, such as ischemia or rejection.

Distribution of peroxisome proliferator-activated receptors (PPARs) in human skeletal muscle and adipose tissue: relation to insulin action.

AIMS/HYPOTHESIS: To evaluate the tissue distribution and possible role of the peroxisome proliferator-activated receptors (PPARs) in insulin action in fat and muscle biopsy specimens from lean, obese and subjects with Type II (non-insulin-dependent) diabetes mellitus. METHODS: We measured PPAR alpha, PPAR beta (delta) and PPAR gamma protein expression by western blot analysis. The PPAR gamma protein was also measured in muscle before and after 3-h hyperinsulinaemic (300 mU.m-2.min-1) euglycaemic clamps. RESULTS: The PPAR alpha protein was expressed preferentially in muscle relative to fat (more than sevenfold). The PPAR beta protein was similar in fat and muscle. The amount of PPAR gamma protein found in muscle was, on average, two-thirds of that present in fat. There was no statistically significant difference between non-diabetic and diabetic subjects in baseline (preclamp) muscle PPAR (alpha, beta or gamma) protein expression. Subgroup analysis showed, however, significantly higher PPAR gamma protein in the most insulin resistant diabetic subjects with glucose disposal rates of 3-6 mg.kg-1.min-1 compared with their age and weight matched counterparts with glucose disposal rates of 6-9 (147 +/- 23 vs 88 +/- 10 AU/microgram protein, p < or = 0.01 in diabetic and vs 94 +/- 15, p < or = 0.04 in non-diabetic subjects). Muscle PPAR gamma protein and glucose disposal rates were inversely correlated in diabetic subjects (r = -0.47, p < or = 0.05). CONCLUSION/INTERPRETATION: All PPARs (alpha, beta or gamma) are present in skeletal muscle and adipose tissue with different relative distributions. The PPAR gamma protein is abundant in skeletal muscle as well as adipose tissue. The altered expression of skeletal muscle PPAR gamma is consistent with a role for this nuclear protein in the impaired insulin action of Type II diabetes.

Nucleotide sequence of a cDNA clone coding for an intestinal-type fatty acid binding protein and its tissue-specific expression in zebrafish (Danio rerio).

We have cloned a cDNA from zebrafish (Danio rerio) that contains an open-reading frame of 132 amino acids coding for a fatty acid binding protein (FABP) of approximately 15 kDa. Multiple sequence alignment revealed extensive amino acid identity between this zebrafish FABP and intestinal-like FABPs (I-FABP) from other species. The zebrafish I-FABP cDNA hybridized to single restriction fragments of total zebrafish genomic DNA digested with the restriction endonucleases PstI Bg/II or EcoRI suggesting that a single copy of the I-FABP gene is present in the zebrafish genome. An oligonucleotide probe complementary to the zebrafish I-FABP mRNA hybridized to an mRNA of approximately 800 bases in Northern blot analysis. In situ hybridization revealed that the I-FABP mRNA was expressed exclusively in the intestine of the adult zebrafish.

Uptake and activation of eicosapentaenoic acid are related to accumulation of triacylglycerol in Ramos cells dying from apoptosis.

The present study investigates the mechanism behind induction of cell death by eicosapentaenoic acid (EPA) in leukemia cells. The PUFA-sensitive cell lines Raji and Ramos, which die by necrosis and apoptosis upon incubation with EPA respectively, had 2- to 3-fold higher uptake rate of EPA than the PUFA-resistant U-698 cell line. Furthermore, Ramos cells contained more lipid bodies and 3-fold more triacylglycerol than U-698 cells after 24 h incubation with 60 microm EPA. The mechanism behind the increased rate of EPA uptake in the PUFA-sensitive cell lines was examined by comparing the expression of 6 different fatty acid binding proteins (FABPs) and 3 acyl-CoA synthetases (ACSs) in U-698 and Ramos cells. Moreover, enzymatic activity of ACS and acyl-CoA:1,2-diacylglycerol acyltransferase (ADGAT) was investigated. The protein expression level of CD36 and p-FABPpm, the mRNA level of FABP, liver-FABP, heart-FABP, intestinal-FABP, ACS1, ACS2, and enzymatic ADGAT activity were similar in the two cell lines. However, an mRNA signal observed with a probe for ACS3 was 1.7 times higher in Ramos than in U-698 cells, and lysate from Ramos cells had a higher capacity to activate EPA to EPA-CoA than U-698 cell lysate. In conclusion, the present findings indicate that cellular uptake, activation and incorporation of EPA into lipids may be related to induction of cell death in leukemia cell lines.

Liver and intestinal fatty acid-binding proteins obtain fatty acids from phospholipid membranes by different mechanisms.

Intestinal enterocytes contain high concentrations of two cytosolic fatty acid-binding proteins (FABP), liver FABP (L-FABP) and intestinal FABP (I-FABP), which are hypothesized to play a role in cellular fatty acid trafficking. The mechanism(s) by which fatty acids move from membranes to each of these proteins is not known. Here we demonstrate that fluorescent anthroyloxy fatty acid analogues (AOFA) are transferred from phospholipid vesicles to L-FABP versus I-FABP by different mechanisms. For L-FABP a diffusion-mediated transfer process is demonstrated. The AOFA transfer rate from phosphatidylcholine-containing vesicles (POPC) to L-FABP is similar to that observed with another diffusional process, namely inter-membrane AOFA transfer. Furthermore, the AOFA transfer rate was modulated by buffer ionic strength and AOFA solubility, while the transfer rate remained relatively unchanged by the presence of anionic phospholipids in vesicles. In contrast, the data for I-FABP suggest that a transient collisional interaction of I-FABP with the phospholipid membrane occurs during AOFA extraction from the vesicles by the protein. In particular, the presence of the anionic phospholipid cardiolipin in donor vesicles increased the rate of AOFA transfer to I-FABP by 15-fold compared with transfer to POPC vesicles. The effects of ionic strength on transfer suggest that the interaction of I-FABP with cardiolipin-containing vesicles is likely to contain an electrostatic component. Finally, based on the regulation of AOFA transfer to I-FABP compared with transfer from I-FABP, it is hypothesized that apo- and holo-I-FABPs adopt conformations which may differentially promote I-FABP-membrane interactions. In summary, the results suggest that I-FABP, but not L-FABP, can directly extract fatty acids from membranes, supporting the concept that I-FABP may increase the cytosolic flux of fatty acids via intermembrane transfer.

Intestinal fatty acid binding protein may favor differential apical fatty acid binding in the intestine.

The intestinal mucosa metabolizes fatty acids differently when presented to the lumenal or basolateral membrane. Expression of both liver and intestinal fatty acid binding proteins (L- and I-FABPs) uniquely in the enterocyte offers a possible explanation of this phenomenon. An organ explant system was used to analyze the relative binding of fatty acids to each protein. More fatty acid was bound to L-FABP than to I-FABPs (28% vs. 6% of cytosolic radioactivity), no matter on which side the fatty acid was added. However, a 2-3-fold increase in fatty acid binding to the intestinal paralog was noted after apical addition of palmitic or oleic acid in mucosa from chow fed rats. When oleic acid was added apically, a 1.4-fold increase in binding to I-FABP was observed in mucosa derived from chronically fat fed rats, consistent with the previously observed 50% increase in the content of that protein. Immunocytochemical localization of both FABPs in vivo demonstrated an apical cytoplasmic localization in the fasting state, and redistribution to the entire cytoplasm after fat feeding. These data are consistent with the hypothesis that I-FABP may contribute to the metabolic compartmentalization of apically presented fatty acids in the intestine.

Beta-sheet proteins with nearly identical structures have different folding intermediates.

The folding mechanisms of two proteins in the family of intracellular lipid binding proteins, ileal lipid binding protein (ILBP) and intestinal fatty acid binding protein (IFABP), were examined. The structures of these all-beta-proteins are very similar, with 123 of the 127 amino acids of ILBP having backbone and C(beta) conformations nearly identical to those of 123 of the 131 residues of IFABP. Despite this structural similarity, the sequences of these proteins have diverged, with 23% sequence identity and an additional 16% sequence similarity. The folding process was completely reversible, and no significant concentrations of intermediates were observed by circular dichroism or fluorescence at equilibrium for either protein. ILBP was less stable than IFABP with a midpoint of 2. 9 M urea compared to 4.0 M urea for IFABP. Stopped-flow kinetic studies showed that both the folding and unfolding of these proteins were not monophasic, suggesting that either multiple paths or intermediate states were present during these processes. Proline isomerization is unlikely to be the cause of the multiphasic kinetics. ILBP had an intermediate state with molten globule-like spectral properties, whereas IFABP had an intermediate state with little if any secondary structure during folding and unfolding. Double-jump experiments showed that these intermediates appear to be on the folding path for each protein. The folding mechanisms of these proteins were markedly different, suggesting that the different sequences of these two proteins dictate different paths through the folding landscape to the same final structure.

Properties and crystal structure of a beta-barrel folding mutant.

A mutant of a beta-barrel protein, rat intestinal fatty acid binding protein, was predicted to be more stable than the wild-type protein due to a novel hydrogen bond. Equilibrium denaturation studies indicated the opposite: the V60N mutant protein was less stable. The folding transitions followed by CD and fluorescence were reversible and two-state for both mutant and wild-type protein. However, the rates of denaturation and renaturation of V60N were faster. During unfolding, the initial rate was associated with 75-80% of the fluorescence and all of the CD amplitude change. A subsequent rate accounted for the remaining fluorescence change for both proteins; thus the intermediate state lacked secondary structure. During folding, one rate was detected by both fluorescence and CD after an initial burst phase for both wild-type and mutant. An additional slower folding rate was detected by fluorescence for the mutant protein. The structure of the V60N mutant has been obtained and is nearly identical to prior crystal structures of IFABP. Analysis of mean differences in hydrogen bond and van der Waals interactions did not readily account for the stability loss due to the mutation. However, significant average differences of the solvent accessible surface and crystallographic displacement factors suggest entropic destabilization.

Secretion of a novel class of iFABPs in nematodes: coordinate use of the Ascaris/Caenorhabditis model systems.

A novel fatty acid binding protein, As-p18, is secreted into both the perivitelline and perienteric fluids of the parasitic nematode, Ascaris suum, and at least eight potential homologues of As-p18 have been identified in the Caenorhabditis elegans genome. The products of the three most closely related homologues are fatty acid binding proteins (LBP-1, LBP-2 and LBP-3) which contain putative secretory signals. Phylogenetic analysis revealed that these secreted fatty acid binding proteins comprise a distinct gene class within the fatty acid binding protein family and are possibly unique to nematodes. To examine the potential sites of As-p18 secretion, the expression of the putative promoters of the C. elegans homologues was examined with GFP reporter constructs. The developmental expression of lbp-1 was identical to that of As-p18 and consistent with the secretion of LBP-1 from the hypodermis to the perivitelline fluid. The expression patterns of lbp-2 and lbp-3 were consistent with the secretion of LBP-2 and LBP-3 from muscle into the perienteric fluid later in development. These studies demonstrate that at least some perivitelline fluid proteins appear to be secreted from the hypodermis prior to the formation of the cuticle and, perhaps more importantly, that this coordinate C. elegans/A. suum approach may be potentially useful for examining a number of key physiological processes in parasitic nematodes.