Proteolysis of the carboxyl-terminal GPI signal independent of GPI modification as a mechanism for selective protein secretion

Variable amounts of soluble forms of a variety of glycosyl-phosphatidylinositol (GPI)-anchored proteins occur extracellularly, but the molecular mechanisms governing their release are not entirely clear. When the GPI-anchored folate receptor (FR) type beta was expressed transiently in human 293 fibroblasts, there was a roughly equal distribution of [3H]folic acid binding protein between the cell surface and the medium after 24 h over a wide range of expression levels of FR-beta. The difference in apparent molecular masses between the soluble FR-beta and the PI-PLC-treated membrane protein indicated that the former was not released from the membrane by the action of phospholipase. Brefeldin A inhibited the release of soluble FR-beta from both the transfected 293 cells and stable recombinant CHO (CHO-FR-beta) cells while pre-existing levels of cell surface FR were unaltered suggesting the absence of a precursor-product relationship between the membrane-associated FR-beta and the soluble protein in the medium. [35S]Cysteine pulse-chase analysis was consistent with this finding. Interchanging of carboxyl-terminal peptides between FR-beta and FR-alpha revealed that the nature of the processed signal for GPI modification was responsible for the quantitative membrane anchoring of FR-alpha and the production of soluble FR-beta. When total cell lysates were analyzed by Western blot, a diffuse band of apparent 41 kDa and three additional sharp bands of apparent 35, 33, and 29.3 kDa were seen. The 41 kDa band was identified as the PI-PLC sensitive cell surface receptor. Several mutant constructs of FR-beta, in which the carboxyl-terminal signal for GPI modification was either disrupted or deleted only gave the three lower bands. The three sharp bands from the wild-type and the mutant forms of FR-beta were identified as nonglycosylated (29.3 kDa) or glycosylated polypeptides in which the carboxyl-terminal peptide was at least partially proteolyzed without GPI modification. All of the mutations in the GPI signal resulted in the recovery of [3H]folic acid binding protein in the media which, similar to the wild-type FR recovered from the media, were converted to the 29.3 kDa band by N-glycanase. The results from this study indicate that a carboxyl-terminal peptide in FR-beta is efficiently proteolyzed intracellularly by a pathway that is independent of GPI signal recognition resulting in proper protein folding and secretion. Such carboxyl-terminal sequences could represent a simple adaptation for proteins whose physiologic functions reside both at the cell surface and in extracellular fluids, allowing their selective and tissue-specific release.     

Characterization of folate receptor from normal and neoplastic murine tissue: influence of dietary folate on folate receptor expression

Membrane-associated folate receptors (FRs) have been detected in many mammalian species, and multiple isoforms have been identified. The pharmacological properties of FRs from murine kidney, liver, and six murine tumors were characterized. Murine kidney expressed primarily folate-binding protein 1, analogous to human FR-alpha, whereas murine liver expressed predominantly folate-binding protein 2, analogous to human FR-beta. Five of six murine tumors expressed high-affinity FRs with pharmacological properties consistent with folate-binding protein 1 isoform expression. Restriction of dietary folate resulted in significant changes in the FR expression in most murine tissues. Kidney and tumor FRs showed a decreased affinity for folic acid, suggesting a change in isoform expression in response to a low folate diet. Density of the FR in the kidney decreased, and, in contrast, density of the FR in all tumors increased. The response of the liver to a low folate diet was unique in that there were no detectable changes in affinity or density of liver FR. Changes in dietary folate that modulate FR isoform expression may have relevance for cancer patients treated with antifolates.     

Structure-activity relationships of a series of analogues of the octadecaneuropeptide ODN on calcium mobilization in rat astrocytes

The octadecaneuropeptide ODN (QATVGDVNTDRPGLLDLK), originally characterized as an endogenous ligand for central-type benzodiazepine receptors, increases intracellular calcium concentration ([Ca2+]i) in rat astroglial cells. A series of ODN analogues was synthesized, and each compound was studied for its ability to induce Ca2+ mobilization in cultured rat astrocytes. Replacement of each amino acid by an L-alanine residue (AlaScan) showed that the N-terminal region of the molecule was relatively tolerant to alanine substitution (2-8, 10), except for the Ala9-substituted analogue (9) which was totally devoid of activity. Pyroglutamization (21) and acetylation (22) of the Gln1 residue reduced the Ca2+ response suggesting that a free N-terminal amine function is required for full activity of ODN. Alanine substitution of the residues in the C-terminal region of the molecule (11-14, 16-18) significantly reduced the biological activity of ODN. In particular, modifications of the Leu15 residue (15, 20) abolished the Ca2+-mobilizing activity. The analogues [Ala9]ODN (9), [Ala15]ODN (15), [D-Thr9]ODN (19), and [D-Leu15]ODN (20) partially antagonized the Ca2+ response evoked by ODN. Most importantly, the octapeptide ODN11-18 (OP, 24) produced a dose-response curve that was superimposable to that obtained with ODN, indicating that the C-terminal region of the molecule possesses full biological activity. Finally, the AlaScan of OP revealed that replacement of the Leu5 residue by Ala (29) or D-Leu (33) totally suppressed the calcium response, confirming the crucial contribution of the Leu15 residue of ODN to the biological activity of the neuropeptide.     

Definition of optimal substrate recognition motifs of Ca2+-calmodulin-dependent protein kinases IV and II reveals shared and distinctive features

The substrate recognition determinants of Ca2+-calmodulin-dependent protein kinase (CaMK) IV and CaMKIIalpha were investigated using peptide substrates modeled on the amino acid sequence encompassing Ser-9 of synapsin I. For both kinases, hydrophobic residues (Leu or Phe) at the -5 position, are well tolerated, whereas non-hydrophobic residues (Arg, Ala, or Asp) decrease Vmax/Km by 55- to >4000-fold. At the -3 position, substitution of Ala for Arg leads to decreases of 99- and 343- fold in Vmax/Km for CaMKIV and CaMKIIalpha, respectively. For both kinases, the nature of the residues occupying the -4, -1, and + 4 positions exerts relatively little influence on phosphorylation kinetics. CaMKIV and CaMKIIalpha respond differently to substitutions at the -2 and +1 positions. Substitution of Arg at the -2 position with non-basic residues (Gln or Ala) leads to 6-fold decreases in Vmax/Km for CaMKIV, but 17-28-fold increases for CaMKIIalpha. Additionally, peptides containing Leu, Asp, or Ala at the +1 position are phosphorylated with similar efficiencies by CaMKIV, whereas the Leu-substituted peptide is preferred by CaMKIIalpha (by a factor of 5.8-9.7-fold). Thus, CaMKIV and CaMKIIalpha preferentially phosphorylate substrates with the motifs: Hyd-X-Arg-X-X-Ser*/Thr*, and Hyd-X-Arg-NB-X-Ser*/Thr*-Hyd, respectively, where Hyd represents a hydrophobic, X any, and NB a non-basic amino acid residue. The different specificities of the two kinases may contribute to their targeting to distinct physiological substrates during Ca2+-dependent cellular events.     

NMDA-receptor antagonist requirements in conantokin-G

A series of variants of the neuroactive 17-residue gamma-carboxyglutamate-(Gla)-containing polypeptide, conantokin-G (con-G), were synthesized with the intention of determining those features that were important for its N-methyl-D-aspartate (NMDA) receptor-targeted antagonist activity and for adoption of its divalent cation-dependent alpha-helical conformation. Employing the binding of [3H]dizolcipine (MK-801) as an assay for open receptor ion channels in rat brain membranes, which displays inhibition by con-G (IC50 = 0.48 microM), it was found that replacement by an Ala residue of Gla4 led to complete inactivation of the peptide, whereas a similar replacement of Gla3 resulted in a 20-fold decreased potency. Ala substitutions for Gla10 and Gla14 did not substantially affect [3H]MK-801 binding. This same substitution at Gla7 appeared to slightly enhance binding. Ala replacements of non-Gla residues demonstrated that four of them, viz. Glu2, Leu5, Gln9, and Ile12, possessed at least 200-fold decreases in inhibitory potency, whereas similar replacements at Gly1, Leu11, and Arg13 resulted in peptides with 8- to 12-fold increases in the IC50 values. The remaining amino acid residues tested in the single Ala replacement series showed no significant changes in the inhibitory characteristics of wild-type con-G. Additional studies with carboxyl-terminal truncated peptides revealed that the carboxyl-terminal 4 amino acids were unimportant for this activity. There was no strict correlation of inhibition of [3H]MK-801 binding with the ability of these peptides to form cation-dependent alpha-helices. Peptides with notably low alpha-helical content in the presence of these cations were lacking at least one, or both, of Gla10 and Gla14. Con-G[Gla3,4,7,10,14E] and con-G[Gla7,10,14E] were the only peptides that remained in a completely random conformation upon metal ion addition.     

The unique nature of the serine interactions for alpha 1-adrenergic receptor agonist binding and activation

Activation of the beta2- and alpha2-adrenergic receptors (AR) involves hydrogen bonding of serine residues in the fifth transmembrane segment (TMV) to the catechol hydroxyls of the endogenous agonists, epinephrine and norepinephrine. With the beta2-AR both Ser204 and Ser207 but not a third TMV serine (Ser203) are required for binding and full agonist activity. However, with the alpha2a-AR only one of two TMV serines (Ser204, equivalent to Ser207 in the beta-AR) appears to contribute partially to agonist-binding and activation. Because the alpha1a-AR uniquely contains only two TMV serines, this subtype was used to systematically evaluate the role of hydrogen bonding in alpha1-AR activation. Binding of epinephrine or its monohydroxyl congeners, phenylephrine and synephrine, was not decreased when tested with alanine- substitution mutants that lacked either Ser188 (Ser188--> Ala) or Ser192 (Ser192-->Ala). With the substitution of both serines in the double mutant, Ser188/192-->Ala, binding of all three ligands was significantly reduced (10- 100-fold) consistent with a single hydrogen bond interaction. However, receptor-mediated inositol phosphate production was markedly attenuated only with the Ser188-->Ala mutation and not with Ser192-->Ala. In support of the importance of Ser188, binding of phenylephrine (meta-hydroxyl only) by Ser192-->Ala increased 7-fold over that observed with either the wild type receptor or the Ser188-->Ala mutation. Binding of synephrine (para-hydroxyl only) was unchanged with the Ser192-->Ala mutation. In addition, when combined with a recently described constitutively active alpha1a-AR mutation (Met292-->Leu), only the Ser188-->Ala mutation and not Ser192-->Ala relieved the high affinity binding and increased agonist potency observed with the Met292-->Leu mutation. A simple interpretation of these findings is that the meta-hydroxyl of the endogenous agonists preferentially binds to Ser188, and it is this hydrogen bond interaction, and not that between the para-hydroxyl and Ser192, that allows receptor activation. Furthermore, since Ser188 and Ser192 are separated by three residues on the TMV alpha-helix, whereas Ser204 and Ser207 of the beta2-AR are separated by only two residues, the orientation of the catechol ring in the alpha1-AR binding pocket appears to be unique and rotated approximately 120 degrees to that in the beta2-AR.     

Receptor-mediated folate uptake is positively regulated by disruption of the actin cytoskeleton

Receptor-mediated folate uptake is initiated by binding of ligand to a glycosyl phosphatidylinositol-anchored protein, folate receptor alpha (FR alpha). This receptor is expressed in a limited number of normal tissues but is overexpressed in a large number of epithelial malignancies. FR alpha synthesis, at least in part, is regulated by endogenous folate and by hormones in some cells, but much less is known about the control of function. Recently, we showed that phorbol 12-myristate 13-acetate increases the rate of receptor cycling, increases the rate of folate delivery, and causes the majority of the receptor to reside on the cell surface in nonmalignant cells in vitro (C. M. Lewis et al., Biochim. Biophys. Acta, 1401: 157-169, 1998). However, based upon effects (or lack of effects) of specific inhibitors of protein kinase C, the mechanism of action of phorbol 12-myristate 13-acetate is not likely via protein kinase C. Because exo- and endocytosis are controlled by the actin cytoskeleton, we tested cytochalasin D and latrunculin B, actin-disrupting agents, on FR alpha-mediated folate uptake. Disruption of the actin cytoskeleton reversibly increases the proportion of receptors on the cell surface and increases the rate of 5-methyltetrahydrofolate delivery. Disrupting microtubules with nocodazole had no effect. The increased rate of folate delivery caused by cytochalasin D is not observed in FR-negative cell lines. Although we have not yet identified the upstream effectors, likely candidates include small G-proteins such as rho, which are known to cause actin polymerization. In addition to identifying the machinery for receptor-mediated folate uptake, it may be important to integrate this new data into studies of FR alpha as a tumor antigen for imaging or delivering molecules via anti-FR antibodies or compounds coupled to folic acid.     

Processing of Escherichia coli alkaline phosphatase: role of the primary structure of the signal peptide cleavage region

A wide range (69) of mutant Escherichia coli alkaline phosphatases with single amino acid substitutions at positions from -5 to +1 of the signal peptide were obtained for studying protein processing as a function of the primary structure of the cleavage region. Amber suppressor mutagenesis, used to create mutant proteins, included: (i) introduction of amber mutations into respective positions of the phoA gene; and (ii) expression of each mutant phoA allele in E. coli strains producing amber suppressor tRNAs specific to Ala, Cys, Gln, Glu, Gly, His, Leu, Lys, Phe, Pro, Ser and Tyr. Most amino acid substitutions at positions -3 and -1 resulted in a complete block of protein processing. These data give new experimental support for the "-3, -1 rule". Only Ala, Gly and Ser at position -1 allowed protein processing, and Ala provided the highest rate of processing. The results revealed the more conservative nature of the amino acids at the -1 position of signal peptides of Gram-negative bacteria as compared with those of eukaryotic organisms. Position -3 was less regular, since not only Ala, Ser and Gly, but also Leu and Cys at this position, allowed the processing. Mutations at position -4 had an insignificant effect on the processing. Surprisingly, efficient processing was provided mainly by large amino acid residues at position -2 and by middle-sized residues at position -5, indicating that the processing rate is affected by the size of amino acid residues not only at positions -1 and -3. Conformation analysis of the cleavage site taken together with the mutation and statistical data suggests an extended beta-conformation of the -5 to -1 region in the signal peptidase binding pocket.     

Pharmaceutical and pharmacological development of antitumor prostaglandins]

The stoichiometry of the interaction between Erythrina variegata chymotrypsin inhibitor ECI and chymotrypsin was reinvestigated by analysis of their complex with ultracentrifugation and with amino acid analysis of the components separated. The amino acid analysis clearly showed that the stoichiometry of ECI and chymotrypsin was 1:1, though the apparent molecular mass of the complex was estimated to be 60 kDa. To examine the contribution of Leu64 (the P1 residue) to the inhibitory activity of ECI, a complete set of mutated inhibitors in which the amino acid at position 64 was replaced by 19 other amino acid residues was constructed by means of site-directed mutagenesis. Potent inhibitory activities (Ki, 1.3-4.6 x 10(-8) M) exceeding that of the wild-type ECI (Ki, 9.8 x 10(-8) M) were present in the mutant proteins L64F, L64M, L64W, and L64Y. The inhibitory activity of the mutant L64R was practically identical to that of the wild-type ECI. All other mutants exhibited slightly decreased inhibitory activities with Ki values of 1.9-4.6 x 10(-7) M. These results indicate that ECI-chymotrypsin interaction involves not only the P1 site residue but also other residue(s) of ECI. A series of individual alanine mutations was then constructed in residues Gln62 (P3), Phe63 (P2), Ser65 (P1'), Thr66 (P2'), and Phe67 (P3') in order to evaluate the contribution of each residue in the primary binding loop to the inhibitory activity. Replacement of Gln62, Phe63, and Phe67 with Ala residues decreased the inhibitory activity, the Ki values being increased by approximately 3-4-fold; but replacement of Ser65 and Thr66 had relatively little effect. This suggests that the P2, P3, and P3' residues, together with the P1 residue, in the primary binding loop play an important role in the inhibitory activity toward chymotrypsin.     

A case of cytophagic histiocytic panniculitis: successful treatment of recurrent attacks with steroid pulse therapy and oral cyclosporin A

Basic fibroblast growth factor (bFGF) is implicated in the pathogenesis of several vascular and connective diseases. A key step in the discovery of bFGF receptor antagonists to mitigate these actions is to define the functional epitope required for receptor binding of the growth factor. In previous studies, we identified Glu96 as an essential residue in this epitope using site-directed mutagenesis. Here we examined the role of solvent accessible neighboring residues of Glu96 of bFGF on receptor binding affinity. Wild-type bFGF and its muteins were cloned and expressed in Escherichia coli and evaluated for FGF receptor binding affinity. Replacement of Asn104 of bFGF by alanine reduced receptor binding affinity over 400-fold compared with wild-type bFGF. We next explored the effect of neighboring residues of Asn104 on receptor binding affinity-Muteins in which Arg97, Leu98, Glu99, Asn101, Asn102, Thr105 and Pro141 were individually replaced by alanine exhibited receptor binding similar to wild-type bFGF. By contrast, substitution of Tyr103 or Leu140 by alanine reduced receptor binding affinity about 400- and 150-fold, respectively, in accord with a previous report. We conclude that at least six solvent-accessible residues in bFGF are crucial for high-affinity receptor binding, as evidenced by at least a 10-fold diminution in the affinity of the corresponding alanine muteins. The polar residues Glu96 and Asn104 appear to form an area important for facilitating the initial contact between ligand and receptor, whereas Tyr24, Tyr103, Leu140 and Met142 form a hydrophobic patch that may stabilize the complex. The detailed structure of this functional epitope can be employed in the discovery and design of bFGF antagonists using computational methods.     

High affinity hormone binding to the extracellular N-terminal exodomain of the follicle-stimulating hormone receptor is critically modulated by exoloop 3

The human follicle-stimulating hormone receptor (FSH-R) consists of two distinct domains of >330 amino acids, the N-terminal extracellular exodomain and membrane-associated endodomain. The exodomain alone binds hormone with high affinity, whereas the endodomain is the site of receptor activation. Coordination of these two domains is essential for successful hormone action but little is known about their functional and structural relationship. In this communication, we report that exoloop 3 of FSH-R constrains follicle-stimulating hormone binding to the exodomain. When the FSH-R exodomain was prepared by truncating its endodomain, the hormone binding affinity of the exodomain was slightly improved, compared with the wild type receptor. The binding affinity was further improved by >3-fold when the exodomain was attached to the membrane-associated domain of CD8. These results suggest that the FSH-R endodomain attenuates hormone binding at the exodomain. As a first step to test this hypothesis, the 11 amino acids except Ala589 of exoloop 3 were individually substituted with Ala. Ala substitution for Leu583 or Ile584 improved the hormone binding affinity by 4-6-fold while totally abolishing cAMP induction, indicating an inverse relationship. The Ala substitution for Lys580 or Pro582 had a similar trend but to a lesser extent. This significant improvement in the binding affinity suggests that the four residues at the N-terminal region of exoloop 3 interact with the exodomain and constrain the hormone binding in the wild type receptor. This effect is specific since substitutions for other than the 4 residues did not improve the hormone binding affinity. Computer modeling shows that the 4 residues can be positioned on one side of exoloop 3. This result and the apparent inverse relationship of hormone binding and cAMP induction suggest that these two essential functions may work against each other. Therefore, hormone binding might be compromised to preserve cAMP inducibility while maintaining a reasonably high, but below maximum, binding affinity.     

Site-directed mutagenesis of the human A1 adenosine receptor: influences of acidic and hydroxy residues in the first four transmembrane domains on ligand binding

To provide new insights into ligand/A1 adenosine receptor (A1 AR) interactions, site-directed mutagenesis was used to test the role of several residues in the first four transmembrane (TM) domains of the human A1 AR. Based on multiple sequence analysis of all known ARs, both acidic (glutamic acid and aspartic acid) and polar hydroxy (serine and threonine) amino acids were identified that could potentially play a role in binding adenosine. Glu16 (TM1), Asp55 (TM2), Ser93 and Ser94 (TM3), Ser135 (TM4), and Thr 141 (TM4) were identified in all ARs, and Ser6 and Ser23 (TM1) were identified in all A1 ARs. To test the role of these residues, each was individually mutated to alanine. When Ala6, Ala23, Ala50, Ala93, Ala135, and Ala141 constructs were tested, affinities for [3H]2-chloro-N6-cyclopentyladenosine (CCPA) and [3H]1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were similar to those seen for the wild-type receptor. After conversion of Glu16 to Ala16, the affinity for [3H]CCPA and other agonists fell 10-100-fold, whereas the affinity for [3H]DPCPX and other antagonists was not affected. After conversion of Asp55 to Ala55, the affinity for [3H]CCPA and other agonists increased < or = 100-fold, whereas the affinity for [3H]DPCPX and other antagonists was not affected. Studies of the Ala55 construct also revealed that Asp55 is responsible for allosteric regulation of binding by sodium because the affinity for [3H]CCPA did not change over broad ranges of sodium concentrations. When Ser94 was converted to Ala94, A1 AR immunoreactivity was present on stable cell lines; however, functional binding sites could not be detected. When Ser94 was converted to Thr94, the affinity for some xanthine antagonists fell. These data show that Glu16 in TM1 and Asp55 in TM2 play important roles in agonist/A1 AR interactions and show that Asp55 is responsible for allosteric regulation of ligand/A1 AR binding by sodium. We also identify Ser94 as an important site for ligand binding.     

Elastin degradation by matrix metalloproteinases. Cleavage site specificity and mechanisms of elastolysis

Insoluble elastin was used as a substrate to characterize the peptide bond specificities of human (HME) and mouse macrophage elastase (MME) and to compare these enzymes with other mammalian metalloproteinases and serine elastases. New amino termini detected by protein sequence analysis in insoluble elastin following proteolytic digestion reveal the P'1 residues in the carboxyl-terminal direction from the scissile bond. The relative proportion of each amino acid in this position reflects the proteolytic preference of the elastolytic enzyme. The predominant amino acids detected by protein sequence analysis following cleavage of insoluble elastin with HME, MME, and 92-kDa gelatinase were Leu, Ile, Ala, Gly, and Val. HME and MME were similar in their substrate specificity and showed a stronger preference for Leu/Ile than did the 92-kDa enzyme. Fibroblast collagenase showed no activity toward elastin. The amino acid residues detected in insoluble elastin following hydrolysis with porcine pancreatic elastase and human neutrophil elastase were predominantly Gly and Ala, with lesser amounts of Val, Phe, Ile, and Leu. There were interesting specificity differences between the two enzymes, however. For both the serine and matrix metalloproteinases, catalysis of peptide bond cleavage in insoluble elastin was characterized by temperature effects and water requirements typical of common enzyme-catalyzed reactions, even those involving soluble substrates. In contrast to what has been observed for collagen, the energy requirements for elastolysis were not extraordinary, consistent with cleavage sites in elastin being readily accessible to enzymatic attack.     

NMR structure of the (52-96) C-terminal domain of the HIV-1 regulatory protein Vpr: molecular insights into its biological functions

The HIV-1 regulatory protein Vpr (96 amino acid residues) is incorporated into the virus particle through a mechanism involving its interaction with the C-terminal portion of Gag. Vpr potentiates virus replication by interrupting cell division in the G2 phase and participates in the nuclear transport of proviral DNA. The domain encompassing the 40 C-terminal residues of Vpr was shown to be involved in cell cycle arrest and binding of nucleocapsid protein NCp7, and suggested to promote nuclear provirus transfer. Accordingly, we show here that the synthetic 52-96 but not 1-51 sequences of Vpr interact with HIV-1 RNA. Based on these results, the structure of (52-96)Vpr was analysed by two-dimensional 1H-NMR in aqueous TFE (30%) solution and refined by restrained molecular dynamics. The structure is characterized by a long (53-78) amphipathic alpha-helix, followed by a less defined (79-96) C-terminal domain. The Leu60 and Leu67 side-chains are located on the hydrophobic side of the helix, suggesting their involvement in Vpr dimerization through a leucine zipper-type mechanism. Accordingly, their replacement by Ala eliminates Vpr dimerization in the two hybrid systems, while mutations of Ile74 and Ile81 have no effect. This was confirmed by gel filtration measurements and circular dichroism, which also showed that the alpha-helix still exists in (52-96)Vpr and its Ala60, Ala67 mutant in the presence and absence of TFE. Based on these results, a model of the coiled-coil Vpr dimer has been described, and its biological relevance as well as that of the structural characteristics of the 52-96 domain for the different functions of Vpr, including HIV-1 RNA binding, are discussed.     

Differential binding of HMG1, HMG2, and a single HMG box to cisplatin-damaged DNA

The HMG box domain is a DNA binding domain present in the nonhistone chromosomal proteins HMG1 and HMG2 and in other proteins involved in the regulation of gene expression. Previous studies have demonstrated that HMG1 and HMG2 bind with high affinity to DNA modified with the cancer chemotherapeutic drug cisplatin (CDDP). In this report, we compare the binding of full-length HMG1 and HMG2 and the HMG boxes present in these proteins to that of CDDP-DNA. Complexes between HMG1, HMG2, or HMG Box A + B and CDDP-DNA were stable at > or = 500 mM salt, while complexes between a single HMG box and CDDP-DNA exhibited decreased stability. Analysis of a series of HMG1 Box A mutant constructs revealed different affinities for CDDP-DNA. Two constructs containing a Phe to Ala substitution at position 19 and a Tyr to Gly substitution at position 71, are noteworthy; these peptides exhibited reduced affinity for CDDP-DNA. We have generated a structure of HMG1 Box A and used it, along with the results of our binding studies, to model its interaction with CDDP-DNA. HMG1 Box A binds in the minor groove of CDDP-DNA, in agreement with earlier studies. Our model predicts that Tyr71 partially intercalates and forms an H bond with the sugar-phosphate backbone. The model also suggests that Phe 19 does not directly interact with DNA, and hence an Ala substitution at position 19 may alter protein structure. This model should provide a framework for future studies examining HMG Box-DNA interactions.     

Quantitative responses of serum folate to increasing intakes of folic acid in healthy women

OBJECTIVES: Health authorities are advising people to increase folate intake and more foods are being fortified with folic acid. Estimation of dietary folate is difficult because nutrient data bases lack this nutrient in many countries and bioavailability is variable. To see if serum folate can be used to reflect effective folate intake, we have measured serum folate after healthy women had taken different doses of pure folic acid supplements in the nutritional range. METHODS: A total of 20 volunteer subjects took part in one or more of three experiments, six of them took part in all three. In each experiment subjects took two different doses of folic acid, each for three weeks. Experiment (1) 100 micrograms then 1000 micrograms/d; experiment (2) 500 micrograms then 1500 micrograms/d; experiment (3) 1000 then 2000 micrograms/d. Serum folate was measured after overnight fast with a homogenous enzyme binding assay kit. In experiment (3) red cell folates were also measured. RESULTS: It took three weeks' of the same supplement for serum folate to reach its full higher value. Mean serum folates of small groups of subjects were significantly higher with each higher dose of supplement. The biggest increase was for the first 100 micrograms that is in the range of intake from unfortified foods. The curve of serum folate against folic acid supplement intake shows that serum folate of groups of people, properly standardized, can provide a practical method for assessing adequacy or change of people's folate intake. Red cell folates were less responsive.     

Localization of ligand-binding domains of human corticotropin-releasing factor receptor: a chimeric receptor approach

Two CRF receptors, CRFR1 and CRFR2, have recently been cloned and characterized. CRFR1 shares 70% sequence identity with CRFR2, yet has much higher affinity for rat/human CRF (r/hCRF) than CRFR2. As a first step toward understanding the interactions between rat/human CRF and its receptor, the regions that are involved in receptor-ligand binding and/or receptor activation were determined by using chimeric receptor constructs of the two human CRFR subtypes, CRFR1 and CRFR2, followed by generating point mutations of the receptor. The EC50 values in stimulation of intracellular cAMP of the chimeric and mutant receptors for the peptide ligand were determined using a cAMP-dependent reporter system. Three regions of the receptor were found to be important for optimal binding of r/hCRF and/or receptor activation. The first region was mapped to the junction of the third extracellular domain and the fifth transmembrane domain; substitution of three amino acids of CRFR1 in this region (Val266, Tyr267, and Thr268) by the corresponding CRFR2 amino acids (Asp266, Leu267, and Val268) increased the EC50 value by approximately 10-fold. The other two regions were localized to the second extracellular domain of the CRFR1 involving amino acids 175-178 and His189 residue. Substitutions in these two regions each increased the EC50 value for r/hCRF by approximately 7- to 8-fold only in the presence of the amino acid 266-268 mutation involving the first region, suggesting that their roles in peptide ligand binding might be secondary.     

Use of high-precision gas isotope ratio mass spectrometry to determine natural abundance 13C in lutein isolated from C3 and C4 plant sources

Previous studies have shown that cholesterol esterification activity by lecithin:cholesterol acyltransferase (LCAT) is progressively inhibited as up to three acidic acid residues are chemically modified. The purpose of this study was to determine whether three glutamic acid residues in LCAT (154, 155, and 165), that align exactly with three acidic acid residues (270, 271, and 281) in the amphipathic phospholipid binding region of apoE, were necessary for enzymatic activity. Site-directed mutagenesis was used to generate mutant constructs of LCAT in which glutamic acid residues 154, 155, and 165 were replaced with glutamine or lysine. Media harvested from transiently transfected COS cells was used as a source of LCAT for cholesterol esterification and phospholipase A2 (PLA2) assays. Cholesterol esterification for all mutant constructs (11-26 nmol CE/h/microg) was similar to or greater than that of wild type LCAT (16 nmol CE/h/microg), except for a triple mutant, in which glutamic acid residues 154, 155, and 165 were changed to lysines (5 nmol CE/h/microg). PLA2 activity followed a similar trend. There was a significant decrease in the cholesterol esterification to PLA2 activity ratio when residue 165 was mutated from its wild type negative charge (E) to an uncharged (Q) or positive (K) charged residue (10.2 vs. 6.0 vs. 4.3, respectively). We conclude that glutamic acid residues 154, 155, and 165 individually or collectively are not necessary for LCAT activity and that residue 165 may be in a region of LCAT that is involved with cholesterol binding or is sensitive to cholesterol binding at the active site of the enzyme.