David Puett

Racial Differences in Plasma Endothelin-1 Concentrations in Individuals With Essential Hypertension

Hypertension is more prevalent in blacks than whites, and the reasons for this difference remain unclear. To test whether endothelin may play a role in these racial variations, we analyzed plasma samples from black and white women and men with high blood pressure by an enzyme-linked immunoassay specific for endothelin-1 (ET-1), a potent vasoconstrictor, and compared them with those obtained from similar subjects with normal blood pressure. Both female and male hypertensive blacks had elevated levels of immunoreactive ET-1 (11.3 +/- 1.0 and 12.3 +/- 1.3 pmol/L, respectively) compared with values in normotensive control blacks (1.5 +/- 0.2 and 1.4 +/- 0.2 pmol/L). Corresponding values in female and male hypertensive whites were 3.8 +/- 0.6 and 3.8 +/- 0.6 pmol/L, respectively, compared with respective values of 1.4 +/- 0.1 and 2.8 +/- 0.4 pmol/L in normotensive control whites. These results indicate that plasma concentrations of immunoreactive ET-1 levels differ significantly between black and white individuals with high blood pressure. This finding may be an important factor in the etiology of racial differences in the prevalence and severity of hypertension and deserves further study [corrected].

An integrated transcriptomic and computational analysis for biomarker identification in gastric cancer

This report describes an integrated study on identification of potential markers for gastric cancer in patients’ cancer tissues and sera based on: (i) genome-scale transcriptomic analyses of 80 paired gastric cancer/reference tissues and (ii) computational prediction of blood-secretory proteins supported by experimental validation. Our findings show that: (i) 715 and 150 genes exhibit significantly differential expressions in all cancers and early-stage cancers versus reference tissues, respectively; and a substantial percentage of the alteration is found to be influenced by age and/or by gender; (ii) 21 co-expressed gene clusters have been identified, some of which are specific to certain subtypes or stages of the cancer; (iii) the top-ranked gene signatures give better than 94% classification accuracy between cancer and the reference tissues, some of which are gender-specific; and (iv) 136 of the differentially expressed genes were predicted to have their proteins secreted into blood, 81 of which were detected experimentally in the sera of 13 validation samples and 29 found to have differential abundances in the sera of cancer patients versus controls. Overall, the novel information obtained in this study has led to identification of promising diagnostic markers for gastric cancer and can benefit further analyses of the key (early) abnormalities during its development.

A model for constitutive lutropin receptor activation based on molecular simulation and engineered mutations in transmembrane helices 6 and 7.

Many naturally occurring and engineered mutations lead to constitutive activation of the G protein-coupled lutropin receptor (LHR), some of which also result in reduced ligand responsiveness. To elucidate the nature of interhelical interactions in this heptahelical receptor and changes thereof accompanying activation, we have utilized site-directed mutagenesis on transmembrane helices 6 and 7 of rat LHR to prepare and characterize a number of single, double, and triple mutants. The potent constitutively activating mutants, D556(6.44)H and D556(6.44)Q, were combined with weaker activating mutants, N593(7.45)R and N597(7.49)Q, and the loss-of-responsiveness mutant, N593(7.45)A. The engineered mutants have also been simulated using a new receptor model based on the crystal structure of rhodopsin. The results suggest that constitutive LHR activation by mutations at Asp-556(6.44) is triggered by the breakage or weakening of the interaction found in the wild type receptor between Asp-556(6.44) and Asn-593(7.45). Whereas this perturbation is unique to the activating mutations at Asp-556(6.44), common features to all of the most active LHR mutants are the breakage of the charge-reinforced H-bonding interaction between Arg-442(3.50) and Asp-542(6.30) and the increase in solvent accessibility of the cytosolic extensions of helices 3 and 6, which probably participate in the receptor-G protein interface. Asn-593(7.45) and Asn-597(7.49) also seem to be necessary for the high constitutive activities of D556(6.44)H and D556(6.44)Q and for full ligand responsiveness. The new theoretical model provides a foundation for further experimental work on the molecular mechanism(s) of receptor activation.

Gonadotropin and subunit conformation

Abstract Circular dichroic spectra have been obtained and resolved for the gonadotropins, ovine pituitary luteinizing hormone and human chorionic (urinary) gonadotropin, their subunits and glycopeptides. Much of the gonadotropin ellipticity above 250 nm can be attributed to the disulfide chromophore, although there are discernible contributions from tyrosyl and phenylalanyl residues as well. Of the two dissimilar subunits, the β-subunit makes the greatest contribution to the near-ultraviolet circular dichroic spectrum of the gonadotropins. From the position of the 0-0 tyrosyl band, i.e., 286–287 nm, one can ascertain that at least some of the tyrosyl residues of the gonadotropins are located in a hydrophobic environment. A positive circular dichroic extremum at 232.5 nm, present in luteinizing hormone but not in chorionic gonadotropin, can be ascribed to the α-subunit and probably results from tyrosines 21 and/or 30 in luteinizing hormone. An analysis of the circular dichroic difference spectrum above 230 nm, generated by subtracting the sum of the molecular ellipticities of the respective subunits from the molecular ellipticities of each gonadotropin, indicates that the local environment of disulfides and of tyrosyl residues is altered when gonadotropins dissociate. Circular dichroic difference spectra between the two α-subunits and between the two β-subunits indicated major contributions from- tyrosyl residues, presumably arising from tyrosyl substitutions. Between 200 and 230 nm, both gonadotropins exhibit negative circular dichroic extrema. The extremum occurs at 210 nm for luteinizing hormone and at 207.5 nm for chorionic gonadotropin. Each extremum can be described by two negative resolved bands, one at 215 nm and the other between 207 and 208.5 nm. The 215-nm resolved band is assigned to the peptide chromophore in a β-pleated sheet conformation and there is no evidence of α-helicity. The lower-wavelength resolved band is believed to have a significant contribution from the N -acetyl groups of glucosamine, galactosamine, and sialic acid, particularly since the glycopeptide fractions, prepared from each gonadotropin by digestion of the S -carboxymethyl derivatives with Pronase, exhibited a negative circular dichroic extremum at about 207 nm. The extent of β-structure in both gonadotropins is estimated to be about 28% whereas the separated subunits contain less β-structure, e.g., about 21 and 13% for the α- and β-subunits, respectively. The sum of the subunit β-structure, corrected for the respective molecular weight of each subunit, is about 17%. This is substantially less than that of the native hormone, thus indicating that significant conformational changes occur during gonadotropin dissociation to the biologically inactive subunits. Also, part of the gonadotropin β-structure may arise from intermolecular hydrogen bonding involving a pleated sheet arrangement between the subunits.

On the conformation of the acetylcholine receptor protein from Torpedo nobiliana.

It is well established that acetylcholine acts as a regulatory ligand controlling selective increase in membrane permeability to cations [ 11. This permeability change is thought to be mediated by a minimum of two distinct structural elements [2] : a ‘receptor’ protein which zzcognizes cholinergic agonists and an ‘ionophore’ that accounts for the selective transition of ions. The latter could be a region of the receptor protein or may constitute a separate but tightly coupled entity [2]. AChRT from several species has been extensively purified by several laboratories [3-91 and its molecular properties are currently under investigation. However, no information is available on the receptor conformation. This report describes for the first time the far ultraviolet CD spectrum of AChR. A detailed analysis of the resolved spectrum indicates that the Torpedo nobiliana AChR contains a particularly high content of ordered secondary structure, e.g., about 34% a-helix and 29% p-structure. The amino acid composition of the receptor is also reported and the overall polarity and hydrophobicity is typical of soluble proteins.

A functional transmembrane complex: The luteinizing hormone receptor with bound ligand and G protein

The luteinizing hormone receptor (LHR) is one of eight members in a cluster of the rhodopsin family of the large G protein-coupled receptor (GPCR) superfamily that contains some 800-900 genes in the human genome. LHR, along with its paralogons, follicle stimulating hormone receptor (FSHR) and thyroid stimulating hormone receptor, form one of the three classes in this cluster; the two other classes contain the relaxin-binding GPCRs and orphan GPCRs. These GPCRs are characterized by a relatively large ectodomain (ECD) containing leucine-rich-repeats (LRRs); in the class of glycoprotein hormone receptors, the LRR region is capped by N-terminal and C-terminal cysteine-rich regions. Binding of human chorionic gonadotropin (hCG) or luteinizing hormone to the LHR-ECD triggers a conformational change of the transmembrane region of the receptor facilitating binding and activation of Gs, followed by effector enzyme activation and subsequent intracellular signaling. Viewing LHR as a transmembrane anchoring protein that sequentially binds hCG and Gs to give the hCG-LHR-Gs complex, numerous interactions and conformational changes must be considered. There is, unfortunately, a paucity of structural data on LHR, but crystal structures exist for hCG, the homologous FSH-FSHR-ECD (N-terminal fragment) complex, rhodopsin (in the inactive state), an active form of Galphas (transducin), and the betagamma heterodimer. Using a combined experimental (site-directed mutagenesis followed by characterization in transfected cells) and computational (homology modeling and molecular dynamics simulations) approach, good working models are being developed for the protein-protein interaction faces and, in some cases, the ensuing conformational changes induced by complex formation. hCG binding to the LHR-ECD appears to involve several LRRs; LHR activation can be described in terms of disrupting a network of H-bonds in the cytosolic halves of helices 1-3, 6, and 7; and binding of LHR to Gs involves, in large part, intracellular loop 2 binding, presumably to Gsalpha at its C-terminus. Major gaps exist in our understanding at the molecular level of the six-polypeptide chain complex, hCG-LHR-Gs, but considerable progress has been made in the past few years.

LYS583 IN THE THIRD EXTRACELLULAR LOOP OF THE LUTROPIN/CHORIOGONADOTROPIN RECEPTOR IS CRITICAL FOR SIGNALING

The lutropin/choriogonadotropin receptor (LH/CG-R) contains a relatively large extracellular domain, in addition to the seven transmembrane helices (TMH), three extracellular loops (ECL), and three intracellular loops typical of G protein-coupled receptors. While high affinity ligand binding has been attributed to the N-terminal extracellular domain, there is evidence that portions of the three ECLs may function in ligand binding and transmembrane signaling. We have investigated the role of several ionizable amino acid residues of rat LH/CG-R in human choriogonadotropin (hCG) binding and hCG-mediated cAMP production. COS-7 cells were transfected with the pSVL expression vector containing cDNAs of either wild-type or mutant rat LH/CG-R. Several point mutants of Lys583, located at the interface of ECL III and TMH VII, bound hCG like wild-type receptor but exhibited greatly diminished ligand-mediated signaling. Neither the point mutant, Lys401 → Asp (ECL I), nor the double mutant, Asp>397ed cells, only the double mutant bound hCG. The mutants Arg341 → Glu (interface of the extracellular domain and TMH I) and Lys488 → Glu (ECL II) proved to be similar to wild-type receptor in binding and signaling. Our results establish that Lys583 is important in signaling but not ligand binding. Its location on the opposite side of the membrane from Gs precludes a direct interaction, thus emphasizing the importance of a conformational change in the receptor and suggesting that ligand binding to receptor and ligand-mediated receptor activation are dissociable phenomena.

Intracellular uptake and catabolism of lutropin by testicular tissue in vivo

The mechanisms involved in the expression of biological activity of lutropin have been extensively studied [l] and, analogous to other peptide hormones, the initial step is believed to involve binding of the hormone to a specific receptor located in the plasma membrane of the target cells. The specificity and characteristics of hormone binding to testicular [ 1 ] and ovarian [2] receptors have been investigated and preparations of partially purified receptor are available [2-41. Little is known, however, about the fate of the hormone-receptor complex and its relationship, if any, to the termination of biological activity. Interestingly, it has recently been reported that the number of rat testicular gonadotropin receptors decreased with time following injection of either lutropin or choriogonadotropin [5]. Studies in this laboratory have been concerned with the kinetics and mechanisms of plasma clearance and biotransformations of lutropin [6-IO]. The results of in vivo studies on the kinetics of lutropin uptake and catabolism by target tissue are presented in the present communication. As expected, tritium labeled ovine lutropin binds specifically to the testes following intravenous injection into mature male rats. From sucrose gradient centrifugation, it appears that at least some of the hormones which are bound to Leydig cells are internalized and eventually degraded by lysosomes. This phenomenon suggests a possible mechanism for terminating hormone action by target cells and may be important in the regulation of lutropin receptors by the gonadotropin itself.

Protein Engineering of a Novel Constitutively Active Hormone-Receptor Complex

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone consisting of an α and a β subunit that stimulates intracellular levels of cAMP via a G protein-coupled receptor. Herein we report the engineering and characterization of a novel molecule in which the receptor and its heterodimeric ligand were covalently linked in a single polypeptide chain. The hormone-receptor complex was expressed in cells transfected with this construct, but the cells were unable to bind significant amounts of exogenous hCG. However, cleavage of the hormone with a site-specific protease rendered the receptor accessible to exogenously added hormone. Cells transfected with the hCG-receptor construct contained elevated basal levels of cAMP; moreover, addition of hormone had no significant effect. These results are consistent with a strong and stable interaction between the single-chain hormone and its covalently linked receptor that results in a constitutively active complex.

Collagen-Mediated Platelet Aggregation EFFECTS OF COLLAGEN MODIFICATION INVOLVING THE PROTEIN AND CARBOHYDRATE MOIETIES

In an effort to elucidate the nature of the collagen-platelet interaction, the effects of collagen modification on platelet aggregation have been studied. We have shown that purified rat skin (salt) soluble collagen is effective at about 20 nM in mediating platelet aggregation in human platelet-rich plasma. This concentration is somewhat greater than that required of several skin insoluble collagens (ca. 10 nM). Both the alpha1(I) and alpha2 chains from rat skin soluble collagen produced platelet aggregation, but only at concentrations of about 13 muM and 55 muM, respectively. In contrast, heat-denatured collagen and chains (e.g., 65 muM alpha1(I) and 160 muM alpha2) failed to induce platelet aggregation and to inhibit platelet aggregation by native collagen. Glycopeptides were prepared from human skin insoluble collagen by extended digestion with bacterial collagenase and trypsin, and were purified by gel filtration into two classes. One class of higher molecular weight contained sialic acid, glucosamine, galactosamine, fucose, mannose, galactose, and glucose, and the other of lower molecular weight consisted primarily of a mixture of galactose and galactosyl-glucose units O-glycosidically linked to hydroxylysine-containing peptides. We found that, after the residual tryptic activity contaminating the higher molecular weight fraction was inhibited, neither of the glycopeptide classes produced nor inhibited native human skin insoluble collagen-mediated platelet aggregation at the highest concentration examined (ca. 1-2 mg glycopeptide per ml of platelet-rich plasma). Highly purified samples of the hydroxylysyl glycosides, hydroxylysylgalactose and hydroxylysylgalactosylglucose (Hyl-Gal and Hyl-Gal-Glc, respectively), were prepared from human urine and labeled at galactose using galactose oxidase followed by reduction with tritiated borohydride. Binding studies with platelet-rich plasma showed that, at concentrations greater than 50 nM, Hyl-Gal gives apparent binding to platelets, but there was no evidence of Hyl-Gal-Glc binding to platelets at concentrations up to 250 nM. At concentrations several hundredfold higher than the equivalents present in the minimum concentration of rat skin soluble collagen required for platelet aggregation, neither Hyl-Gal (at 29 muM) nor Hyl-Gal-Glc (at 18 muM) caused platelet aggregation or inhibited platelet aggregation by native collagen. Also, at a concentration of 85 muM (which represents a concentration about two thousandfold higher than the equivalents in the minimum concentration of soluble collagen required for platelet aggregation) the Gal-Glc-containing 36 residue rat skin soluble collagen alpha1(I)cyanogen bromide #5 peptide had no platelet aggregating or inhibiting activity. Modification of at least 90% of the rat skin soluble collagen carbohydrate by mild periodate oxidation had no effect on the platelet aggregating activity. Human skin insoluble collagen was reacted with periodate under the same conditions, and this had no demonstrable effect on its ability to induce platelet aggregation. This indicates that the normal carbohydrate side chains of these collagens are not required for the platelet interaction that produces the release of ADP and other metabolic constituents and leads to aggregation.Thus, collagen-platelet interactions appear to involve at least two distinct binding sites on the platelet plasma membrane. One is a protein binding site that activates platelet aggregation and has high specificity and affinity for the collagen triple-helical fold or perhaps even for a particular amino acid sequence in the triple helix.