Molecular interaction of Agouti protein and Agouti-related protein with human melanocortin receptors

Agouti protein and the Agouti-related protein (AGRP) are antagonists of the melanocortin-3 receptor and melanocortin-4 receptor. Both proteins contain 10 cysteines in the C-terminal domain arranged in five disulfide bonds. One possible arrangement of the disulfide bonds predicts an octapeptide loop, and the chemical properties of four residues within this loop (residues 111-114 in human AGRP) bear striking resemblance to those of several melanocortin peptides, including alpha-MSH, MT-II, and SHU-9119. We showed that cyclic synthetic octapeptides based on the sequence of this loop from Agouti protein or human AGRP are functional antagonists of the human melanocortin-4 receptor. All peptides had a lower affinity for the melanocortin-3 receptor than for the melanocortin-4 receptor. Substitution of serines for cysteines resulted in linear peptides which had reduced binding affinities for both receptors. Mutational analysis of human AGRP indicated that its C-terminal domain is functionally equivalent to the intact human AGRP. The RFF111-113 triplet appears to be the most critical portion of AGRP in determining the binding affinity for both melanocortin-3 and melanocortin-4 receptors. These data strongly suggest that the loop defined by Cys-110 and Cys-117 is critical in determining the antagonist activity of human AGRP. Our data provide indirect evidence for the suggestion that the Cys-110 to Cys-117 octapeptide loop of human AGRP mimics the conformation of alpha-MSH, MT-II, and SHU-9119.     

Molecular basis for p38 protein kinase inhibitor specificity

p38 is a member of the mitogen-activated protein (MAP) kinase family and is a critical enzyme in the proinflammatory cytokine pathway. Other MAP kinase group members that share both structural and functional homology to p38 include the c-Jun NH2-terminal kinases (JNKs or SAPKs) and the extracellular-regulated protein kinases (ERKs). In this study, we determined the molecular basis for p38alpha inhibitor specificity exhibited by five compounds in the diarylimidazole, triarylimidazole, and triarylpyrrole classes of protein kinase inhibitors. These compounds are significantly more potent inhibitors of p38 compared to the JNKs and ERKs. Three active site ATP-binding domain residues in p38, T106, M109, and A157, selected based on primary sequence alignment, molecular modeling, and X-ray crystal structure data, were mutated to assess their role in inhibitor binding and enzymatic catalysis. All mutants, with the exception of T106M, had kinase activity within 3-fold of wild-type p38. Mutation of T106 to glutamine, the residue present at the corresponding position in ERK-2, or methionine, the corresponding residue in p38gamma, p38delta, and the JNKs, rendered all five inhibitors ineffective. The diarylimidazoles had approximately a 6-fold decrease in potency toward M109A p38. For the mutant A157V, all diarylimidazoles and triarylimidazoles tested were 5-10-fold more potent compared with wild-type p38. In contrast, two triarylpyrroles were 15-40-fold less potent versus A157V p38. These results showed that the molecular basis for the specificity of the p38 inhibitors was attributed largely to threonine 106 in p38 and that methionine 109 contributes to increased binding affinity for imidazole based inhibitors.     

Molecular interaction between the Fyn-associated protein SKAP55 and the SLP-76-associated phosphoprotein SLAP-130

It has previously been reported that in resting T-lymphocytes the protein tyrosine kinase p59 constitutively co-precipitates with four phosphoproteins of 43, 55, 85, and 120 kDa, respectively. We have recently cloned the 55-kDa protein that was termed Src kinase-associated phosphoprotein of 55 kDa (SKAP55). Here we demonstrate that the recently characterized SH2-domain-containing leukocyte protein 76-associated phosphoprotein of 130 kDa (SLAP-130) is one of the components of the Fyn complex and that it also co-precipitates with SKAP55 in human T-cells. We establish that SKAP55 and SLAP-130 associate with each other when both molecules are co-expressed in COS cells. By co-transfection of truncated mutants of SKAP55 and SLAP-130 as well as by using the two-hybrid selection system, we further demonstrate that the association between SLAP-130 and SKAP55 is direct and involves the Src homology 3 domain of SKAP55 and the proline-rich sequence of SLAP-130.     

Studies on lead-binding protein and interaction between lead and selenium in the human erythrocytes

Transmission of respiratory syncytial virus is thought to be highly seasonal based on reported clinical cases, although transmission resulting in mild disease in all age groups has been little studied. This has been investigated in a seroepidemiological survey using sera from S?o Paulo, Brazil. Seroprevalence was found to increase rapidly with age, reaching over 90% by three years of age. This is typical of viral infections, which produce life-long immunity following primary infection. One-hundred percent seropositivity was attained by five years of age and maintained throughout adulthood, whereas mean antibody titers continued to increase with age. The mean duration of maternal antibodies was estimated to be 3.3 months with antibody decay demonstrated in paired samples from infants. The results are discussed in relation to possible mechanisms generating such a profile.     

Virus-specific interaction between the human cytomegalovirus major capsid protein and the C terminus of the assembly protein precursor

We previously identified a minimal 12-amino-acid domain in the C terminus of the herpes simplex virus type 1 (HSV-1) scaffolding protein which is required for interaction with the HSV-1 major capsid protein. An alpha-helical structure which maximizes the hydropathicity of the minimal domain is required for the interaction. To address whether cytomegalovirus (CMV) utilizes the same strategy for capsid assembly, several glutathione S-transferase fusion proteins to the C terminus of the CMV assembly protein precursor were produced and purified from bacterial cells. The study showed that the glutathione S-transferase fusion containing 16 amino acids near the C-terminal end was sufficient to interact with the major capsid protein. Interestingly, no cross-interaction between HSV-1 and CMV could be detected. Mutation analysis revealed that a three-amino-acid region at the N-terminal side of the central Phe residue of the CMV interaction domain played a role in determining the viral specificity of the interaction. When this region was converted so as to correspond to that of HSV-1, the CMV assembly protein domain lost its ability to interact with the CMV major capsid protein but gained full interaction with the HSV-1 major capsid protein. To address whether the minimal interaction domain of the CMV assembly protein forms an alpha-helical structure similar to that in HSV-1, peptide competition experiments were carried out. The results showed that a cyclic peptide derived from the interaction domain with a constrained (alpha-helical structure competed for interaction with the major capsid protein much more efficiently than the unconstrained linear peptide. In contrast, a cyclic peptide containing an Ala substitution for the critical Phe residue did not compete for the interaction at all. The results of this study suggest that (i) CMV may have developed a strategy similar to that of HSV-1 for capsid assembly; (ii) the minimal interaction motif in the CMV assembly protein requires an alpha-helix for efficient interaction with the major capsid protein; and (iii) the Phe residue in the CMV minimal interaction domain is critical for interaction with the major capsid protein.     

Single-tube mixed agglutination test for the detection of staphylococcal protein A

Intra-arterial and intravenous catheters were inserted in six fetal lambs at 125-130 days of gestation. On the following day, fetal arterial pressures and blood gases were monitored and fetal cardiac output and its distribution were measured by injection of radionuclide-labeled microspheres 15 mum in diameter. Acetylsalicylic acid, 55-90 mg/kg of estimated fetal weight, then was administered into the fetal stomach. Fetal pulmonary arterial pressure rose significantly after an average of 58 minutes, increasing the pressure difference between the pulmonary artery and the aorta from 2 +/- 0.3 (SEM) mm Hg during control to 11.2 +/- 1.6 mm Hg. Resistance across the ductus arteriosus rose from 4.2 +/- 0.5 (SEM) to 27.4 +/- 4.01 units, and flow fell from 495 +/- 44 (SEM) to 409 +/- 20 ml/minute. The proportion of combined ventricular output distributed to the placenta, adrenals, heart, and lungs increased, whereas the proportion of combined ventricular output distributed to the brain, liver, intestine, kidneys, and upper and lower body fell. In two fetuses infusion of prostaglandin E1 reversed the pulmonary hypertension. Inhibition of prostaglandin synthesis in fetal lambs produced constriction of the ductus arteriosus and redistribution of cardiac output. It is probable that prostaglandins, particularly E1, are involved in regulation of blood flow through the ductus arteriosus and various vascular beds in the normal resting fetus.     

Molecular basis for interactions of G protein betagamma subunits with effectors

Both the alpha and betagamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) communicate signals from receptors to effectors. Gbetagamma subunits can regulate a diverse array of effectors, including ion channels and enzymes. Galpha subunits bound to guanine diphosphate (Galpha-GDP) inhibit signal transduction through Gbetagamma subunits, suggesting a common interface on Gbetagamma subunits for Galpha binding and effector interaction. The molecular basis for interaction of Gbetagamma with effectors was characterized by mutational analysis of Gbeta residues that make contact with Galpha-GDP. Analysis of the ability of these mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2, phospholipase C-beta2, and beta-adrenergic receptor kinase revealed the Gbeta residues required for activation of each effector and provides evidence for partially overlapping domains on Gbeta for regulation of these effectors. This organization of interaction regions on Gbeta for different effectors and Galpha explains why subunit dissociation is crucial for signal transmission through Gbetagamma subunits.     

Structural basis of the RNA-binding specificity of human U1A protein

The RNP domain is a very common eukaryotic protein domain involved in recognition of a wide range of RNA structures and sequences. Two structures of human U1A in complex with distinct RNA substrates have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning the origin of binding specificity. The beta-sheet of the domain provides an extensive RNA-binding platform for packing aromatic RNA bases and hydrophobic protein side chains. However, many interactions between functional groups on the single-stranded nucleotides and residues on the beta-sheet surface are potentially common to RNP proteins with diverse specificity and therefore make only limited contribution to molecular discrimination. The refined structure of the U1A complex with the RNA polyadenylation inhibition element reported here clarifies the role of the RNP domain principal specificity determinants (the variable loops) in molecular recognition. The most variable region of RNP proteins, loop 3, plays a crucial role in defining the global geometry of the intermolecular interface. Electrostatic interactions with the RNA phosphodiester backbone involve protein side chains that are unique to U1A and are likely to be important for discrimination. This analysis provides a novel picture of RNA-protein recognition, much closer to our current understanding of protein-protein recognition than that of DNA-protein recognition.     

Molecular basis for cardiac functions

The cause of mechanical failure of the fixture component of an osseointegrated dental implant was investigated. The surfaces of five clinical specimens that had fractured were compared to those of new specimens fractured in the laboratory under monotonic and cyclic loads. Scanning electron microscopy revealed striations on the fractured surfaces of the clinical specimens, similar to striations on the laboratory-fatigued specimens and in contrast to the dimpled surfaces on the overloaded specimens. The investigation demonstrated that fractures of the fixture component of this dental implant occurred by fatigue under physiologic loads, with marginal alveolar bone loss around the fixture.     

Molecular basis of drug interaction with L-type Ca2+ channels

This article seeks to identify mental health issues in regard to violence among Hispanics and Latinos relative to other groups in the United States. A review of the literature provides prevalence and incidence rates, population and demographic information, and unique issues in regard to violence for Latinos and Latinas, including in the areas of battering and wife abuse, child and sexual abuse, sexual harassment, and the complicating effects of racial-ethnic discrimination. Aspects of cultural ideology, including the social construction of gender and its relevance to violence, are discussed, as are theoretical explorations of the impact of violence on familism, a major Hispanic cultural value. Finally, therapeutic implications for clinicians are explored, structured by the American Psychological Association (1993).     

Phosphorylation of LukS by protein kinase A is crucial for the LukS-specific function of the staphylococcal leukocidin on human polymorphonuclear leukocytes

Staphylococcal leukocidin (Luk) consists of two protein components, LukF and LukS, which cooperatively lyse human and rabbit polymorphonuclear leukocytes. Here, we demonstrate that the phosphorylation of LukS by protein kinase A is crucial for the LukS-specific leukocytolytic function of Luk on HPMNLs by using N-[2(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), which is a potent and selective inhibitor of protein kinase A. At 0.5 microM H-89 completely prevented the Luk-induced cell lysis accompanied by blocking of the incorporation of exogenous 32P-H3PO4 into LukS on HPMNLs. However, with LukS and LukF together, 0.5 microM H-89 did not inhibit the cell swelling which takes place before the cell lysis. HPMNLs also became swollen upon treating with both LukF and LukS mutants which could not be phosphorylated.     

Molecular basis of human complement C1s deficiency

This is the first report on the molecular basis of human complement C1s deficiency. Two abnormalities in the C1s gene were identified in a Japanese family, including one patient, by using exon-specific PCR, single-strand conformation polymorphism analysis, and nucleotide sequencing. A deletion of 4 bp, TTTG, was identified in exon X when using genomic DNA from the patient, his father, and his paternal grandmother. They were all heterozygous for the mutation. The mutant gene encodes a truncated C1s from the N terminus to the short consensus repeat domain. By further sequencing the PCR products, a nonsense mutation from G to T was identified at codon 608 in exon XII in the patient, his mother, and his sister. They were all heterozygous for the nonsense mutation. The mutant gene encodes a truncated form of C1s that lacks the C-terminal 80 amino acids. These results indicate that the patient was a compound heterozygote with the 4-bp deletion on the paternal allele and the nonsense mutation on the maternal allele. The levels of serum C1s seem to be correlated to the genotypes of the C1s gene in which no C1s was detected in the patient, and one-half of the normal level in the family members who are heterozygous for either mutation. The present study demonstrates that the disease is inherited in an autosomal recessive mode.     

Molecular basis of IsK protein regulation by oxidation or chelation

Slowly activating IsK channels were expressed in Xenopus oocytes and exposed to oxidative agents. Oxidative treatment reduced the resulting current IsK, while no inhibition was observed for IsK protein mutants carrying a Ser mutation instead of a highly conserved Cys residue in the intracellular domain. In contrast, Hg2+, which may not only oxidize thiol groups but also form chelates with dibasic amino acids, caused a use-dependent, positive regulation of IsK. This effect was reversed in an IsK protein mutant with a deletion in the extracellular domain. These data suggest opposite effects of peroxides and Hg2+ on IsK, a peroxide-mediated IsK inhibition by intracellular oxidation and a Hg(2+)-mediated IsK increase, caused by extracellular Hg2+ chelation of the IsK protein.     

The specific interaction between LSD and serotonin-binding protein

In this report, we describe the chemical interaction between lysergic acid (LSD) and serotonin-binding protein (SBP) by a three-dimensional spectroscopic technique. This instrument records simultaneously the activation and fluorescence spectra, and plots fluorescence intensity level by a series of isointensity contours. Free LSD exhibits maximum fluorescence at 435 nm with excitation maximum at 330 nm. While SBP-bound LSD shifted its fluoresence and excitation maximum to 465 nm and 375 nm respectively. These results suggest that the drug-protein interaction caused an extensive delocalization of the molecular orbital electrons and thereby lengthen the electronic conjugation of the drug molecule. Such shift was not observed when LSD was incubated with bovine serum albumin under the same condition. This technique is sensitive and requires a small volume of samples. It permits us to determine both free and bound drug in the same sample and provides us specific information about the drug and protein interaction.     

Enzyme-linked immunosorbent assay, using staphylococcal protein A for detecting virus antibodies

A modification of the indirect enzyme-linked immunosorbent assay (ELISA) was developed which used staphylococcal protein A linked to horseradish peroxidase. Virus antibodies in equine, bovine, porcine, feline, canine, lagomorphic (rabbit), and human sera were detected, using the indirect ELISA in which the antiglobulin enzyme conjugate was replaced by protein A linked to horseradish peroxidase. Results of the ELISA were compared with the results of the serum-virus neutralization test. The application of the test in laboratories performing serologic assays with sera from diverse animal species is discussed.     

Structural basis for the interaction of Ras with RalGDS

The Malthus-AT system provided a satisfactory method for examining the effects of permeabilizing agents on the activity of sub-inhibitory concentrations of antibacterial agents against Pseudomonas aeruginosa G48. Under this system, disodium edetate potentiated the activity of chlorhexidine diacetate (CHA), cetylpyridinium chloride, para-chlorometaxylenol and triclosan. Nitrilotriacetic acid enhanced the activity of some of the antibacterials tested, whereas sodium polyphosphate markedly reduced the efficacy of CHA.     

Molecular basis for species and ligand specificity of a monoclonal antibody raised against human IGF-I

The anti-hIGF-I monoclonal antibody, alpha-sm1.2, was found to have substantial crossreactivity with human and rat IGF-II, but recognized rat IGF-I only when this ligand was present at very high concentration. (E50 for hIGF-I approximately 3.5 ng/tube vs. approximately 12,000 ng/tube for rat IGF-I). In the context of previous studies to define the epitope(s) of alpha-sm1.2, these findings point to the critical importance of aspartic acid at residue 20 in the B domain in determining the species and ligand specificity of this antibody. Previous studies using this antibody in rodent tissues may require reinterpretation in the light of these findings.