Biomedical subjects

J A Wells

Publications and source records attributed to J A Wells.

At least 19 recordsLinked to original sources

PubMed1995

Minimization of a polypeptide hormone.

A stepwise approach for reducing the size of a polypeptide hormone, atrial natriuretic peptide (ANP), from 28 residues to 15 while retaining high biopotency is described. Systematic structural and functional analysis identified a discontinuous functional epitope for receptor binding and activation, most of which was placed onto a smaller ring (Cys6 to Cys17) that was created by repositioning the ANP native disulfide bond (Cys7 to Cys23). High affinity was subsequently restored by optimizing the remaining noncritical residues by means of phage display. Residues that flanked the mini-ring structure were then deleted in stages, and affinity losses were rectified by additional phage-sorting experiments. Thus, structural and functional data on hormones, coupled with phage display methods, can be used to shrink the hormones to moieties more amendable to small-molecule design.

Amino Acid Sequence↗

PubMed1995

Mutations of the growth hormone receptor in children with idiopathic short stature. The Growth Hormone Insensitivity Study Group.

BACKGROUND: Short stature in children who are not deficient in growth hormone (GH) is probably caused by a variety of defects. Some children with idiopathic short stature have low serum concentrations of GH-binding protein, which is derived from the GH receptor. The possibility that low serum concentrations of GH-binding protein might indicate partial insensitivity to GH led us to investigate possible defects in the gene for the GH receptor in children with idiopathic short stature and low serum concentrations of GH-binding protein. METHODS: We studied 14 children with idiopathic short stature who were selected on the basis of normal GH secretion and low serum concentrations of GH-binding protein. Analysis of single-strand conformation polymorphisms and DNA sequencing were both used to identify mutations in the GH-receptor gene. RESULTS: Mutations in the region of the GH-receptor gene that codes for the extracellular domain of the receptor were found in 4 of the 14 children, but in none of 24 normal subjects. One of the four children with mutations was a compound heterozygote, with one mutation that reduced the affinity of the receptor for GH and a second mutation that may affect a function other than ligand binding. The remaining three children had single mutations in one allele of the gene. One mutation introduced a premature termination codon, and two caused substitutions of single amino acids in a structurally conserved domain of the receptor. CONCLUSIONS: Some children with idiopathic short stature may have partial insensitivity to GH due to mutations in the GH-receptor gene.

Adolescent↗

PubMed1995

Designing subtilisin BPN' to cleave substrates containing dibasic residues.

The bacterial serine protease, subtilisin BPN', has been mutated so that it will efficiently and selectively cleave substrates containing two consecutive basic (dibasic) residues. Mutants were designed on the basis of both the structure of subtilisin BPN' and considerations of sequence differences between it and eukaryotic homologs, Kex2, PC2, and furin, which are known to cleave dibasic substrates. These eukaryotic proteases have high sequence homology to one another but differ substantially from subtilisin BPN' in loops that interact with the substrate. When these loops were grafted into subtilisin BPN', the mutated enzyme could not be expressed, presumably due to destabilization of the folded enzyme. We noted that several neutral residues in subtilisin BPN' (Gly 166, Ser 33, and Asn 62) that are positioned to interact with a dibasic substrate are acidic residues at analogous positions in Kex2. Mutating these residues individually to either Glu or Asp in subtilisin BPN' resulted in systematic shifts in substrate specificity (kcat/Km) toward basic residues and away from the natural preference for hydrophobic substrates. A combination mutant, where Asn 62 was changed to Asp and Gly 166 was changed to Asp (N62D/G166D), had a larger than additive shift in specificity toward dibasic substrates. This unexpectedly large change was confirmed by detailed analysis with a variety of synthetic substrates. Additional substrate determinants were revealed by sorting a library of phage particles (substrate phage) containing five contiguous randomized residues. This method identified a particularly good substrate (Asn-Leu-Met-Arg-Lys) that was selectively cleaved in the context of a fusion protein by the N62D/G166D subtilisin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Prolactin receptor antagonists that inhibit the growth of breast cancer cell lines.

We investigated the mechanism of action of the human prolactin (hPRL) receptor on four different breast cancer cell lines, T-47D, MCF-7, BT-474, and SK-BR3, that express elevated levels of the receptor compared with normal cells. Cells treated with human growth hormone (hGH), which binds and activates the hPRL receptor, exhibited bell-shaped dose-response growth curves consistent with the sequential dimerization mechanism proposed for the hPRL receptor (Fuh, G., Colosi, P., Wood, W.I., and Wells, J.A. (1993) J. Biol. Chem. 268, 5376-5381). Growth stimulation was enhanced by Zn2+, which preferentially increases the affinity of hGH for the hPRL receptor. Furthermore, receptor-selective variants of hGH that bind the hPRL receptor but not the hGH receptor were agonistic, providing additional support that specific binding to the hPRL receptor can stimulate these breast cancer cells to grow. On this basis we produced variants of hGH and human placental lactogen (hPL) that were potential antagonists because they bind but do not dimerize the hPRL receptor. The hPL-based antagonist was less potent than the hGH-based antagonist toward the growth of MCF-7 cells, consistent with the lower affinity of hPL for hPRL receptor than for hGH. However, the hPL-based antagonist was more potent than the hGH antagonist for BT-474 cells. Antibodies to the hPRL receptor inhibited growth of FDC-P1 cells transfected with the hPRL receptor; these also inhibited MCF-7 cells and T47D cells but not BT-474 cells. A unique feature of BT-474 cells was found when screening its cDNA revealed the presence of a novel alternative splice of the hPRL receptor that codes for the soluble extracellular domain; this may explain these differential inhibitory effects. These studies provide further molecular insight into the potential role of the hPRL receptor in breast cancer and demonstrate that hPRL receptor antagonists can inhibit the growth of breast cancer cells.

Alternative Splicing↗

PubMed1995

A hot spot of binding energy in a hormone-receptor interface.

The x-ray crystal structure of the complex between human growth hormone (hGH) and the extracellular domian of its first bound receptor (hGHbp) shows that about 30 side chains from each protein make contact. Individual replacement of contact residues in the hGHbp with alanine showed that a central hydrophobic region, dominated by two tryptophan residues, accounts for more than three-quarters of the binding free energy. This "functional epitope" is surrounded by less important contact residues that are generally hydrophilic and partially hydrated, so that the interface resembles a cross section through a globular protein. The functionally important residues on the hGHbp directly contact those on hGH. Thus, only a small and complementary set of contact residues maintains binding affinity, a property that may be general to protein-protein interfaces.

Carrier Proteins↗

PubMed1995

The prevalence of homosexual behavior and attraction in the United States, the United Kingdom and France: results of national population-based samples.

Researchers determining the prevalence of homosexuality in nationally representative samples have focused upon determining the prevalence of homosexual behavior, ignoring those individuals whose sexual attraction to the same sex had not resulted in sexual behavior. We examine the use of sexual attraction as well as sexual behavior to estimate the prevalence of homosexuality in the United States, the United Kingdom, and France using the Project HOPE International Survey of AIDS-Risk Behaviors. We find that 8.7, 7.9, and 8.5% of males and 11.1, 8.6, and 11.7% of females in the United States, the United Kingdom, and France, respectively, report some homosexual attraction but no homosexual behavior since age 15. Further, considering homosexual behavior and homosexual attraction as different but overlapping dimensions of homosexuality, we find 20.8, 16.3, and 18.5% of males, and 17.8, 18.6, and 18.5% of females in the United States, the United Kingdom, and France report either homosexual behavior or homosexual attraction since age 15. Examination of homosexual behavior separately finds that 6.2, 4.5, and 10.7% of males and 3.6, 2.1, and 3.3% of females in the United States, the United Kingdom, and France, respectively, report having had sexual contact with someone of the same sex in the previous 5 years. Our findings highlight the importance of using more than just homosexual behavior to examine the prevalence of homosexuality.

Adolescent↗

PubMed1994

Subtiligase: a tool for semisynthesis of proteins.

A variant of subtilisin BPN', which we call subtiligase, has been used to ligate esterified peptides site-specifically onto the N termini of proteins or peptides in aqueous solution and in high yield. We have produced biotinylated or heavy-atom derivatives of methionyl-extended human growth hormone (Met-hGH) by ligating it onto synthetic peptides containing biotin or mercury. Polyethylene glycol (PEG)-modified atrial natriuretic peptide (ANP) was produced by ligating ANP onto peptides containing sites for PEG modification. We have established the N-terminal sequence requirements for efficient ligation onto proteins, using either synthetic substrates or pools of filamentous phage containing Met-hGH with random N-terminal sequences (substrate phage). To facilitate ligations involving proteins with highly structured or buried N termini, a more stable subtiligase was designed that effectively ligates peptides onto Met-hGH even in 4 M guanidine hydrochloride. The use of subtiligase should expand the possibilities for protein semisynthesis and rational protein design.

Amino Acid Sequence↗

PubMed1994

A designed peptide ligase for total synthesis of ribonuclease A with unnatural catalytic residues.

An engineered variant of subtilisin BPN', termed subtiligase, which efficiently ligates esterified peptides in aqueous solution, was used for the complete synthesis of ribonuclease (RNase) A that contains unnatural catalytic residues. Fully active RNase A (124 residues long) was produced in milligram quantities by stepwise ligation of six esterified peptide fragments (each 12 to 30 residues long) at yields averaging 70 percent per ligation. Variants of RNase A were produced in which the catalytic histidines at positions 12 and 119 were substituted with the unnatural amino acid 4-fluorohistidine, which has a pKa of 3.5 compared to 6.8 for histidine. Large changes in the profile of the pH as it affects rate occurred for the single and double mutants with surprisingly little change in the kcat for either the RNA cleavage or hydrolysis steps. The data indicate that these imidazoles function as general acids and bases, but that the proton transfer steps are not rate-limiting when the imidazoles are present in their correct protonation states. These studies indicate the potential of subtiligase for the blockwise synthesis of large proteins.

Amino Acid Sequence↗

PubMed1994

Production of an atrial natriuretic peptide variant that is specific for type A receptor.

Receptor-specific variants of atrial natriuretic peptide (ANP) were selected from libraries of filamentous phage particles that displayed single copies of random ANP mutants fused to gene III protein. These ANP variants were differentially selected by binding to immobilized natriuretic peptide receptor A (NPR-A) over competing receptor C (NPR-C) in solution. This method also selected ANP variants with improved secretion expression in Escherichia coli. Several of the identified mutations were combined to produce an efficiently expressed ANP analog that was as potent as wild-type ANP in stimulating NPR-A guanylyl cyclase activity but resistant to inactivation mediated by NPR-C. Such NPR-A-selective analogs should be useful for correlating the various activities of ANP to the relevant receptor and may also be more potent therapeutics in the targeting of NPR-A.

Amino Acid Sequence↗

PubMed1994

Structure from function: screening structural models with functional data.

Structural constraints derived from different antibody epitopes on human growth hormone (hGH) were used to screen three-dimensional models of hGH that were generated by computer algorithms. Previously, alanine-scanning mutagenesis defined the residues that modulate binding to 21 different monoclonal antibodies to hGH. These functional epitopes were composed of 4-14 side chains whose alpha-carbons clustered within 4-23 A. Distance and topographic constraints for these functional epitopes were virtually the same as constraints derived from known x-ray structures of protein-antigen complexes. The constraints were used to evaluate about 1400 models of hGH that were computer-generated by a secondary-structure prediction and packing algorithm. On average each functional epitope reduced the number of models in the pool by a factor of 2, so that 8 monoclonal antibodies could reduce the number of possible models to < 10. The average root-mean-square deviation of alpha-carbon coordinates between the x-ray structure and either the pool of starting models or final models ranged from 13 to 16 A or 4 to 7 A, respectively, depending on the pool of starting models and the level of constraints imposed. All of the final models had the correct folding topography, and the best model was within 3.8 A root-mean-square deviation of the x-ray coordinates. This model was as close as it could have been because the models were built by using ideal helices and those in the x-ray structure are not. Our studies suggest that epitope mapping data can effectively screen structural models and, when coupled to predictive algorithms, can help to generate low-resolution models of a protein.

Amino Acid Sequence↗

PubMed1994

A survey of furin substrate specificity using substrate phage display.

The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.

Amino Acid Sequence↗

PubMed1994

Dissecting the energetics of an antibody-antigen interface by alanine shaving and molecular grafting.

Alanine-scanning mutagenesis on human growth hormone (hGH) identified 5 primary determinants (Arg 8, Asn 12, Arg 16, Asp 112, and Asp 116) for binding to a monoclonal antibody (MAb 3) (Jin L, Fendly BM, Wells JA, 1992, J Mol Biol 226:851-865). To further analyze the energetic importance of residues surrounding these five, we mutated all neighboring residues to alanine in groups of 7-16 (a procedure we call alanine shaving). Even the most extremely mutated variant, with 16 alanine substitutions, caused less than a 10-fold reduction in binding affinity to MAb3. By comparison, mutating any 1 of the 5 primary determinants to alanine caused a 6- to > 500-fold reduction in affinity. Replacing any of the 4 charged residues (Arg 8, Arg 16, Asp 112, and Asp 116) with a homologous residue (i.e., Arg to Lys or Asp to Glu) caused nearly as large a reduction in affinity as the corresponding alanine replacement. It was possible to graft the 5 primary binding determinants onto a nonbinding homologue of hGH, human placental lactogen (hPL), which has 86% sequence identity to hGH. The grafted hPL mutant bound 10-fold less tightly than hGH to MAb3 but bound as well as hGH when 2 additional framework mutations were introduced. Attempts to recover binding affinity by grafting the MAb3 epitope onto more distantly related scaffolds having a similar 4-helix bundle motif, such as human prolactin (23% sequence identity) or granulocyte colony-stimulating factor, were unsuccessful.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗

PubMed1994

In vitro selection from protein and peptide libraries.

In vitro selection from molecular libraries has rapidly come of age as a protein-engineering tool. Dramatic increases in protein affinity can be engineered using phage-display libraries, and specific antibodies can be selected directly from a single 'naïve' library of their genes. Repertoires of small molecules are a potentially valuable resource for drug discovery. Libraries of linear peptides provide ligands for proteins that recognize continuous epitopes, and low-affinity mimics of some small molecules, but generally do not contain mimics of large molecular interfaces. Switching to constrained peptide formats, and deploying more diverse, non-peptide chemical libraries, may bring greater success.

Animals↗

PubMed1994

Readability of HIV/AIDS educational materials: the role of the medium of communication, target audience, and producer characteristics.

The reading difficulty of many HIV/AIDS brochures and pamphlets limits their effectiveness. This analysis addresses correlates of readability in 136 HIV/AIDS educational items. Readability is measured using the SMOG Index. The medium of communication is significantly related to readability: comic books and brochures are, on average, more readable than books and pamphlets (10.9 versus 11.9). The target audience also differentiates readability. Materials for HIV antibody test seekers, the general community, and sexually active adults have a more difficult reading grade, averaging 12.1, whereas materials for ethnic minorities average a more readable 9.2. The producer organization's type and location are unrelated to readability, but an AIDS-specific organizational focus correlates with better readability (grade 10.8 vs. 11.8). These findings remain significant in multivariate analysis. The results indicate that brochures and comics are more likely to be comprehended by low-literacy populations, that an understanding of the literacy of target audiences is needed to produce materials with appropriate reading levels, and that policies to influence producer organizations may result in the creation of more readable materials.

Adolescent↗

PubMed1994

Structural and functional basis for hormone binding and receptor oligomerization.

Most single-pass transmembrane receptors undergo a change in oligomeric state upon hormone binding. Recent mutational, biophysical and structural studies of the human growth hormone and tumor necrosis factor receptor complexes have revealed much about the mechanisms and molecular bases for binding and oligomerization. Principles learned from these examples and others should apply to many other hormone-receptor complexes.

Animals↗

PubMed1994

Bilateral macular drusen in age-related macular degeneration. Prognosis and risk factors.

BACKGROUND: In patients with unilateral visual loss related to age-related macular disease, the risk of visual loss in the second eye is documented as being between 7% and 10% per year. The risk is uncertain in those with good vision with each eye and bilateral macular drusen. METHODS: In a prospective study, 126 patients with bilateral drusen were reviewed annually for up to 3 years. Serial fundus photographs and fluorescein angiograms were analyzed independently by two readers in a masked fashion using a standardized grading scheme, including size, number, density, and fluorescence angiographic behavior of drusen. RESULTS: New lesions occurred in one or both eyes of 17 (13.5%) of the 126 patients. The cumulative incidence of exudative or nonexudative lesions was 8.55% at 1 year, at 2 years 16.37%, and 23.52% at 3 years for patients older than 65 years of age. Significant risk factors included the degree of confluence of drusen within 1600 microns of the center of the fovea (P = 0.023), focal hyperpigmentation (P = 0.004), slow choroidal filling (P = 0.023), and focal extrafoveal areas of atrophy of the retinal pigment epithelium (P = 0.042). CONCLUSIONS: The results give an estimate for the incidence of complicating lesions in patients with bilateral drusen and identify those features indicating higher than average risk of visual loss.

Aged↗

PubMed1994

Corneal opacities in spondyloepiphyseal dysplasia tarda.

Spondyloepiphyseal dysplasia tarda is an inherited skeletal dysplasia involving the spine and epiphyses of long bones with onset in childhood, giving rise to disproportionate short stature and degenerative spine and hip disease. Associated ocular disease is not commonly recognized. We report a patient with spondyloepiphyseal dysplasia tarda and a unique pattern of corneal opacities. Bilateral, irregularly shaped, nodular, deeply posterior opacities confined to the peripheral cornea were noted in this patient. A central stromal granularity was also seen. Minimal visual loss was associated with these findings. An X-linked inheritance pattern is presumed but could not be confirmed.

Aged↗

PubMed1994

Ocular findings associated with a 3 base pair deletion in the peripherin-RDS gene in autosomal dominant retinitis pigmentosa.

Affected members of a family with autosomal dominant retinitis pigmentosa were found to have a 3 base pair deletion at codon 118 or 119 of the retinal degeneration slow gene. This mutation causes the loss of a highly conserved cysteine residue in the predicted third transmembrane domain of peripherin-rds, a photo-receptor specific structural glycoprotein localised to both rod and cone outer segment disc membranes. Four of these individuals underwent detailed clinical, psychophysical, and electroretinographic testing in order to characterise their photoreceptor dysfunction. Nyctalopia was reported early in the second decade by all patients. Global rod and cone dysfunction was recorded by the third decade with severe reduction of both photopic and scotopic function by age 30 years. This retinal degeneration slow gene mutation may lead to the primary loss of both rod and cone photo-receptor function.

Adult↗