Tanya MacNeil

Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector

An integration vector for gene analysis in Streptomyces has been constructed. This vector replicates in Escherichia coli, and integrates into Streptomyces by homologous recombination between a cloned fragment and the genome. To overcome methylation-specific restriction barriers, an E. coli mutant triply defective in DNA methylation was constructed as a source for the integration plasmids. The frequency of integration of pVE616 derivatives into the Streptomyces avermitilis genome was proportional to the size of the cloned DNA. Derivatives of pVE616, containing fragments from pVE650, a plasmid with a 24-kb insert of S. avermitilis DNA, were used in complementation analyses of seven S. avermitilis mutants defective in glycosylation of avermectin (Av). Three complementation groups, located in a 7-kb region, were identified. Derivatives of pVE616, containing fragments from the 18-kb of DNA adjacent to the glycosylation region, were integrated into an Av producer. Av produced from the integrants was substantially reduced, indicating that the 18 kb also encodes gene products which are involved in Av biosynthesis.

A role for the melanocortin 4 receptor in sexual function

By using a combination of genetic, pharmacological, and anatomical approaches, we show that the melanocortin 4 receptor (MC4R), implicated in the control of food intake and energy expenditure, also modulates erectile function and sexual behavior. Evidence supporting this notion is based on several findings: (i) a highly selective non-peptide MC4R agonist augments erectile activity initiated by electrical stimulation of the cavernous nerve in wild-type but not Mc4r-null mice; (ii) copulatory behavior is enhanced by administration of a selective MC4R agonist and is diminished in mice lacking Mc4r; (iii) reverse transcription (RT)-PCR and non-PCR based methods demonstrate MC4R expression in rat and human penis, and rat spinal cord, hypothalamus, brainstem, pelvic ganglion (major autonomic relay center to the penis), but not in rat primary corpus smooth muscle cavernosum cells; and (iv) in situ hybridization of glans tissue from the human and rat penis reveal MC4R expression in nerve fibers and mechanoreceptors in the glans of the penis. Collectively, these data implicate the MC4R in the modulation of penile erectile function and provide evidence that MC4R-mediated proerectile responses may be activated through neuronal circuitry in spinal cord erectile centers and somatosensory afferent nerve terminals of the penis. Our results provide a basis for the existence of MC4R-controlled neuronal pathways that control sexual function.

Complex organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase

The avermectin (Av) polyketide synthase (PKS) and erythromycin (Er) PKS are encoded by modular repeats of DNA, but the genetic organization of the modules encoding Av PKS is more complex than Er PKS. Sequencing of several related DNA fragments from Streptomyces avermitilis that are part of the Av biosynthetic gene cluster, revealed that they encode parts of large multifunctional PKS proteins. The Av PKS proteins show strong similarity to each other, as well as similarity to Er PKS proteins [Donadio et al., Science 252 (1991) 675-679] and fatty acid synthases. Partial DNA sequencing of the 65-kb region containing all the related sequence elements in the avr genes provides evidence for twelve modular repeats encoding FAS-like domains. The genes encoding the Av PKS are organized as two sets of six modular repeats which are convergently transcribed.

The role of melanocortins in body weight regulation: opportunities for the treatment of obesity

Five G-protein-coupled melanocortin receptors (MC(1)-MC(5)) are expressed in mammalian tissues. The melanocortin receptors support diverse physiological functions, including the regulation of hair color, adrenal function, energy homeostasis, feed efficiency, sebaceous gland lipid production and immune and sexual function. The melanocortins (adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), beta-MSH and gamma-MSH) are agonist peptide ligands for the melanocortin receptors and these peptides are processed from the pre-prohormone proopiomelanocortin (POMC). Peptide antagonists for the melanocortin MC(1), MC(3) and MC(4) receptors include agouti-related protein (AgRP) and agouti. Diverse lines of evidence, including genetic and pharmacological data obtained in rodents and humans, support a role for the melanocortin MC(3) and MC(4) receptors in the regulation of energy homeostasis. Recent advances in the development of potent and selective peptide and non-peptide melanocortin receptor ligands are anticipated to help unravel the roles for the melanocortin receptors in humans and to accelerate the clinical use of small molecule melanocortin mimetics.

Cloning and pharmacological characterization of a rabbit bradykinin B1 receptor

A rabbit B1 bradykinin receptor cDNA was isolated from a rabbit aorta smooth muscle cell library. The 1223 bp cDNA clone encodes a protein of 352 amino acids which is 78% identical to the human bradykinin B1(3) receptor protein. Heterologous expression of the rabbit B1 receptor cDNA in COS-7 cells imparts a high affinity specific binding for 3H-labeled [des-Arg10,Leu9]kallidin. Scatchard analysis indicates that the receptor binds the radiolabeled ligand with a Kd of 0.5 nM. The ability of kallidin (Lys-bradykinin) and bradykinin analogues to compete with binding of 3H-labeled [des-Arg10,Leu9]kallidin was determined and defined a rank order of potency: [des-Arg10,Leu9]kallidin = [des-Arg10]kallidin > [des- Arg9]bradykinin = kallidin >> bradykinin. This receptor exhibits the classical B1 pharmacological property of preferentially binding to kinin analogues which lack the C-terminal arginine. In addition, the affinities for [des-Arg10]kallidin and [des-Arg10,Leu9]kallidin are 100-fold higher than those for the corresponding bradykinin analogues [des-Arg9]bradykinin and [des-Arg9,Leu8]bradykinin which lack the N-terminal lysine. This pharmacological profile is characteristic of the B1 receptor subtype.

Structure-function studies on the cyclic peptide MT-II, lactam derivative of α-melanotropin

The alanine-substituted and the retro, enantio, and retro-enantio analogs of MT-II, a potent agonist at melanocortin (MC) receptors, were prepared by solid-phase synthesis and evaluated for their ability to bind and activate human MC3, MC4, and MC5 receptors. Replacement of His with Ala resulted in [Ala6]-MT-II with affinity and agonist potency at human MC3, MC4, and MC5 receptors similar to MT-II. Substitution of Arg with Ala gave compound 100-fold less potent than MT-II, but replacement of Phe or Trp with Ala led to inactive compounds (at the micromolar concentrations). The significant drop of potency of the retro, enantio, and retro-enantio analogs of MT-II, demonstrated a crucial role of side-chain topology, and to a lesser degree, of peptide backbone in interactions of MT-II with the melanocortin receptors. The nuclear magnetic resonance analysis of MT-II suggested involvement of Phe and Arg residues in H-bonds stabilizing the bent conformations of the peptide backbone.

The products of glnL and glnG are bifunctional regulatory proteins

SummaryThe role of the two glnA linked genes, glnL and glnG, in regulation of glnA expression and nitrogen metabolism in Escherichia coli has been studied by analysis of 131 glnL and 164 glnG genetically characterized mutations. A comparison of phenotypes with genetic position was performed for all mutations in glnL and glnG. We determined the ability of mutants to derepress GS, to grow on a variety of nitrogen sources in the absence of glutamine, and to suppress the glutamine requirement caused by a glnF mutation. The results indicate that both glnL and glnG products mediate negative regulation of glnA. The glnG product, but not that of glnL, is required for derepression of glnA. Both glnL and glnG products are required for positive regulation of gene products involved in the utilization of poor nitrogen sources. In each gene, point mutations were found which confer a phenotype dramatically different than that caused by insertion mutations. These point mutations fall into several frequently occurring classes. The phenotypes of these classes suggests that each gene product has bifunctional regulatory properties. Further, each class tends to be located in only a portion of a gene suggesting that the region encoding each function is genetically distinct.The role of glutamine synthetase in the regulation of glnA expression was investigated using two-dimensional polyacrylamide gel electrophoresis on extracts of 38 GlnA- mutants. Results of this analysis argue that glutamine synthetase is not structurally involved in the regulation of glnA expression.

Molecular analysis of a new splice variant of the human melanocortin-1 receptor

The primary hormonal regulator of pigmentation is melanocyte stimulating hormone derived from proopiomelanocortin by proteolytic processing. The melanocortin‐1 receptor serves a key role in the regulation of pigmentation. We describe the identification of the first intron within a melanocortin receptor. A new melanocortin‐1 receptor isoform, generated by alternative mRNA splicing, encodes an additional 65 amino acids at the predicted intracellular, C‐terminal tail of the melanocortin‐1 receptor. When expressed in heterologous cells, the new spliced form of the melanocortin‐1 receptor (melanocortin‐1 receptor B) appears pharmacologically similar to the non‐spliced melanocortin‐1 receptor. Melanocortin‐1 receptor B is expressed in testis, fetal heart and melanomas.

Extensive structure–activity studies of lactam derivatives of MT‐II and SHU‐9119: their activity and selectivity at human melanocortin receptors 3, 4, and 5

The melanocortin system is involved in the regulation of several diverse physiologic pathways. Recently we have demonstrated that replacing His6 by Pro6 in the well-known antagonist SHU-9119 resulted in a potent agonist at the hMC5R (EC50 = 0.072 nm) with full antagonist activity at the hMC3R and the hMC4R. We have designed, synthesized, and pharmacologically characterized a series of peptide analogs of MT-II and SHU-9119 at the human melanocortin receptors MC3R, MC4R and MC5R. All these peptides were modified at position 6 with a Pro instead of a His residue. In this study, we have identified new scaffolds which are antagonists at the hMC4R and hMC3R. Additionally, we have discovered a new selective agonist at the hMC4R, Ac-Nle-c[Asp-Pro-D-Phe-Arg-Trp-Lys]-Pro-Val-NH2 (6, PG-931) which will be useful in further biologic investigations of the hMC4R. PG-931 was about 100-fold more selective for the hMC4R vs. the hMC3R (IC50 = 0.58 and 55 nm, respectively). Some of these new analogs have exceptional biologic potencies at the hMC5R and will be useful in further efforts to differentiate the substructural features responsible for selectivity at the hMC3R, hMC4R, and hMC5R.

Correlation of the Avermectin Polyketide Synthase Genes to the Avermectin Structure: Implications for Designing Novel Avermectins

Streptomyces avermitilis produces a series of eight potent anthelmintic compounds called avermectins (AVM). AVM are pentacyclic, macrocyclic lactone compounds containing an oleandrose disaccharide. Labeling studies have shown that AVM is a polyketide derived from the condensation of 12 acyl units (five propionates and seven acetates) to an isobutyl or 2-methylbutyryl starter unit. The genes required for AVM biosynthesis have been cloned, and deletion mapping has located the AVM gene cluster to a 95-kb region. Partial DNA sequencing of this region indicates two 30-kb segments encode large, multifunctional peptides of the AVM polyketide synthase (PKS). The PKS proteins contain at least 49 domains with homology to the domains in fatty acid synthase and erythromycin PKS. These domains are arranged as 12 modular repeats that each encode a PKS unit with various subsets of the FAS-like functions. The predicted functions required to form the side groups on the AVM macrocyclic ring were compared to the functions found in the 12 PKS units. This comparison suggests that each PKS unit is specific for condensation and reduction of one acyl unit. If the various domains can be manipulated without disrupting the PKS, it may be possible to synthesize a variety of AVM derivatives.