Darren A. Thompson

A β-Defensin Mutation Causes Black Coat Color in Domestic Dogs

Genetic analysis of mammalian color variation has provided fundamental insight into human biology and disease. In most vertebrates, two key genes, Agouti and Melanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls pigment type-switching, but in domestic dogs, a third gene is implicated, the K locus, whose genetic characteristics predict a previously unrecognized component of the melanocortin pathway. We identify the K locus as β-defensin 103 (CBD103) and show that its protein product binds with high affinity to the Mc1r and has a simple and strong effect on pigment type-switching in domestic dogs and transgenic mice. These results expand the functional role of β-defensins, a protein family previously implicated in innate immunity, and identify an additional class of ligands for signaling through melanocortin receptors.

Selecting Improved Peptidyl Motifs for Cytosolic Delivery of Disparate Protein and Nanoparticle Materials

Cell penetrating peptides facilitate efficient intracellular uptake of diverse materials ranging from small contrast agents to larger proteins and nanoparticles. However, a significant impediment remains in the subsequent compartmentalization/endosomal sequestration of most of these cargoes. Previous functional screening suggested that a modular peptide originally designed to deliver palmitoyl-protein thioesterase inhibitors to neurons could mediate endosomal escape in cultured cells. Here, we detail properties relevant to this peptides ability to mediate cytosolic delivery of quantum dots (QDs) to a wide range of cell-types, brain tissue culture and a developing chick embryo in a remarkably nontoxic manner. The peptide further facilitated efficient endosomal escape of large proteins, dendrimers and other nanoparticle materials. We undertook an iterative structure-activity relationship analysis of the peptide by discretely modifying key components including length, charge, fatty acid content and their order using a comparative, semiquantitative assay. This approach allowed us to define the key motifs required for endosomal escape, to select more efficient escape sequences, along with unexpectedly identifying a sequence modified by one methylene group that specifically targeted QDs to cellular membranes. We interpret our results within a model of peptide function and highlight implications for in vivo labeling and nanoparticle-mediated drug delivery by using different peptides to co-deliver cargoes to cells and engage in multifunctional labeling.

Inverse agonist activity of agouti and agouti-related protein

Agouti and agouti-related protein (AgRP) are endogenous antagonists of the melanocortin receptors (MCxR). Previous data showed that recombinant full-length agouti and a synthetic fragment of AgRP, AgRP (83-132), are inverse agonists at the MC1R and MC4R, respectively. This study demonstrates the smaller analogs AgRP (87-120) and ASIP [90-132 (L89Y)], and short peptides Yc[CRFFNAFC]Y and Qc[CRFFRSAC]S are also MC4R inverse agonists. Furthermore, the relative affinity of the series of MC4R ligands for displacement of radiolabeled antagonist 125I-AgRP (86-132) versus radiolabeled agonist 125I-NDP-MSH did not correlate with ligand efficacy, which is more consistent with an induced-fit model than a simple two-state model of MC4R activation. These data shed new light on the determinants and mechanism of inverse agonism at the MC4R.

NMR structure of a minimized human agouti related protein prepared by total chemical synthesis.

The structure of the chemically synthesized C‐terminal region of the human agouti related protein (AGRP) was determined by 2D 1H NMR. Referred to as inimized gouti elated rotein, MARP is a 46 residue polypeptide containing 10 Cys residues involved in five disulfide bonds that retains the biological activity of full length AGRP. AGRP is a mammalian signaling molecule, involved in weight homeostasis, that causes adult onset obesity when overexpressed in mice. AGRP was originally identified by homology to the agouti protein, another potent signaling molecule involved in obesity disorders in mice. While AGRPs exact mechanism of action is unknown, it has been identified as a competitive antagonist of melanocortin receptors 3 and 4 (MC3r, MC4r), and MC4r in particular is implicated in the hypothalamic control of feeding behavior. Full length agouti and AGRP are only 25% homologous, however, their active C‐terminal regions are ∼40% homologous, with nine out of the 10 Cys residues spatially conserved. Until now, 3D structures have not been available for either agouti, AGRP or their C‐terminal regions. The NMR structure of MARP reported here can be characterized as three major loops, with four of the five disulfide bridges at the base of the structure. Though its fold is well defined, no canonical secondary structure is identified. While previously reported structural models of the C‐terminal region of AGRP were attempted based on Cys homology between AGRP and certain toxin proteins, we find that Cys spacing is not sufficient to correctly determine the 3D fold of the molecule.

Phosphodiesterase inhibitor-dependent inverse agonism of agouti-related protein on melanocortin 4 receptor in sea bass ( Dicentrarchus labrax )

The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor mainly expressed in the central nervous system of vertebrates. Activation of the MC4R leads to a decrease in food intake, whereas inactivating mutations are a genetic cause of obesity. The binding of agouti-related protein (AGRP) reduces not only agonist-stimulated cAMP production (competitive antagonist) but also the basal activity of the receptor, as an inverse agonist. Transgenic zebrafish overexpressing AGRP display increased food intake and linear growth, indicative of a physiological role for the melanocortin system in the control of the energy balance in fish. We report on the cloning, pharmacological characterization, tissue distribution, and detailed brain mapping of a sea bass (Dicentrarchus labrax) MC4R ortholog. Sea bass MC4R is profusely expressed within food intake-controlling pathways of the fish brain. However, the activity of the melanocortin system during progressive fasting does not depend on the hypothalamic/pituitary proopiomelanocortin (POMC) and MC4R expression, which suggests that sea bass MC4R is constitutively activated and regulated by AGRP binding. We demonstrate that AGRP acts as competitive antagonist and reduces MTII-induced cAMP production. AGRP also decreases the basal activity of the receptor as an inverse agonist. This observation suggests that MC4R is constitutively active and supports the evolutionary conservation of the AGRP/MC4R interactions. The inverse agonism, but not the competitive antagonism, depends on the presence of a phosphodiesterase inhibitor (IBMX). This suggests that inverse agonism and competitive antagonism operate through different intracellular signaling pathways, a view that opens up new targets for the treatment of melanocortin-induced metabolic syndrome.

Pineal-specific agouti protein regulates teleost background adaptation

Background adaptation is used by teleosts as one of a variety of camouflage mechanisms for avoidance of predation. Background adaptation is known to involve light sensing by the retina and subsequent regulation of melanophore dispersion or contraction in melanocytes, mediated by α-melanocyte–stimulating hormone and melanin-concentrating hormone, respectively. Here, we demonstrate that an agouti gene unique to teleosts, agrp2, is specifically expressed in the pineal and is required for up-regulation of hypothalamic pmch and pmchl mRNA and melanosome contraction in dermal melanocytes in response to a white background. floating head, a mutant with defective pineal development, exhibits defective up-regulation of mch mRNAs by white background, whereas nrc, a blind mutant, exhibits a normal response. These studies identify a role for the pineal in background adaptation in teleosts, a unique physiological function for the agouti family of proteins, and define a neuroendocrine axis by which environmental background regulates pigmentation.

Mechanism of N-terminal modulation of activity at the melanocortin-4 receptor GPCR.

Most of our understanding of G protein-coupled receptor (GPCR) activation has been focused on the direct interaction between diffusible ligands and their seven-transmembrane domains. However, a number of these receptors depend on their extracellular N-terminal domain for ligand recognition and activation. To dissect the molecular interactions underlying both modes of activation at a single receptor, we used the unique properties of the melanocortin-4 receptor (MC4R), a GPCR that shows constitutive activity maintained by its N-terminal domain and is physiologically activated by the peptide α-melanocyte stimulating hormone (αMSH). We find that activation by the N-terminal domain and αMSH relies on different key residues in the transmembrane region. We also demonstrate that agouti-related protein, a physiological antagonist of MC4R, acts as an inverse agonist by inhibiting N terminus-mediated activation, leading to the speculation that a number of constitutively active orphan GPCRs could have physiological inverse agonists as sole regulators.

New ligands for melanocortin receptors

Named originally for their effects on peripheral end organs, the melanocortin system controls a diverse set of physiological processes through a series of five G-protein-coupled receptors and several sets of small peptide ligands. The central melanocortin system plays an essential role in homeostatic regulation of body weight, in which two alternative ligands, α-melanocyte-stimulating hormone and agouti-related protein, stimulate and inhibit receptor signaling in several key brain regions that ultimately affect food intake and energy expenditure. Much of what we know about the relationship between central melanocortin signaling and body weight regulation stems from genetic studies. Comparative genomic studies indicate that melanocortin receptors used for controlling pigmentation and body weight regulation existed more than 500 million years ago in primitive vertebrates, but that fine-grained control of melanocortin receptors through neuropeptides and endogenous antagonists developed more recently. Recent studies based on dog coat-color genetics revealed a new class of melanocortin ligands, the β-defensins, which reveal the potential for cross talk between the melanocortin and the immune systems.

Peptoid mimics of agouti related protein

The Agouti Related Protein (AGRP) is an endogenous antagonist of melanocortin-3 and -4 receptors, each of which plays a key role in body weight homeostasis. We designed a peptoid trimer based on AGRP 111-113 in which a single chiral atom is used to partially restrain the backbone structure. Peptoid 5 displaced both radiolabeled Nle4-alpha-MSH (IC(50)=3.1 microM) and AGRP (86-132) (IC(50)=1.9 microM) from the human melanocortin-4 receptor and functioned as an antagonist of alpha-MSH stimulated cAMP generation, thus providing an important lead in the development of AGRP mimetics.

Accessibility of selenomethionine proteins by total chemical synthesis: structural studies of human herpesvirus-8 MIP-II

The determination of high resolution three‐dimensional structures by X‐ray crystallography or nuclear magnetic resonance (NMR) is a time‐consuming process. Here we describe an approach to circumvent the cloning and expression of a recombinant protein as well as screening for heavy atom derivatives. The selenomethionine‐modified chemokine macrophage inflammatory protein‐II (MIP‐II) from human herpesvirus‐8 has been produced by total chemical synthesis, crystallized, and characterized by NMR. The protein has a secondary structure typical of other chemokines and forms a monomer in solution. These results indicate that total chemical synthesis can be used to accelerate the determination of three‐dimensional structures of new proteins identified in genome programs.