Derril H. Willard

Functional Dissection of Osteoprotegerin and Its Interaction with Receptor Activator of NF-κB Ligand

The receptor activator of NF-κB (RANK) belongs to the neuregulin/tumor necrosis factor (TNF) receptor superfamily and is activated by RANK ligand (RANK-L), a homotrimeric, TNF-like cytokine. RANK is present on the surface of osteoclast cell precursors, where its interaction with RANK-L induces their terminal differentiation into osteoclasts, thus increasing bone breakdown. The secreted, soluble receptor osteoprotegerin (OPG) interrupts this activation by binding directly to RANK-L. Therefore, osteoclast maturation (and bone homeostasis) is regulated in vivo by OPG levels of expression. We have studied the assembly state and affinity of OPG for RANK-L by sedimentation analyses and surface plasmon resonance (Biacore). Full-length, homodimeric OPG binds to RANK-L with a KD of 10 nm. OPG is also a member of the TNF receptor superfamily and contains four disulfide-rich ligand-binding domains, yet lacks a transmembrane region separating the ligand-binding region from the two death domains, as observed for other receptor family members. We showed that dimerization of OPG results from noncovalent interactions mediated by the death domains and to a lesser extent by a C-terminal heparin-binding region. In contrast, a C-terminal intermolecular disulfide bond does not contribute to the formation or stability of OPG dimers. A truncate of osteoprotegerin, containing the ligand-binding domains but lacking the dimerization domains, bound RANK-L with a KD of ∼3 μm, indicating that monomer oligomerization for the OPG provides an increase of 3 orders of magnitude in the affinity for RANK-L. Therefore, OPG dimer formation is required for the mechanism of inhibition of the RANK-L/RANK receptor interaction.

INTERACTIONS OF ALPHA -MELANOTROPIN AND AGOUTI ON B16 MELANOMA CELLS : EVIDENCE FOR INVERSE AGONISM OF AGOUTI

alpha-Melanocyte-stimulating hormone (alpha-MSH, alpha-melanotropin) and agouti control the switch between eumelanin and pheomelanin synthesis in mammalian melanocytes. Here we investigated interactions between alpha-MSH, agouti protein, cAMP elevating agents and phorbol ester on mouse B16 melanoma cells. Agouti (Kd 3.7 nmol/l) and alpha-MSH (Kd 2.3 nmol/l) had similar affinities to the MC1 melanocortin receptor. Both alpha-MSH and agouti induced MC1 receptor down-regulation. Agouti antagonized melanogenesis induced by alpha-MSH, forskolin, cholera toxin (CT), and pertussis toxin (PT). It also reduced the constitutive melanin formation of long-term cultures. Cell proliferation was inhibited by agouti (43% at 100 nM). This effect was reversed by alpha-MSH, forskolin, or CT. B16-G4F cells, a cell variant that lacks the MC1 receptor, did not respond to agouti. From these results we conclude that agouti shows the characteristics of an inverse agonist acting through the MC1 receptor.

Product Release Is the Major Contributor tok cat for the Hepatitis C Virus Helicase-catalyzed Strand Separation of Short Duplex DNA

Hepatitis C virus (HCV) helicase catalyzes the ATP-dependent strand separation of duplex RNA and DNA containing a 3′ single-stranded tail. Equilibrium and velocity sedimentation centrifugation experiments demonstrated that the enzyme was monomeric in the presence of DNA and ATP analogues. Steady-state and pre-steady-state kinetics for helicase activity were monitored by the fluorescence changes associated with strand separation of F21:HF31 that was formed from a 5′-hexachlorofluorescein-tagged 31-mer (HF31) and a complementary 3′-fluorescein-tagged 21-mer (F21).k cat for this reaction was 0.12 s−1. The fluorescence change associated with strand separation of F21:HF31 by excess enzyme and ATP was a biphasic process. The time course of the early phase (duplex unwinding) suggested only a few base pairs (∼2) were disrupted concertedly. The maximal value of the rate constant (k eff) describing the late phase of the reaction (strand separation) was 0.5 s−1, which was 4-fold greater than k cat. Release of HF31 from E·HF31 in the presence of ATP (0.21 s−1) was the major contributor tok cat. At saturating ATP and competitor DNA concentrations, the enzyme unwound 44% of F21:HF31 that was initially bound to the enzyme (low processivity). These results are consistent with a passive mechanism for strand separation of F21:HF31 by HCV helicase.

Agouti Antagonism of Melanocortin-4 Receptor: Greater Effect with Desacetyl-α-Melanocyte-Stimulating Hormone (MSH) than withα -MSH

Desacetyl-alpha-MSH is more abundant than alpha-MSH in the brain, the fetus, human blood, and amniotic fluid, but there is little information on its ability to interact with melanocortin receptors. The aim of this study is to compare and contrast the ability of desacetyl-alpha-MSH and alpha-MSH to couple melanocortin receptors stably expressed in HEK293 cells, to the protein kinase A (PKA) signaling pathway. Desacetyl-alpha-MSH activated mouse MC1, MC3, MC4 and MC5 receptors with EC50s = 0.13, 0.96, 0.53, and 0.84 nM, and alpha-MSH activated these receptors with EC50s = 0.17, 0.88, 1.05, and 1.34 nM, respectively. Mouse agouti protein competitively antagonized alpha-MSH and desacetyl-alpha-MSH coupling to the MC1-R similarly. In contrast, mouse agouti protein antagonized desacetyl-alpha-MSH much more effectively and potently than alpha-MSH coupling the MC4-R to the PKA signaling pathway. Furthermore, mouse agouti protein (10 nM) significantly reduced (1.4-fold) the maximum response of mMC4-R to desacetyl-alpha-MSH and 100 nM mouse agouti significantly increased (4.8-fold) the EC50. Minimal antagonism of alpha-MSH coupling mMC4-R to the PKA signaling pathway was observed with 10 nM mouse agouti, whereas both 50 and 100 nM mouse agouti appeared to reduce the maximum reponse (1.1- and 1.3-fold, respectively) and increase the EC50 (2.5- and 3.4-fold respectively). Mouse agouti protein did not significantly antagonize either alpha-MSH or desacetyl-alpha-MSH coupling mouse MC3 and MC5 receptors. Understanding the similarities and differences in activation of melanocortin receptors by desacetyl-alpha-MSH and alpha-MSH will contribute to delineating the functional roles for these endogenous melanocortin peptides.

RAPID PURIFICATION, SEPARATION AND IDENTIFICATION OF PROTEINS AND ENZYME DIGESTS USING PACKED CAPILLARY PERFUSION COLUMN LC AND LC/MS

Publisher Summary This chapter presents a study incorporating rapid purification, separation, and identification of proteins and enzyme digests using packed capillary perfusion column liquid chromatography (LC) and LC/mass spectrometry (MS). In this study, C-src tyrosine kinase SH2 domain was placed in a pGEX-3X vector and expressed in XLI Blue cells. E. coli cells obtained from a 5-liter fermentation were thawed in a 1:5 dilution of lysis buffer. The resulting sample was passed through a 40,000 psi piston 2X in a SLM Amicon French Press. The disrupted cells were centrifuged at 20,000g to remove cellular debris. The supernatant was diluted twofold with lysis buffer and dounce homogenized for two minutes before centrifugation at 48,000i. The resulting supernatant contained the GST–SH2 fusion. A total of 30 mg of fusion product was purified using a glutathione–agarose affinity column. The study demonstrated the first successful coupling of custom packed 320 μm i.d fused silica capillaries with reverse-phase perfusion packing to electrospray ionization mass spectrometry.

A strategy for predicting the ligand binding competence of recombinant orphan nuclear receptors using biophysical characterization

Publisher Summary Nuclear receptors have been historically associated with the steroid hormone receptors, for example, estrogen and glucocorticoid receptors, by virtue of DNA binding domain sequence homology comprising two zinc finger motifs. Many of these are orphan receptors, having no defined ligand. The nuclear receptors present tempting targets in the pursuit of a systems based research approach as so many at present have been cloned. However, when recombinant forms of a receptor are available before its cognate ligand has been identified, confusion arises on weather an orphan receptor is active for use in vitro assays. Researchers now have access to unparalleled amounts of DNA sequence and genetic data. Families of homologous gene products can be studied with the intent of connecting specific proteins to various disease conditions. Specifically, to apply this type of strategy in studies, the problem has been approached with two premises in the chapter. First, the recombinant constructs of orphan nuclear receptors are engineered to contain domains with hypothetical functional homology to receptors with known activities and ligands. In particular, a lot of knowledge has been gained concerning retinoid X receptor a (RXRα) and the domains necessary for DNA binding, retinoid binding, and self/hetero-association. For the purpose of this study, PPARα, PPARδ, PPARγ, RXRα, and LXRα constructs were created to contain the putative ligand binding domains (LBD). The amino acid residues within this conserved contiguous region have been shown to be both necessary and sufficient to demonstrate ligand binding competence for RXRα and other nuclear receptors. Structurally, the LBDs are composed primarily of multiple α-helicies. Second, each nuclear receptor is characterized using a variety of biophysical techniques. This chapter has presented data that suggests that the usefulness of a recombinant protein can be determined before an appropriate ligand is available. These results are an encouraging start in the attempt to predict the binding competency of recombinant orphan nuclear receptors.