Dean E. McNulty

Orexins and Orexin Receptors: A Family of Hypothalamic Neuropeptides and G Protein-Coupled Receptors that Regulate Feeding Behavior

The hypothalamus plays a central role in the integrated control of feeding and energy homeostasis. We have identified two novel neuropeptides, both derived from the same precursor by proteolytic processing, that bind and activate two closely related (previously) orphan G protein-coupled receptors. These peptides, termed orexin-A and -B, have no significant structural similarities to known families of regulatory peptides. prepro-orexin mRNA and immunoreactive orexin-A are localized in neurons within and around the lateral and posterior hypothalamus in the adult rat brain. When administered centrally to rats, these peptides stimulate food consumption. prepro-orexin mRNA level is up-regulated upon fasting, suggesting a physiological role for the peptides as mediators in the central feedback mechanism that regulates feeding behavior.

Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14

Urotensin-II (U-II) is a vasoactive ‘somatostatin-like’ cyclic peptide which was originally isolated from fish spinal cords, and which has recently been cloned from man. Here we describe the identification of an orphan human G-protein-coupled receptor homologous to rat GPR14 (refs 4, 5) and expressed predominantly in cardiovascular tissue, which functions as a U-II receptor. Goby and human U-II bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates. The potency of vasoconstriction of U-II is an order of magnitude greater than that of endothelin-1, making human U-II the most potent mammalian vasoconstrictor identified so far. In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction. Furthermore, as U-II immunoreactivity is also found within central nervous system and endocrine tissues, it may have additional activities.

Proteolytic Activity of Human Osteoclast Cathepsin K EXPRESSION, PURIFICATION, ACTIVATION, AND SUBSTRATE IDENTIFICATION

Human cathepsin K is a recently identified protein with high primary sequence homology to members of the papain cysteine protease superfamily including cathepsins S, L, and B and is selectively expressed in osteoclasts (Drake, F. H., Dodds, R., James, I., Connor, J., Debouck, C., Richardson, S., Lee, E., Rieman, D., Barthlow, R., Hastings, G., and Gowen, M.(1996) J. Biol. Chem. 271, 12511-12516). To characterize its catalytic properties, cathepsin K has been expressed in baculovirus-infected SF21 cells and the soluble recombinant protein isolated from growth media was purified. Purified protein includes an inhibitory pro-leader sequence common to this family of protease. Conditions for enzyme activation upon removal of the pro-sequence have been identified. Fluorogenic peptides have been identified as substrates for mature cathepsin K. In addition, two protein components of bone matrix, collagen and osteonectin, have been shown to be substrates of the activated protease. Cathepsin K is inhibited by E-64 and leupeptin, but not by pepstatin, EDTA, phenylmethylsulfonyl fluoride, or phenanthroline, consistent with its classification within the cysteine protease class. Leupeptin has been characterized as a slow binding inhibitor of cathepsin K (k/[I] = 273,000 M•s). Cathepsin K may represent the elusive protease implicated in degradation of protein matrix during bone resorption and represents a novel molecular target in treatment of disease states associated with excessive bone loss such as osteoporosis.

Hydrophilic Interaction Chromatography Reduces the Complexity of the Phosphoproteome and Improves Global Phosphopeptide Isolation and Detection

The diversity and complexity of proteins and peptides in biological systems requires powerful liquid chromatography-based separations to optimize resolution and detection of components. Proteomics strategies often combine two orthogonal separation modes to meet this challenge. In nearly all cases, the second dimension is a reverse phase separation interfaced directly to a mass spectrometer. Here we report on the use of hydrophilic interaction chromatography (HILIC) as part of a multidimensional chromatography strategy for proteomics. Tryptic peptides are separated on TSKgel Amide-80 columns using a shallow inverse organic gradient. Under these conditions, peptide retention is based on overall hydrophilicity, and a separation truly orthogonal to reverse phase is produced. Analysis of tryptic digests from HeLa cells yielded numbers of protein identifications comparable to that obtained using strong cation exchange. We also demonstrate that HILIC represents a significant advance in phosphoproteomics analysis. We exploited the strong hydrophilicity of the phosphate group to selectively enrich and fractionate phosphopeptides based on their increased retention under HILIC conditions. Subsequent IMAC enrichment of phosphopeptides from HILIC fractions showed better than 99% selectivity. This was achieved without the use of derivatization or chemical modifiers. In a 300-μg equivalent of HeLa cell lysate we identified over 1000 unique phosphorylation sites. More than 700 novel sites were added to the HeLa phosphoproteome.

Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.

Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin‐2, MPIF‐2, or CKβ‐6, is a human CC chemokine with low amino acid sequence identity to other chemo‐ kines. Eotaxin‐2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross‐desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP‐4, but not RANTES, MIP‐lα, or MCP‐3, can completely cross‐desensitize the calcium response to eotaxin‐2 on these cells, indicating that eotaxin‐2 shares the same receptor used by eotaxin and MCP‐4. Eotaxin‐2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin‐2 also displaced 125I‐eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO‐CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP‐4. l25I‐Eotaxin‐2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin‐2 displace the ligand with equal affinity. Eotaxin and eotaxin‐2 promote a Ca2+ transient in RBL‐2H3 cells stably transfected with CCR3 (RBL‐2H3‐CCR3) and both ligands cross‐desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin‐2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor. J. Leukoc. Biol. 62: 667–675; 1997.

Antitumor activity of an allosteric inhibitor of centromere-associated protein-E

Centromere-associated protein-E (CENP-E) is a kinetochore-associated mitotic kinesin that is thought to function as the key receptor responsible for mitotic checkpoint signal transduction after interaction with spindle microtubules. We have identified GSK923295, an allosteric inhibitor of CENP-E kinesin motor ATPase activity, and mapped the inhibitor binding site to a region similar to that bound by loop-5 inhibitors of the kinesin KSP/Eg5. Unlike these KSP inhibitors, which block release of ADP and destabilize motor-microtubule interaction, GSK923295 inhibited release of inorganic phosphate and stabilized CENP-E motor domain interaction with microtubules. Inhibition of CENP-E motor activity in cultured cells and tumor xenografts caused failure of metaphase chromosome alignment and induced mitotic arrest, indicating that tight binding of CENP-E to microtubules is insufficient to satisfy the mitotic checkpoint. Consistent with genetic studies in mice suggesting that decreased CENP-E function can have a tumor-suppressive effect, inhibition of CENP-E induced tumor cell apoptosis and tumor regression.

Mistranslational errors associated with the rare arginine codon CGG in Escherichia coli

In Escherichia coli, CGG is a rare arginine codon occurring at a frequency of 0.54% in all E. coli mRNAs or 9.8% when an arginine residue is encoded for. When present in high numbers or in clusters in highly expressed recombinant mRNA, rare codons can cause expression problems compromising product yield and translational fidelity. The coding region for an N-terminally polyhistidine tagged p27 protease domain from Herpes Simplex Virus 2 (HSV-2) contains 11 of these rare arginine codons, with 3 occurring in tandem near the C-terminus of the protein. When expressed in E. coli, the majority of the recombinant material produced had an apparent molecular mass of 31 kDa by SDS-PAGE gels or 3 kDa higher than predicted. Detailed biochemical analysis was performed on chemical and enzymatic digests of the protein and peptide fragments were characterized by Edman and MS/MS sequencing approaches. Two major species were isolated comprising +1 frameshift events at both the second and third CGG codons in the triplet cluster. Translation proceeded in the missense frame to the next termination codon. In addition, significant levels of glutamine misincorporating for arginine were discovered, suggesting second base misreading of CGG as CAG. Coexpression of the argX gene, which encodes the cognate tRNA for CGG codons, largely eliminated both the frameshift and misincorporation events, and increased expression levels of authentic product by up to 7-fold. We conclude that supplementation of the rare arginyl tRNA(CGG) levels by coexpression of the argX gene can largely alleviate the CGG codon bias present in E. coli, allowing for efficient and accurate translation of heterologous gene products.

Cloning, in vitro expression, and functional characterization of a novel human CC chemokine of the monocyte chemotactic protein (MCP) family (MCP-4) that binds and signals through the CC chemokine receptor 2B.

Here we describe the characterization of a novel human CC chemokine, tentatively named monocyte chemotactic protein (MCP-4). This chemokine was detected by random sequencing of expressed sequence tags in cDNA libraries. The full-length cDNA revealed an open reading frame for a 98-amino acid residue protein, and a sequence alignment with known CC chemokines showed high levels of similarity (59–62%) with MCP-1, MCP-3, and eotaxin. MCP-4 cDNA was cloned into Drosophila S2 cells, and the mature protein (residues 24–98) was purified from the conditioned medium. Recombinant MCP-4 induced a potent chemotactic response (EC50 = 2.88 ± 0.15 nm) and a transient rise in cytosolic calcium concentration in fresh human peripheral blood monocytes but not in neutrophils. Binding studies in monocytes showed that MCP-4 and MCP-3 were very potent in displacing high affinity binding of125I-MCP-1 (IC50 for MCP-4, MCP-3, and unlabeled MCP-1 of 2.1 ± 1.4, 0.85–1.6, and 0.7 ± 0.2 nm respectively), suggesting that all three chemokines interact with the CC chemokine receptor-2 (MCP-1 receptor). This was confirmed in binding studies with Chinese hamster ovary cells, stably transfected with the CC chemokine 2B receptor. Northern blot analysis in extracts of normal human tissues showed expression of mRNA for MCP-4 in small intestine, thymus, and colon, but the level of protein expression was too low to be detected in Western blot analysis. However, expression of MCP-4 protein was demonstrated by immunohistochemistry in human atherosclerotic lesion and found to be associated with endothelial cells and macrophages.

Binding Interactions of Human Interleukin 5 with Its Receptor α Subunit LARGE SCALE PRODUCTION, STRUCTURAL, AND FUNCTIONAL STUDIES OF DROSOPHILA-EXPRESSED RECOMBINANT PROTEINS

Human interleukin 5 (hIL5) and soluble forms of its receptor α subunit were expressed in Drosophila cells and purified to homogeneity, allowing a detailed structural and functional analysis. B cell proliferation confirmed that the hIL5 was biologically active. Deglycosylated hIL5 remained active, while similarly deglycosylated receptor α subunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-Å resolution and found to be similar to that of the protein produced in Escherichia coli. Human IL5 was shown by analytical ultracentrifugation to form a 1:1 complex with the soluble domain of the hIL5 receptor α subunit (shIL5Rα). Additionally, the relative abundance of ligand and receptor in the hIL5·shIL5Rα complex was determined to be 1:1 by both titration calorimetry and SDS-polyacrylamide gel electrophoresis analysis of dissolved cocrystals of the complex. Titration microcalorimetry yielded equilibrium dissociation constants of 3.1 and 2.0 n M, respectively, for the binding of hIL5 to shIL5Rα and to a chimeric form of the receptor containing shIL5Rα fused to the immunoglobulin Fc domain (shIL5Rα-Fc). Analysis of the binding thermodynamics of IL5 and its soluble receptor indicates that conformational changes are coupled to the binding reaction. Kinetic analysis using surface plasmon resonance yielded data consistent with the Kdvalues from calorimetry and also with the possibility of conformational isomerization in the interaction of hIL5 with the receptor α subunit. Using a radioligand binding assay, the affinity of hIL5 with full-length hIL5Rα in Drosophila membranes was found to be 6 n M, in accord with the affinities measured for the soluble receptor forms. Hence, most of the binding energy of the α receptor is supplied by the soluble domain. Taken with other aspects of hIL5 structure and biological activity, the data obtained allow a prediction for how 1:1 stoichiometry and conformational change can lead to the formation of hIL5·receptor αβ complex and signal transduction.

An abundantly secreted glycoprotein from Drosophila melanogaster is related to mammalian secretory proteins produced in rheumatoid tissues and by activated macrophages

An abundantly secreted 47-kDa glycoprotein, DS47, was purified from Drosophila melanogaster (Dm) Schneider line-2 cells, a line exhibiting macrophage-like properties. DS47 is also secreted from several Dm cell lines resembling S2 but not from lines that are morphologically distinct. A cDNA cline was isolated from an S2 cell cDNA library using oligodeoxyribonucleotide probes based on the DS47 amino acid (aa) sequence and found to encode a novel secretory glycoprotein of 452 aa. Analysis of DS47 protein production and mRNA expression during fly development indicates that both are present throughout the entire Dm life cycle, suggesting that DS47 may be important at all developmental stages. In larvae, the DS47 message is made in the fat body and by hemocytes, and secreted into the hemolymph. DS47 is related to a human cartilage glycoprotein, HC gp-39, that is secreted from cell types associated with the arthritic joint, such as synovial cells and activated macrophages. Interestingly, the HC gp-39 message is most readily detected in the human liver, an organ that is somewhat analogous to the Dm fat body. DS47 also shares homology to a mouse secretory glycoprotein, YM-1, identified in activated macrophages. These homologies extend to the chitinase gene family and include a conserved cysteine aa motif, as well as two blocks of aa within the enzymatic active site, although neither DS-47 nor HC gp-39 exhibit chitinase activity. Potential functions of this conserved protein family are discussed.