John D. Martin

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.

Differential Calcitonin Gene-Related Peptide (CGRP) and Amylin Binding Sites in Nucleus Accumbens and Lung: Potential Models for Studying CGRP/Amylin Receptor Subtypes

Abstract: Calcitonin gene‐related peptide (CGRP), a 37‐amino‐acid peptide, is a member of a small family of peptides including amylin or islet amyloid polypeptide and salmon calcitonin. These related peptides have been shown to display similar effects on in vitro and in vivo carbohydrate metabolism. The present study was initiated to identify and characterize the binding sites for these peptides in lung and nucleus accumbens membranes prepared from pig and guinea pig. Both tissues in either species displayed high‐affinity (2‐[125I]iodohistidyl10)humanCGRPα ([125I]hCGRPα) binding (IC50 = 0.4–7.7 nM), which was displaced by hCGRP8–37α with equally high affinity (IC50 = 0.4–7.3 nM). High‐affinity binding for [125I]Bolton‐Hunter human amylin ([125I]BH‐h‐amylin) was also observed in these tissues (IC50 = 0.2–6.0 nM). In membranes from the nucleus accumbens of both species, salmon calcitonin competed for amylin binding sites with high affinity (IC50 = 0.1 nM) but was poor in competing for amylin binding in lung membranes. Rat amylin8–37 competed for [125I]hCGRPα binding with higher affinity (IC50 = 5.4 nM) compared with [125I]BH‐h‐amylin binding (IC50 = 200 nM) in porcine nucleus accumbens, whereas in guinea pig nucleus accumbens, the IC50 values for rat amylin8–37 were 117 and 12 nM against [125I]hCGRPα and [125I]BH‐h‐amylin, respectively. Also, functional studies evaluating the activation of adenylate cyclase and generation of cyclic AMP in response to these agonists indicated that hCGRPα (EC50 = 0.3 nM), h‐amylin (EC50 = 150 nM), and salmon calcitonin (EC50 = 1,000 nM) activated adenylate cyclase, resulting in increased cyclic AMP production in porcine lung membranes that was antagonized by hCGRP8–37α. The affinity of hCGRP8–37α was similar for all three peptides. The cyclic AMP responses to amylin and salmon calcitonin were significantly (p < 0.05) lower than that of hCGRPα and not additive, suggesting that they are acting as partial agonists at the same CGRP1‐type receptor in porcine lung membranes. Similar observations were made for guinea pig lung membranes. However, human amylin and salmon calcitonin were weaker than hCGRPα in activating lung adenylate cyclase. None of these peptides activated adenylate cyclase in membranes prepared from the nucleus accumbens of both species. The data from these studies demonstrate both species and tissue differences in the existence of distinct CGRP and amylin binding sites and present a potential opportunity to study further CGRP and amylin receptor subtypes.

C-terminal region of USP7/HAUSP is critical for deubiquitination activity and contains a second mdm2/p53 binding site

USP7, also known as the hepes simplex virus associated ubiquitin-specific protease (HAUSP), deubiquitinates both mdm2 and p53, and plays an important role in regulating the level and activity of p53. Here, we report that deletion of the TRAF-like domain at the N-terminus of USP7, previously reported to contain the mdm2/p53 binding site, has no effect on USP7 mediated deubiquitination of Ub(n)-mdm2 and Ub(n)-p53. Amino acids 208-1102 were identified to be the minimal length of USP7 that retains proteolytic activity, similar to full length enzyme, towards not only a truncated model substrate Ub-AFC, but also Ub(n)-mdm2, Ub(n)-p53. In contrast, the catalytic domain of USP7 (amino acids 208-560) has 50-700 fold less proteolytic activity towards different substrates. Moreover, inhibition of the catalytic domain of USP7 by Ubal is also different from the full length or TRAF-like domain deleted proteins. Using glutathione pull-down methods, we demonstrate that the C-terminal domain of USP7 contains additional binding sites, a.a. 801-1050 and a.a. 880-1050 for mdm2 and p53, respectively. The additional USP7 binding site on mdm2 is mapped to be the C-terminal RING finger domain (a.a. 425-491). We propose that the C-terminal domain of USP7 is responsible for maintaining the active conformation for catalysis and inhibitor binding, and contains the prime side of the proteolytic active site.

Biochemical characterization of human HIF hydroxylases using HIF protein substrates that contain all three hydroxylation sites

The HIF (hypoxia-inducible factor) plays a central regulatory role in oxygen homoeostasis. HIF proteins are regulated by three Fe(II)- and α-KG (α-ketoglutarate)-dependent prolyl hydroxylase enzymes [PHD (prolyl hydroxylase domain) isoenzymes 1-3 or PHD1, PHD2 and PHD3] and one asparaginyl hydroxylase [FIH (factor inhibiting HIF)]. The prolyl hydroxylases control the abundance of HIF through oxygen-dependent hydroxylation of specific proline residues in HIF proteins, triggering subsequent ubiquitination and proteasomal degradation. FIH inhibits the HIF transcription activation through asparagine hydroxylation. Understanding the precise roles and regulation of these four Fe(II)- and α-KG-dependent hydroxylases is of great importance. In the present paper, we report the biochemical characterization of the first HIF protein substrates that contain the CODDD (C-terminal oxygen-dependent degradation domain), the NODDD (N-terminal oxygen-dependent degradation domain) and the CAD (C-terminal transactivation domain). Using LC-MS/MS (liquid chromatography-tandem MS) detection, we show that all three PHD isoenzymes have a strong preference for hydroxylation of the CODDD proline residue over the NODDD proline residue and the preference is observed for both HIF1α and HIF2α protein substrates. In addition, steady-state kinetic analyses show differential substrate selectivity for HIF and α-KG in reference to the three PHD isoforms and FIH.

A High-Throughput Screen Measuring Ubiquitination of p53 by Human mdm2

Tumor suppressor p53 is typically maintained at low levels in normal cells. In response to cellular stresses, such as DNA damage, p53 is stabilized and can stimulate responses leading to cell cycle arrest or apoptosis. Corresponding to its central role in preventing propagation of damaged cells, mutation or deletion of p53 is found in nearly 50% of all human tumors. Mdm2 (mouse-d-minute 2) and its human ortholog (hmdm2 or hdm2) catalyze the ubiquitination of p53, targeting it for degradation via the proteosome. Thus, the activity of mdm2 is inversely correlated with p53 levels. Based on this, inhibition of human mdm2 activity by a small-molecule therapeutic will lead to net stabilization of p53 and be the basis for development of a novel cancer therapeutic. Previous high-throughput screening assays of mdm2 measured the autoubiquitination activity of mdm2, which occurs in the absence of an acceptor substrate such as p53. The major drawback to this approach is that inhibitors of mdm2 autoubiquitination may lead to a net stabilization of mdm2 and thus have the opposite effect of inhibitors that interfere with p53 ubiquitination. The authors describe the development, validation, and execution of a high-throughput screening measuring the ubiquitination of p53 by mdm2, with p53 labeled with europium and the other substrate (Ub-UbcH5b) labeled with a Cy5 on the ubiquitin. After confirming that known inhibitors are detected with this assay, it was successfully automated and used to query >600,000 compounds from the GlaxoSmithKline collection for mdm2 inhibitors. (Journal of Biomolecular Screening 2007:1050-1058)

Biochemical Characterization of Human Prolyl Hydroxylase Domain Protein 2 Variants Associated with Erythrocytosis

Prolyl hydroxylase domain proteins (PHD isozymes 1-3) regulate levels of the alpha-subunit of the hypoxia inducible factor (HIF) through proline hydroxylation, earmarking HIFalpha for proteosome-mediated degradation. Under hypoxic conditions, HIF stabilization leads to enhanced transcription and regulation of a multitude of processes, including erythropoiesis. Herein, we examine the biochemical characterization of PHD2 variants, Arg371His and Pro317Arg, identified from patients with familial erythrocytosis. The variants display differential effects on catalytic rate and substrate binding, implying that partial inhibition or selective inhibition with regard to HIFalpha isoforms of PHD2 could result in the phenotype displayed by patients with familial erythrocytosis.

Production and characterization of monoclonal antibodies against the vasoconstrictive peptide human urotensin-II.

We report the production and characterization of four monoclonal antibodies (MAbs) against human urotensin-II (hU-II). The antibodies were raised against human hU-II, which contains the C-terminus cyclic ring (CFWKYC) that is conserved across species. Multiple selection assays were applied to ensure antibody potency and reactivity against the ring structure. The MAbs reacted via ELISA with hU-II bound to plastic, immunoprecipitated [(125)I-Y(9)] hU-II, bound to biotinylated hU-II in BIAcore analysis and, by Western analysis, recognized the full-length human preprourotensin-II expressed in transfected HEK293 cells. All four MAbs cross-reacted with porcine A, porcine B, rat, mouse, and goby U-II in ELISA. By competitive RIA, hU-II(5-11) (identical to the C-terminus of goby U-II) reacted equivalently to hU-II and goby U-II. The IC(50)s were 0.8 nM for one MAb and 1.6 nM for the others. All four MAbs reacted 15-fold less potently with hU-II(5-10) and 50-fold less potently with hU-II(5-10) amide. Thus, the ring structure and terminal Val/Ile comprise the binding site for this group of MAbs. This panel of antibodies could be useful tools to help delineate the biology and pharmacology of U-II. They may also be of diagnostic value in monitoring hU-II in body fluids.