Anne Wisner

Snake venom proteins acting on hemostasis.

The venoms of Viperidae and Crotalidae snakes are a rich source of proteins with activity against various factors involved in coagulation and fibrinolysis. These proteins are very specific for their molecular targets, resistant to physiological inhibitors and stable in vitro and in vivo. They have therefore proved to be useful for diagnostic tests. Based on sequence similarities, these snake venom proteins have been classified into various families, such as serine proteinases, metalloproteinases, C-type lectins, disintegrins and phospholipases A(2). The various members of a given family, although structurally similar, act selectively on different blood coagulation factors. This opens up the possibility of characterizing the structural elements involved in target molecule recognition. Thus, snake venom proteins provide excellent models for studies of structure-function relationships.

Human Opiorphin, a natural antinociceptive modulator of opioid-dependent pathways

Mammalian zinc ectopeptidases play important roles in turning off neural and hormonal peptide signals at the cell surface, notably those processing sensory information. We report here the discovery of a previously uncharacterized physiological inhibitor of enkephalin-inactivating zinc ectopeptidases in humans, which we have named Opiorphin. It is a QRFSR peptide that inhibits two enkephalin-catabolizing ectoenzymes, human neutral ecto-endopeptidase, hNEP (EC 3.4.24.11), and human ecto-aminopeptidase, hAP-N (EC 3.4.11.2). Opiorphin displays potent analgesic activity in chemical and mechanical pain models by activating endogenous opioid-dependent transmission. Its function is closely related to the rat sialorphin peptide, which is an inhibitor of pain perception and acts by potentiating endogenous μ- and δ-opioid receptor-dependent enkephalinergic pathways. Here we demonstrate the functional specificity in vivo of human Opiorphin. The pain-suppressive potency of Opiorphin is as effective as morphine in the behavioral rat model of acute mechanical pain, the pin-pain test. Thus, our discovery of Opiorphin is extremely exciting from a physiological point of view in the context of endogenous opioidergic pathways, notably in modulating mood-related states and pain sensation. Furthermore, because of its in vivo properties, Opiorphin may have therapeutic implications.

The crystal structure of the novel snake venom plasminogen activator TSV-PA: a prototype structure for snake venom serine proteinases.

BACKGROUND Trimeresurus stejnejeri venom plasminogen activator (TSV-PA) is a snake venom serine proteinase that specifically activates plasminogen. Snake venom serine proteinases form a subfamily of trypsin-like proteinases that are characterised by a high substrate specificity and resistance to inhibition. Many of these venom enzymes specifically interfere with haemostatic mechanisms and display a long circulating half-life. For these reasons several of them have commercial applications and are potentially attractive pharmacological tools. RESULTS The crystal structure of TSV-PA has been determined to 2.5 A resolution and refined to an R factor of 17.8 (R free, 24.4). The enzyme, showing the overall polypeptide fold of trypsin-like serine proteinases, displays unique structural elements such as the presence of a phenylalanine at position 193, a C-terminal tail clamped via a disulphide bridge to the 99-loop, and a structurally conserved Asp97 residue. The presence of a cis proline at position 218 is in agreement with evolutionary relationships to glandular kallikrein. CONCLUSIONS We postulate that Phe 193 accounts for the high substrate specificity of TSV-PA and renders it incapable of forming a stable complex with bovine pancreatic trypsin inhibitor and other extended substrates and inhibitors. Mutational studies previously showed that Asp97 is crucial for the plasminogenolytic activity of TSV-PA, here we identify the conservation of Asp97 in both types of mammalian plasminogen activator - tissue-type (tPA) and urokinase-type (uPA). It seems likely that Asp97 of tPA and uPA will have a similar role in plasminogen recognition. The C-terminal extension of TSV-PA is conserved among snake venom serine proteinases, although its function is unknown. The three-dimensional structure presented here is the first of a snake venom serine proteinase and provides an excellent template for modelling other homologous family members.

Snake venom proteinases as tools in hemostasis studies: structure-function relationship of a plasminogen activator purified from Trimeresurus stejnegeri venom.

Snake venom serine proteinases affect many steps of the blood coagulation cascade. Each of them usually acts selectively on one coagulation factor. They are therefore potentially useful components to study the mechanisms of action, the regulation and the structure-function relationships of human serine proteinase coagulation factors. This strategy is illustrated for a plasminogen activator purified from Trimeresurus stejnegeri venom.

Hypothalamic prostaglandin E2 receptors coupled to an adenylyl cyclase

We show that the effect of prostaglandin (PG) E2 on luteinizing hormone-releasing hormone (LHRH) release involves a receptor-mediated process coupled to an adenylyl cyclase system. The adenylyl cyclase activity in rat hypothalamus synaptic membrane preparations was stimulated by PGE2 and this stimulation was directly related to the presence of guanine nucleotide (GTP). PGE2 specifically bound to P2 membranes from rat and porcine hypothalami with similar characteristics. Computer-fitted saturation curves provided evidence for two binding components which may be two states of the same receptor (RH and RL). Experiments with Gpp(NH)p, a non-metabolizable analogue of GTP, suggested the interconversion of RH and RL. These results may reflect different states of the ternary complex (hormone-receptor-guanine binding protein). Magnesium (Mg2+) can modify the RH and RL binding parameters, but seems to act directly on the PGE2 receptor site.

Circadian and Estral Changes in the Hypothalamic Prostaglandin E2 Content and [3H]Prostaglandin E2 Binding in Female Rats

Prostaglandin E2, (PGE2) is involved in the luteinizing hormone‐releasing hormone‐stimulated luteinizing hormone surge in female rats and may act via specific membrane receptors. The following studies were performed to determine whether there were any changes in the hypothalamic PGE2 binding and/or PGE2 content which were specific to proestrus and not to the rest of the estrous cycle. Groups of female Wistar rats were sacrificed at 3‐h intervals throughout the estrous cycle to determine both the circadian and circaestral changes in the hypothalamic PGE2 content and [3H]PGE2 binding. The hypothalamic PGE2 content was maximal at 1700 h on each of the 4 consecutive days of the estrous cycle but was independent of the stage of the cycle. [3H]PGE2 binding also displayed a circadian rhythm; the lowest binding occurred near the circadian peak of PGE2, suggesting that the PGE2 binding sites were occupied by endogenous PGE2. Since such circadian rhythms were not observed in the hypothalamus of male rats, they may be under the control of ovarian steroids. Also, since PGE2 binding and the PGE2 content both exhibit a diurnal pattern independent of the day of the cycle, there may be changes in the PGE2 receptor‐mediated process coupled to an adenylyl cyclase which could explain the luteinizing hormone surge in proestrus.