Degradation Of Gonadotropin-releasing Hormone Research Articles

Thimet oligopeptidase and prolyl endopeptidase of spotted scat Scatophagus argus: characterization, tissue distribution, expression at different ovarian stages and down-regulation by estradiol

Thimet oligopeptidase (Top) is a thiol-sensitive metallopeptidase with increased concentrations in brain and reproductive tissues. Prolyl endopeptidase (Pep) is another endopeptidase with high enzymatic activity in the brain. The two neuropeptidases are involved in the degradation of gonadotropin-releasing hormone. To clarify their roles in reproductive regulation, the spotted scat top (sstop) and pep (sspep) genes were cloned and analyzed. Tissue distribution by RT-PCR showed that sstop and sspep were more highly expressed in reproduction-related tissues than in other tissues. The sstop and sspep mRNA was detected in female hypothalamuses at phase II (oocytes at the perinuclear stage), phase III (oocytes forming the vitelline vesicle) and phase IV (oocytes at lipidic and proteic vitellogenesis stages). Both sstop and sspep were highly expressed in phase III, moderately in phase IV, and the lowest in phase II. The sstop and sspep genes were down-regulated in hypothalamuses after treatment with estradiol both in vitro and in vivo. It is suggested that Sstop and Sspep play significant roles in the regulation of reproduction and these results suggested an ssTop- and ssPep-mediated positive feedback mechanism of ovarian function.

Estradiol Regulates Neuropeptidase Activity in Hypothalamic Regions Involved in Regulating Gonadotropin‐Releasing Hormone

Thimet oligopeptidase (TOP) and prolyl endopeptidase (PEP) are neuropeptidases involved in the degradation of gonadotropin‐releasing hormone (GnRH), an important regulator of the ovarian steroid hormone estradiol (E) via the hypothalamic‐pituitary‐gonadal axis. PEP's action to remove the N‐terminal glycinamide of GnRH occurs prior to hydrolysis of the Try5‐Gly6 bond, the rate‐determining step catalyzed by TOP. E levels in turn regulate GnRH by exertion of positive and negative feedback control. TOP and PEP may potentiate GnRH effects and therefore may be regulated by E. We have previously shown that TOP and the estrogen receptor co‐localize within individual hypothalamic cells, and that levels of TOP in specific regions are down‐regulated by E. In order to determine whether E regulates TOP or PEP enzymatic activity in brain regions associated with reproduction, homogenized hypothalamic brain samples of the medial preoptic area (MPA), periaqueductal gray (PAG), and ventral medial nucleus of the hypothalamus (VMN) were collected from ovariectomized mice treated with E or oil control. Fluorometric kinetic assays were conducted to analyze differences in levels of enzymatic activity between treatments. Kinetic assay data showed that E alters TOP and PEP activity in both the VMN and mPOA, but not in the PAG. These results suggests that, in female mice, TOP and PEP are regulated by E in the hypothalamic regions involved in reproductive physiology.

Pyroglutamyl peptidase type I from Trypanosoma brucei: a new virulence factor from African trypanosomes that de-blocks regulatory peptides in the plasma of infected hosts

Peptidases of parasitic protozoans are emerging as novel virulence factors and therapeutic targets in parasitic infections. A trypanosome-derived aminopeptidase that exclusively hydrolysed substrates with Glp (pyroglutamic acid) in P1 was purified 9248-fold from the plasma of rats infected with Trypanosoma brucei brucei. The enzyme responsible was cloned from a T. brucei brucei genomic DNA library and identified as type I PGP (pyroglutamyl peptidase), belonging to the C15 family of cysteine peptidases. We showed that PGP is expressed in all life cycle stages of T. brucei brucei and is expressed in four other blood-stream-form African trypanosomes. Trypanosome PGP was optimally active and stable at bloodstream pH, and was insensitive to host plasma cysteine peptidase inhibitors. Native purified and recombinant hyper-expressed trypanosome PGP removed the N-terminal Glp blocking groups from TRH (thyrotrophin-releasing hormone) and GnRH (gonadotropin-releasing hormone) with a k(cat)/K(m) value of 0.5 and 0.1 s(-1) x microM(-1) respectively. The half-life of TRH and GnRH was dramatically reduced in the plasma of trypanosome-infected rats, both in vitro and in vivo. Employing an activity-neutralizing anti-trypanosome PGP antibody, and pyroglutamyl diazomethyl ketone, a specific inhibitor of type I PGP, we demonstrated that trypanosome PGP is entirely responsible for the reduced plasma half-life of TRH, and partially responsible for the reduced plasma half-life of GnRH in a rodent model of African trypanosomiasis. The abnormal degradation of TRH and GnRH, and perhaps other neuropeptides N-terminally blocked with a pyroglutamyl moiety, by trypanosome PGP, may contribute to some of the endocrine lesions observed in African trypanosomiasis.

Sequential degradation of the neuropeptide gonadotropin-releasing hormone by the 20 S granulosa cell proteasomes

The decapeptide gonadotropin-releasing hormone (GnRH) is degraded by the 20 S multicatalytic proteinase complex (proteasome EC 3.4.99.46), purified from ovarian granulosa cells, at the Tyr 5 Gly 6 bond and to a lesser extent at the Gly 6 Leu 7 bond, when incubated for 2 h at 37°C. Further cleavage, at Trp 3 Ser 4 and Ser 4 Tyr 5 bonds of the neurohormone occurs only subsequently to the appearance of the initial N-terminal degradation products, (1–5)GnRH and (1–6)GnRH. Our results suggest that the sequential degradation of GnRH can serve as an important mechanism for the rapid termination of its biological activity in target cells.

Characterization of Membrane‐Associated Peptidase Activities Expressed by Endothelial Cells of the Ovine Median Eminence

The capillary endothelial cells of the median eminence represent a potential site for the degradation/modification of both circulating and hypothalamic peptides passing through the hypophysial portal system toward the pituitary. This study examines endothelial cell peptidase expression in vitro by monitoring the metabolism of gonadotropin-releasing hormone (GnRH) by cultured endothelial cells from sheep median eminence. Cleavage of GnRH by median eminence endothelial cell membranes generated GnRH1-5 as the primary stable product, which was then degraded to GnRH1-3 and free amino acids. Degradation of GnRH was completely inhibited by TPCK, ZnCl2 and N-ethylmaleimide, and partially inhibited by EDTA and by a specific inhibitor of the metalloendopeptidase EC 3.4.24.15, CFP-AAY-pAB. Interestingly, an increase in GnRH1-9 production was seen with the latter inhibitors, suggesting a two-step mechanism of GnRH degradation involving a primary cleavage at the Pro9-Gly10-NH2 bond, inhibitable by TPCK, ZnCl2, and NEM, followed by cleavage by EC 3.4.24.15 to generate GnRH1-5. Phosphoramidon and angiotensin converting enzyme inhibitors (as well as other non-specific inhibitors) were without effect, indicating that endopeptidase EC 3.4.24.11 and angiotensin converting enzyme are not involved. Neither bovine aortic endothelial cell nor AtT-20 cell membranes exhibited this pattern of peptidase activity. Degradation of GnRH by intact median eminence endothelial cells in culture was also observed, suggesting an extracellular orientation for these enzymes; the potential role of such peptidases in the fine regulation of both pituitary function and local blood flow is currently under investigation.

Structural and functional studies of the metalloendopeptidase (EC 3.4.24.15) involved in degrading gonadotropin releasing hormone

Converging biophysical and molecular biological techniques including genetic and computer aided engineering are being utilized in the study of zinc-metalloendopeptidase E.C. 3.4.24.15 (EP 24.15), a peptidase involved in the degradation of Gonadotropin releasing hormone (GnRH), the master regulatory decapeptide involved in reproduction. This 71 kDa enzyme rapidly cleaves the Tyr-Gly6 bond in GnRH, the rate limiting reaction in hormone inactivation. EP 24.15 has been identified and isolated (1, 2) and the full length rat cDNA has recently been sequenced, and used to direct the expression of the functional 645 amino acid protein (3). The sequence of EP 24.15 shows no sequence identity with any known metalloendopeptidases beyond the commonly shared active site motif, -H-E-x-x-H-, found in this family of enzymes. EP 24.15 is a predominantly cytosolic enzyme that is stable, not glycosylated, and can be modeled with other globular proteins. Hydrophobic Cluster Analysis, (HCA) (4), is a heuristic algorithm ascertaining structural domains by detecting patterns of secondary structure and sequence homology in a two-dimensional analysis. This method has been used to determine if thermolysin may be used in modeling EP 24.15, and extending this analysis to enkephalinase and angiotensin converting enzyme two other members of the zinc-metalloendopeptidase family. Information gained from this study would be a step towards modeling substrate and inhibitor interactions to elucidate the conformation of this enzyme, as a probe for the function of this enzyme in vivo, and as a target for pharmacological intervention.

Degradation of gonadotropin-releasing hormones in the gilthead seabream, Sparus aurata: II. Cleavage of native salmon GnRH, mammalian LHRH, and their analogs in the pituitary, kidney, and liver

The pattern and kinetics of degradation of native salmon gonadotropin-releasing hormone (sGnRH), mammalian luteinizing hormone-releasing hormone (LHRH), and some of their analogs by cytosolic enzymes of pituitary, kidney, and liver were studied in the gilthead seabream, Sparus aurata. The native peptides sGnRH and LHRH are rapidly degraded by all three tissues, LHRH being degraded faster than sGnRH. The kinetics of production of the peptide fragments suggest that initial cleavage of sGnRH and LHRH in the three studied tissues occurs at the 5–6 and 9–10 bonds. This indicates the initial activity of a Tyr 5-Gly 6 endopeptidase and a Pro 9-Gly 10NH 2 peptidase or postproline cleaving enzyme. Secondary degradation of the main initial fragments (1–5, 6–10, and 1–9) is more intensive in the kidney than in the pituitary or liver. Substitution of the position 6 amino acid glycine by a dextrorotatory ( d) amino acid such as in the d-Trp 6-LHRH renders the 5–6 bond resistant to cleavage. However, whereas [ d-Trp 6]-LHRH is intensively cleaved at the Pro 9-Gly 10NH 2 bond by the pituitary, its cleavage at this site by the kidney and liver is slow. This suggests a low activity of the Pro 9-Gly 10NH 2 peptidase in the kidney and liver as compared to the pituitary. When, in addition to the position 6 substitution, the carboxy terminus Pro 9-Gly 10NH 2 is modified to Pro 9NET, such as in the [ d-Ala 6-Pro 9NET]-LHRH and the [ d-Arg 6-Pro 9NET]-sGnRH, the 9–10 cleavage site is also blocked, resulting in GnRH analogs highly resistant to degradation. The relationships between susceptibility of the different forms of GnRH to enzymatic degradation by the pituitary, kidney, and liver and their relative biological activities in S. aurata are discussed. We conclude that increased resistance of GnRH analogs to enzymatic degradation contributes to their superactivity.

Degradation of gonadotropin-releasing hormones in the gilthead seabream, Sparus aurata: I. Cleavage of native salmon GnRH and mammalian LHRH in the pituitary

The pattern and kinetics of degradation of native salmon gonadotropin-releasing hormone (sGnRH) and mammalian luteinizing hormone-releasing hormone (LHRH) by pituitary bound enzymes were studied in the gilthead seabream, Sparus aurata. sGnRH and LHRH were incubated for different periods of time with membrane or cytosolic fractions of pituitary homogenates. At the end of the incubation, the degradation mixture was fractionated on reverse-phase high-pressure liquid chromatography. The degradation products were identified by comparing their retention times to those of synthesized GnRH fragments and by analyzing their amino acid composition. The main GnRH degradative activity resides in the cytosolic fraction of the pituitary homogenate. Both sGnRH and LHRH are rapidly degraded by pituitary cytosol, with 78.3 and 87.7% of the peptides, respectively, cleaved after 3 hr of incubation. Maximal degradation of sGnRH occurred at a pH range of 7 to 8. The main initial products of degradation of sGnRH and LHRH are the 1–5, 6–10, and 1–9 fragments. This suggests the involvement of two site-specific peptidases, a Tyr 5-Gly 6 endopeptidase and a Pro 9-Gly 10NH 2 peptidase or postproline cleaving enzyme. While the 1–6 and 1–9 fragments undergo rapid secondary degradation, the 1–5 is relatively stable. Competition experiments suggest that the endopeptidase cleaving the sGnRH at the Tyr 5-Gly 6 bond is not specific to the neuropeptide and is probably a general proteolitic enzyme. However, the cleavage at the 9–10 bond has a high degree of specificity to the Pro 9-Gly 10NH 2 sequence found in sGnRH. The two proposed pituitary peptidases of S. aurata have some characteristics similar to those of rat hypophyseal and hypothalamic GnRH cleaving enzymes. No differences are found in hypophyseal GnRH degradative activity between females with occytes undergoing previtellogenesis or advanced stages of vitellogenesis.

Gonadotropin releasing hormone (GnRH) is not degraded by intact pituitary tissue [formula omitted

The possible degradation of GnRH by intact pituitary tissue was investigated. Trypsin-, collagenase- and mechanically dispersed pituitary cells in culture and fresh pituitaries cut into eight segments were incubated with specifically tritiated GnRH or unlabeled GnRH which were detected by HPLC and RIA in incubation media. Degradation of GnRH could not be detected by either method during incubations with any of the pituitary cell cultures or fresh tissue. The results suggest that pituitary degradation is not involved in the regulation of circulating levels of GnRH.

Dopamine stimulates the degradation of gonadotropin releasing hormone by rat synaptosomes.

Immunohistochemical studies have shown the existence of synapses between nerve endings containing dopamine and gonadotropin releasing hormone (LH-RH) respectively in the hypothalamic median eminence. It is generally accepted that this transmitter is involved in the regulation of gonadotropin secretion as a modulator of LH-RH release, although the evidence is conflicting and both inhibitory and excitatory actions of dopamine have been reported. In vitro studies support the excitatory effect and low doses of dopamine have been shown to release LH-RH from incubated hypothalamic fragments and synaptosomes. This report describes studies with nerve endings isolated from the rat hypothalamus in which dopamine at higher concentrations than those used previously has been found to stimulate the degradation of LH-RH but not thyrotropin releasing hormone (TRH). This mechanism seems to be physiological in that it is calcium-dependent, requires the structural integrity of the nerve endings and fluctuates with the reproductive state of the animal.

Purification of the Gonadotroptn-Releasing Hormone-Degrading Enzyme by Affinity Chromatography

A crude preparation of Kallikrein inactivator, which inhibits the gonadotropin-releasing hormone (GnRH)-degrading enzyme(s) from rat hypothalamus and anterior pituitary, was fractionated by passage through an ion-exchange column. The enzyme-inhibiting fraction was coupled to Sepharose and the resin obtained was used for, affinity-chromatography purification of the GnRH-degrading enzyme. The enzyme from crude tissue preparations was retained on this column and eluted by 0.05 M phosphate buffer. A 9-12 fold increase in the specific activity of the enzyme was achieved. Bacitracin, an effective peptide inhibitor of the degradation of GnRH, was also coupled to Sepharose. Three different such Sepharose-bacitracin conjugates were synthesized, two of which inhibited the degradation of GnRH by hypothalamic and pituitary extracts. They all failed, however, to separate the active enzymic fraction from the bulk of accompanying proteins, using affinity chromatographic techniques.