Thimet Oligopeptidase Research Articles

Expression of an angiotensin‐(1‐7) endopeptidase in proximal tubules of the sheep and human kidney (1088.10)

Angiotensin‐(1‐7) [Ang 7] is a an active peptide of the renin‐angiotensin system (RAS) that generally antagonizes the actions of Ang II. Within the kidney, Ang 7 is derived from Ang I by endopeptidases including neprilysin (NEP) and thimet oligopeptidase (TOP) or from Ang II by the carboxypeptidase ACE2. Ang 7 is primarily degraded by ACE to the Ang‐(1‐5) in the circulation and tissues; however, we identified an endopeptidase in sheep CSF that efficiently degraded Ang 7 to Ang‐(1‐4) (Ang 4) and accounted for the majority of Ang 7 metabolism in this compartment (Marshall et al AJP 2013). The current study determined whether the Ang 7 peptidase is expressed in the kidney, specifically in isolated proximal tubules of the sheep and human renal epithelial cells. Activity was determined by the conversion of 125I‐Ang 7 to 125I‐Ang 4 in a 100,000 xg supernatant fraction. Both the sheep proximal tubules and the human epithelial cells expressed Ang 7 endopeptidase activity. Kinetic analysis revealed an apparent Km of 2.0 ± 1.0 µM and apparent Vmax’ of 93 ± 37 pmol/min/mg protein [n=3]. Peptidase activity in the isolated tubules was attenuated by the metalloendopeptidase inhibitor JMV‐390 with an IC50 of 0.81 ± 0.12 nM [n=3] that is ~50‐fold more potent than that of NEP and TOP. We conclude that an Ang 7 endopeptidase may be a novel component of the intrarenal RAS to regulate the tubular expression of Ang 7.Grant Funding Source: Supported by NIH grants HL56973, HL52972, HL112237, and HD047584

Sleep loss changes neuropeptidase expression and activty in rat brain (876.3)

The effect of sleep deprivation on proteolytic activity in the central nervous system has not been frequently addressed in the literature. We have started to study the changes on peptidase activities in animal models of paradoxical sleep deprivation (PSD), mainly focusing on metallopeptidases like thimet oligopeptidase (EP24.15) known to process neuropeptides. In the present study, we demonstrated that PSD in rats induces changes both in the expression and activity of EP24.15 in the male rat hypothalamus and striatum tissue extracts. In the hypothalamus, the significant decrease in activity and mRNA levels, after PSD, was only totally reversed after 96h of sleep recovery. In the striatum, albeit significative, changes in mRNA and activity do not parallel changes on protein content. Neurotensin, GnRH, dynorphins, enkephalins, orphanin/nociceptin are among the many neuropeptides whose balance can be modified by the modifications on EP24.15 activity and can be, at least partially, related with many of the physiological changes observed in PSD and during the sleep recovery periods.Grant Funding Source: Supported by FAPESP, CNPq and AFIP

Evidence for an angiotensin‐(1‐7) neuropeptidase in the brain medulla of sheep (1140.6)

Angiotensin‐(1‐7) [Ang 7] is an alternative product of the renin‐angiotensin system (RAS). In contrast to Ang II, Ang 7 exhibits central actions to lower blood pressure and improve baroreflex sensitivity. We identified a thiol‐sensitive peptidase that metabolized Ang 7 to Ang‐(1‐4) (Ang 4) in CSF of adult sheep. This activity was 2.5‐fold higher in sheep with antenatal glucocorticoid exposure while CSF Ang 7 levels were 50% lower compared to controls. To characterize this peptidase, we purified the protein 1400‐fold from the brain medulla by multiple chromatography steps monitored by 125I‐Ang 7 conversion to Ang 4. Chelating agents o‐phenanthroline and EDTA, as well as the mercury compounds APMA and PCMB, but not the cysteine protease inhibitor E64 blocked peptidase activity. Specific inhibitors to neprilysin, neurolysin, or thimet oligopeptidase were ineffective while the activity was abolished by the metallopeptidase inhibitor JMV 390 (Ki = 0.7 nM). HPLC analysis confirmed the processing of unlabeled Ang 7 to Ang 4 by the peptidase, but revealed <5% hydrolysis of Ang II or Ang I and no hydrolysis of neurotensin, bradykinin or apelin. Kinetic studies using 125I‐labeled Ang 7 and Ang I show similar Kms (2.6 and 4.3 μM), but a higher Vmax for Ang 7 (72 vs. 6 nmol/min/mg for Ang I, p<0.01, n=3). We conclude that evidence for an Ang 7 neuropeptidase may portend a novel pathway to influence actions of the peptide within the brain.Grant Funding Source: Supported by HD47584

Intracellular peptides: From discovery to function

Peptidomics techniques have identified hundreds of peptides that are derived from proteins present mainly in the cytosol, mitochondria, and/or nucleus; these are termed intracellular peptides to distinguish them from secretory pathway peptides that function primarily outside of the cell. The proteasome and thimet oligopeptidase participate in the production and metabolism of intracellular peptides. Many of the intracellular peptides are common among mouse tissues and human cell lines analyzed and likely to perform a variety of functions within cells. Demonstrated functions include the modulation of signal transduction, mitochondrial stress, and development; additional functions will likely be found for intracellular peptides.

Expression Analysis of THOP1 in Background Liver, a Prognostic Predictive Factor in Hepatocellular Carcinoma, Extracted by Multiarray Analysis

Hepatocellular carcinoma (HCC) often recurs and multicentric occurrence is more common than intrahepatic metastases after surgery. Prognostic prediction is insufficient when considering only factors in resected primary tumor. Control samples, termed supernormal (SN) liver, were taken from 11 cases of metastatic secondary malignancies of the liver. We selected adjacent nonneoplastic liver tissue from a patient with HCC and liver cirrhosis by hepatitis C (CN) for comparison. Expression profiling and methylation arrays were performed. We identified genes showing differences in both arrays. Prognosis was predicted for 179 cases of HCC based on gene expression. Expression profiling showed that expression of thimet oligopeptidase (THOP1) gene was decreased 4.119-fold in CN. Methylation array showed a higher value for CN (0.869) than SN (0.488). We studied THOP1 gene expression by real-time reverse transcriptase polymerase chain reaction. The average expression level of THOP1 (THOP1 value × 10(3)/GAPDH) decreased in matching normal tissue (14.53 ± 10.14) relative to SN (78.14 ± 44.50). The group with higher than average THOP1 expression (n = 74) showed significant correlations with prolonged survival (P = 0.0383). Strongly reduced THOP1 expression (<3.0, n = 50) was shown to be an independent prognostic factor by multivariate analysis (P = 0.0024). Expression of the THOP1 gene in the background liver of HCC is likely to be a good biomarker for risk of HCC development. When assessing HCC, it is important to extract prognostic factors from background liver tissue as well as considering malignant factors of the primary cancer lesion.

Angiotensin II–Independent Angiotensin-(1–7) Formation in Rat Hippocampus

The involvement and relevance of the renin–angiotensin system have been established clearly in cardiovascular diseases, and renin–angiotensin system involvement has also been investigated extensively in the central nervous system. Angiotensin II acts classically by binding to the AT 1 and AT 2 receptors. However, other pathways within the renin–angiotensin system have been described more recently, such as one in which angiotensin-(1–7) (Ang-(1–7)) binds to the receptor Mas. In the central nervous system specifically, it has been reported that this heptapeptide is involved in learning and memory processes that occur in central limbic regions, such as the hippocampus. Therefore, this prompted us to investigate the possible role of the Ang-(1–7)–receptor Mas pathway in epileptic seizures, which are also known to recruit limbic areas. In the present study, we show that Ang-(1–7) is the main metabolite of angiotensin I in rat hippocampi, and, strikingly, that thimet oligopeptidase is the main enzyme involved in the generation of Ang-(1–7). Furthermore, elevations in the levels of thimet oligopeptidase, Ang-(1–7), and of receptor Mas transcripts are observed in chronically stimulated epileptic rats, which suggest that the thimet oligopeptidase–Ang-(1–7)–receptor Mas axis may have a functional relevance in the pathophysiology of these animals. In summary, our data, which describe a new preferential biochemical pathway for the generation of Ang-(1–7) in the central nervous system and an increase in the levels of various elements of the related thimet oligopeptidase–Ang-(1–7)–receptor Mas pathway, unveil potential new roles of the renin–angiotensin system in central nervous system pathophysiology.

Recycling of the High Valence States of Heme Proteins by Cysteine Residues of Thimet-Oligopeptidase

The peptidolytic enzyme THIMET-oligopeptidase (TOP) is able to act as a reducing agent in the peroxidase cycle of myoglobin (Mb) and horseradish peroxidase (HRP). The TOP-promoted recycling of the high valence states of the peroxidases to the respective resting form was accompanied by a significant decrease in the thiol content of the peptidolytic enzyme. EPR (electron paramagnetic resonance) analysis using DBNBS spin trapping revealed that TOP also prevented the formation of tryptophanyl radical in Mb challenged by H2O2. The oxidation of TOP thiol groups by peroxidases did not promote the inactivating oligomerization observed in the oxidation promoted by the enzyme aging. These findings are discussed towards a possible occurrence of these reactions in cells.

Peptidases released by necrotic cells control CD8+ T cell cross-priming

Cross-priming of CD8+ T cells and generation of effector immune responses is pivotal for tumor immunity as well as for successful anticancer vaccination and therapy. Dead and dying cells produce signals that can influence Ag processing and presentation; however, there is conflicting evidence regarding the immunogenicity of necrotic cell death. We used a mouse model of sterile necrosis, in which mice were injected with sterile primary necrotic cells, to investigate a role of these cells in priming of CD8+ T cells. We discovered a molecular mechanism operating in Ag donor cells that regulates cross-priming of CD8+ T cells during primary sterile necrosis and thereby controls adaptive immune responses. We found that the cellular peptidases dipeptidyl peptidase 3 (DPP-3) and thimet oligopeptidase 1 (TOP-1), both of which are present in nonimmunogenic necrotic cells, eliminated proteasomal degradation products and blocked Ag cross-presentation. While sterile necrotic tumor cells failed to induce CD8+ T cell responses, their nonimmunogenicity could be reversed in vitro and in vivo by inactivation of DPP-3 and TOP-1. These results indicate that control of cross-priming and thereby immunogenicity of primary sterile necrosis relies on proteasome-dependent oligopeptide generation and functional status of peptidases in Ag donor cells.

AGH is a new hemoglobin alpha-chain fragment with antinociceptive activity

Limited proteolysis of certain proteins leads to the release of endogenous bioactive peptides. Hemoglobin-derived peptides such as hemorphins and hemopressins are examples of intracellular protein-derived peptides that have antinociceptive effects by modulating G-protein coupled receptors activities. In the present study, a previously characterized substrate capture assay that uses a catalytically inactive form of the thimet oligopeptidase was combined with isotopic labeling and mass spectrometry in order to identify new bioactive peptides. Indeed, we have identified the peptide AGHLDDLPGALSAL (AGH), a fragment of the hemoglobin alpha-chain, which specifically bind to the inactive thimet oligopeptidase in the substrate capture assay. Previous peptidomics studies have identified the AGH as well as many other natural peptides derived from hemoglobin alpha-chain containing this sequence, further suggesting that AGH is a natural endogenous peptide. Pharmacological assays suggest that AGH inhibits peripheral inflammatory hyperalgesic responses through indirect activation of mu opioid receptors, without having a central nervous system activity. Therefore, we have successfully used the substrate capture assay to identify a new endogenous bioactive peptide derived from hemoglobin alpha-chain.

Shear stress is a positive regulator of thimet oligopeptidase (EC3.4.24.15) in vascular endothelial cells: consequences for MHC1 levels

Thimet oligopeptidase (TOP, endopeptidase EC3.4.24.15) is a soluble metallopeptidase known to be expressed within the mammalian vasculature. We examine for the first time the relationship between TOP expression and laminar shear stress, a haemodynamic force associated with endothelium-mediated vascular homeostasis. Human and bovine aortic endothelial cells were exposed to physiological levels of laminar shear (0-10 dynes/cm², 24-48 h) and monitored for TOP expression using promoter activity assay, qRT-PCR, western blotting, and immunocytochemistry. Using a luciferase reporter encoding the full-length rat TOP promoter, initial studies demonstrated shear-dependent promoter activation (~five-fold). TOP mRNA and protein were also consistently up-regulated by shear, events which could be completely prevented by pre-treatment of cells with either N-acetylcysteine, superoxide dismutase, or catalase, confirming ROS involvement. Consistent with this, targeted inhibition of NADPH oxidase (apocynin, NSC23766, NOX4 siRNA) had a similar blocking effect. Finally, in view of its pivotal role in cellular antigen presentation and major histocompatibility complex (MHC) class-1 regulation, we hypothesized that the shear-dependent induction of TOP may lower MHC1 expression. In this respect, we observed that recombinant TOP over-expression in static HAECs dose-dependently depleted MHC1 (>60%), while siRNA-mediated blockade of TOP induction in sheared HAECs led to substantially elevated MHC1 (~66%). Our findings demonstrate that laminar shear positively regulates endothelial TOP expression. Moreover, a role for ROS production by NADPH oxidase is indicated. Finally, our studies suggest that shear-dependent TOP induction down-regulates MHC1 levels, pointing to a role for TOP in the flow-mediated regulation of endothelial immunogenicity.

Extracellular Thimet Oligopeptidase is carried by cell membrane microvesicles of prostate cancer cells

Peptidases often generate bioactive peptides that regulate and mediate intercellular communication. Although processing of peptide precursors is initiated intracellularly, additional modifications by peptidases are often completed extracellularly. Plasma membrane microvesicles (MVs or microparticles) can carry membrane‐associated molecules, including proteins, when released from their parental cells. Thimet oligopeptidase (TOP) is a biologically significant peptidase that is known to be secreted to the extracellular space. Since TOP has been localized on the external face of the cell plasma membrane, we asked whether TOP might be carried by cellular MVs into the extracellular space, and whether this process is influenced by steroid hormones. DU145 androgen‐sensitive prostate cancer cells were treated with dihydrotestosterone (DHT). The cell culture supernatant was subjected to ultracentrifugation to pellet MVs. Western blot analysis of the homogenized MV sample revealed that TOP protein is, indeed, carried by the MVs of both DHT‐treated and control cells. MVs isolated from PC3 androgen‐sensitive prostate cancer cells treated with a calcium ionophore also carry TOP. Thus, our studies strongly suggest that, in addition to the soluble form, extracellular TOP also exists in MVs. This is a novel form of the extracellular peptidase that may play important pathophysiological roles.

The effects of para-chloromercuribenzoic acid and different oxidative and sulfhydryl agents on a novel, non-AT1, non-AT2 angiotensin binding site identified as neurolysin

A novel, non-AT1, non-AT2 brain binding site for angiotensin peptides that is unmasked by p-chloromercuribenzoate (PCMB) has been identified as a membrane associated variant of neurolysin. The ability of different organic and inorganic oxidative and sulfhydryl reactive agents to unmask or inhibit 125I-Sar1Ile8 angiotensin II (SI-Ang II) binding to this site was presently examined. In tissue membranes from homogenates of rat brain and testis incubated in assay buffer containing losartan (10μM) and PD123319 (10μM) plus 100μM PCMB, 5 of the 39 compounds tested inhibited 125I-SI Ang II binding in brain and testis. Mersalyl acid, mercuric chloride (HgCl2) and silver nitrate (AgNO3) most potently inhibited 125I-SI Ang II binding with IC50s ~1–20μM. This HgCl2 inhibition was independent of any interaction of HgCl2 with angiotensin II (Ang II) based on the lack of effect of HgCl2 on the dipsogenic effects of intracerebroventricularly administered Ang II and 125I-SI Ang II binding to AT1 receptors in the liver. Among sulfhydryl reagents, cysteamine and reduced glutathione (GSH), but not oxidized glutathione (GSSG) up to 1mM, inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis. Thimerosal and 4-hydroxymercuribenzoate moderately inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis at 100μM; however, they also unmasked non-AT1, non-AT2 binding independent of PCMB. 4-Hydroxybenzoic acid did not promote 125 I-SI Ang II binding to this binding site indicating that only specific organomercurial compounds can unmask the binding site. The common denominator for all of these interacting substances is the ability to bind to protein cysteine sulfur. Comparison of cysteines between neurolysin and the closely related enzyme thimet oligopeptidase revealed an unconserved cysteine (cys650, based on the full length variant) in the proposed ligand binding channel (Brown et al., 2001) [45] near the active site of neurolysin. It is proposed that the mercuric ion in PCMB and closely related organomercurial compounds binds to cys650, while the acidic anion forms an ionic bond with a nearby arginine or lysine along the channel to effect a conformational change in neurolysin that promotes Ang II binding.

Mycoplasma hyopneumoniae in vitro peptidase activities: Identification and cleavage of kallikrein-kinin system-like substrates

Bacterial proteases are important for metabolic processes and pathogenesis in host organisms. The bacterial swine pathogen Mycoplasma hyopneumoniae has 15 putative protease-encoding genes annotated, but none of them have been functionally characterized. To identify and characterize peptidases that could be relevant for infection of swine hosts, we investigated the peptidase activity present in the pathogenic 7448 strain of M. hyopneumoniae. Combinatorial libraries of fluorescence resonance energy transfer peptides, specific inhibitors and pH profiling were used to screen and characterize endopeptidase, aminopeptidase and carboxypeptidase activities in cell lysates. One metalloendopeptidase, one serine endopeptidase, and one aminopeptidase were detected. The detected metalloendopeptidase activity, prominent at neutral and basic pH ranges, was due to a thimet oligopeptidase family member (M3 family), likely an oligoendopeptidase F (PepF), which cleaved the peptide Abz-GFSPFRQ-EDDnp at the F-S bond. A chymotrypsin-like serine endopeptidase activity, possibly a subtilisin-like serine protease, was prominent at higher pH levels, and was characterized by its preference for a Phe residue at the P1 position of the substrate. The aminopeptidase P (APP) activity showed a similar profile to that of human membrane-bound APP. Genes coding for these three peptidases were identified and their transcription was confirmed in the 7448 strain. Furthermore, M. hyopneumoniae cell lysate peptidases showed effects on kallikrein-kinin system-like substrates, such as bradykinin-derived substrates and human high molecular weight kininogen. The M. hyopneumoniae peptidase activities, here characterized for the first time, may be important for bacterial survival strategies and thus represent possible targets for drug development against M. hyopneumoniae swine infections.

Abstract B30: Cross-priming of CD8+ T cells is controlled by dipeptidyl peptidase 3 and thimet oligopeptidase 1 present in necrotic cells.

Abstract Cross-priming of CD8+ T cells and generation of effector immune responses is pivotal for tumor immunity, successful anticancer vaccination and therapy. We have discovered a novel molecular mechanism operating in antigen donor cells, which regulates cross-priming of CD8+ T cells during primary sterile necrosis and thereby controls adaptive immune responses. We show that cellular peptidases, dipeptidyl peptidase 3 (DPP-3) and thimet oligopeptidase 1 (TOP-1) present in non-immunogenic necrotic cells eliminate proteasomal degradation products and block antigen cross-presentation. While sterile necrotic tumor cells fail to induce CD8+ T cell responses, their non-immunogenicity can be reversed in vitro and in vivo by inactivation of peptidases, DPP-3 and TOP-1. Thus control of cross-priming and thereby immunogenicity of primary sterile necrosis relies on the proteasome dependent oligopeptide generation and functional status of peptidases in antigen donor cells. Citation Format: Jaba Gamrekelashvili, Tamar Kapanadze, Josef Wissing, Chi Ma, Lothar Jaensch, Firouzeh Korangy, Tim F. Greten. Cross-priming of CD8+ T cells is controlled by dipeptidyl peptidase 3 and thimet oligopeptidase 1 present in necrotic cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B30.

Dipeptidyl peptidase 3 and thimet oligopeptidase 1 knockdown support tumor-specific immune responses to whole cell cancer vaccines and tumor cell death in vivo

Meeting abstracts Tumor cell vaccines are widely used as a potential therapeutic. Different approaches are currently being applied to kill tumor cell vaccines prior to injection to prevent outgrowth of metastasis at the site of injection. We and others have previously shown that the mode by which

Insights into scorpion venom peptides: Alternative processing of β-KTx propeptide from Tityus serrulatus venom results in a new naturally occurring thimet oligopeptidase inhibitor

Most functions attributed to Tityus serrulatus venom (TsV) are related to active molecules on ion-channels; however, here we describe a new pentapeptide that was discovered through enzymatic assay selection using EP24.15. The primary structure analysis revealed the sequence KEXXG (X means Ile or Leu), similar to the sequence present in the β-KTX propeptide described from the venom of Tityus spp. We confirmed through HPLC analysis that KEILG is the peptide present in TsV, but that KELLG also inhibits EP24.15 although through different mechanisms.

Natural intracellular peptides can modulate the interactions of mouse brain proteins and thimet oligopeptidase with 14‐3‐3ε and calmodulin

Protein interactions are crucial for most cellular process. Thus, rationally designed peptides that act as competitive assembly inhibitors of protein interactions by mimicking specific, determined structural elements have been extensively used in clinical and basic research. Recently, mammalian cells have been shown to contain a large number of intracellular peptides of unknown function. Here, we investigate the role of several of these natural intracellular peptides as putative modulators of protein interactions that are related to Ca(2+) -calmodulin (CaM) and 14-3-3ε, which are proteins that are related to the spatial organization of signal transduction within cells. At concentrations of 1-50 μM, most of the peptides that are investigated in this study modulate the interactions of CaM and 14-3-3ε with proteins from the mouse brain cytoplasm or recombinant thimet oligopeptidase (EP24.15) in vitro, as measured by surface plasmon resonance. One of these peptides (VFDVELL; VFD-7) increases the cytosolic Ca(2+) concentration in a dose-dependent manner but only if introduced into HEK293 cells, which suggests a wide biological function of this peptide. Therefore, it is exciting to suggest that natural intracellular peptides are novel modulators of protein interactions and have biological functions within cells.

Inhibition of thimet oligopeptidase by siRNA alters specific intracellular peptides and potentiates isoproterenol signal transduction

Mammalian cells have a large number of intracellular peptides that are generated by extralysosomal proteases. In this study, the enzymatic activity of thimet oligopeptidase (EP24.15) was inhibited in human embryonic kidney (HEK) 293 cells using a specific siRNA sequence. The semi-quantitative intracellular peptidome analyses of siRNA-transfected HEK293 cells shows that the levels of specific intracellular peptides are either increased or decreased upon EP24.15 inhibition. Decreased expression of EP24.15 was sufficient to potentiate luciferase gene reporter activation by isoproterenol (1–10μM). The protein kinase A inhibitor KT5720 (1μM) reduced the positive effect of the EP24.15 siRNA on isoproterenol signaling. Thus, EP24.15 inhibition by siRNA modulates the levels of specific intracellular peptides and isoproterenol signal transduction.

The Cysteine-Rich Protein Thimet Oligopeptidase as a Model of the Structural Requirements for S-glutathiolation and Oxidative Oligomerization

Thimet oligopeptidase (EP24.15) is a cysteine-rich metallopeptidase containing fifteen Cys residues and no intra-protein disulfide bonds. Previous work on this enzyme revealed that the oxidative oligomerization of EP24.15 is triggered by S-glutathiolation at physiological GSSG levels (10–50 µM) via a mechanism based on thiol-disulfide exchange. In the present work, our aim was to identify EP24.15 Cys residues that are prone to S-glutathiolation and to determine which structural features in the cysteinyl bulk are responsible for the formation of mixed disulfides through the reaction with GSSG and, in this particular case, the Cys residues within EP24.15 that favor either S-glutathiolation or inter-protein thiol-disulfide exchange. These studies were conducted by in silico structural analyses and simulations as well as site-specific mutation. S-glutathiolation was determined by mass spectrometric analyses and western blotting with anti-glutathione antibody. The results indicated that the stabilization of a thiolate sulfhydryl and the solvent accessibility of the cysteines are necessary for S-thiolation. The Solvent Access Surface analysis of the Cys residues prone to glutathione modification showed that the S-glutathiolated Cys residues are located inside pockets where the sulfur atom comes into contact with the solvent and that the positively charged amino acids are directed toward these Cys residues. The simulation of a covalent glutathione docking onto the same Cys residues allowed for perfect glutathione posing. A mutation of the Arg residue 263 that forms a saline bridge to the Cys residue 175 significantly decreased the overall S-glutathiolation and oligomerization of EP24.15. The present results show for the first time the structural requirements for protein S-glutathiolation by GSSG and are consistent with our previous hypothesis that EP24.15 oligomerization is dependent on the electron transfer from specific protonated Cys residues of one molecule to previously S-glutathionylated Cys residues of another one.

Neuropeptidase activity is down-regulated by estradiol in steroid-sensitive regions of the hypothalamus in female mice

Thimet oligopeptidase (TOP) and prolyl endopeptidase (PEP) are neuropeptidases involved in the hydrolysis of gonadotropin-releasing hormone, a key component of the hypothalamic-pituitary-gonadal axis. GnRH is regulated in part by feedback from steroid hormones such as estradiol. Previously, we demonstrated that TOP levels are down-regulated by estradiol in reproductively-relevant regions of the female rodent brain. The present study supports these findings by showing that TOP enzyme activity, as well as protein levels, in the ventromedial hypothalamic nucleus of female mice is controlled by estradiol. We further demonstrate that PEP levels in this same brain region are down-regulated by estradiol in parallel with those of TOP. These findings provide evidence that these neuropeptidases are part of the fine control of hormone levels in the HPG axis.