Thimet Oligopeptidase Research Articles

Abstract 2177 Coordinated activity of a thimet oligopeptidase proteolytic couple generates the oxidation-reduction waves required for priming and execution of systemic acquired resistance

Abstract 2177 Coordinated activity of a thimet oligopeptidase proteolytic couple generates the oxidation-reduction waves required for priming and execution of systemic acquired resistance

Thimet oligopeptidase as a potential CSF biomarker for Alzheimer's disease: A cross-platform validation study.

Our previous antibody-based cerebrospinal fluid (CSF) proteomics study showed that Thimet oligopeptidase (THOP1), an amyloid beta (Aβ) neuropeptidase, was increased in mild cognitive impairment with amyloid pathology (MCI-Aβ+) and Alzheimer's disease (AD) dementia compared with controls and dementia with Lewy bodies (DLB), highlighting the potential of CSF THOP1 as an early specific biomarker for AD. We aimed to develop THOP1 immunoassays for large-scale analysis and validate our proteomics findings in two independent cohorts. We developed in-house CSF THOP1 immunoassays on automated Ella and Simoa platforms. The performance of the different assays were compared using Passing-Bablok regression analysis in a subset of CSF samples from the discovery cohort (n = 72). Clinical validation was performed in two independent cohorts (cohort 1: n = 200; cohort 2: n = 165) using the Ella platform. THOP1 concentrations moderately correlated between proteomics analysis and our novel assays (Rho>0.580). In both validation cohorts, CSF THOP1 was increased in MCI-Aβ+ (>1.3-fold) and AD (>1.2-fold) compared with controls; and between MCI-Aβ+ and DLB (>1.2-fold). Higher THOP1 concentrations were detected in AD compared with DLB only when both cohorts were analyzed together. In both cohorts, THOP1 correlated with CSF total tau (t-tau), phosphorylated tau (p-tau), and Aβ40 (Rho>0.540) but not Aβ42. Validation of our proteomics findings underpins the potential of CSF THOP1 as an early specific biomarker associated with AD pathology. The use of antibody-based platforms in both the discovery and validation phases facilitated the translation of proteomics findings, providing an additional workflow that may accelerate the development of biofluid-based biomarkers.

Expression of thimet oligopeptidase (THOP) modulated by oxidative stress in human multidrug resistant (MDR) leukemia cells

Thimet oligopeptidase (THOP) is a cytosolic metallopeptidase known to regulate the fate of post-proteasomal peptides, protein turnover and peptide selection in the antigen presentation machinery (APM) system. Oxidative stress influences THOP expression and regulates its proteolytic activity, generating variable cytosolic peptide levels, possibly affecting the immune evasion of tumor cells. In the present work, we examined the association between THOP expression/activity and stress oxidative resistance in human leukemia cells using the K562 cell line, a chronic myeloid leukemia (CML), and the multidrug-resistant (MDR) Lucena 1 (K562-derived MDR cell line) as model. The Lucena 1 phenotype was validated under vincristine treatment and the relative THOP1 mRNA levels and protein expression compared to K562 cell line. Our data demonstrated increased THOP1 gene and protein levels in K562 cells in contrast to the oxidative-resistant Lucena 1, even after H2O2 treatment, suggesting an oxidative stress dependence in THOP regulation. Further, it was observed higher basal levels of reactive oxygen species (ROS) in K562 compared to Lucena 1 cell line using DHE fluorescent probe. Since THOP activity is dependent on its oligomeric state, we also compared its proteolytic activity under reducing agent treatment, which demonstrated that its function modulation with respect to changes in redox state. Finally, the mRNA expression and FACS analyses demonstrated a reduced expression of MHC I only in K562 cell line. In conclusion, our results highlight THOP redox modulation, which could influence antigen presentation in multidrug resistant leukemia cells.

Thimet Oligopeptidase is a potential CSF biomarker for Alzheimer’s Disease

AbstractBackgroundUsing antibody‐based Olink proteomics, we recently established that Thimet Oligopeptidase (THOP1) is a potential cerebrospinal fluid (CSF) biomarker for Alzheimer’s Disease (AD). THOP1 is a neuropeptidase able to degradate Aβ peptides. Here, we aimed to develop specific THOP1 immunoassays for further clinical validation and to enable future implementation of this biomarker.MethodWe developed and compared novel immunoassays for CSF THOP1 analysis on automated Ella™ and Simoa™ platforms. After analytical validation, THOP1 levels were measured in a selection of CSF samples from the original discovery study (24 cognitively unimpaired controls, 24 AD and 24 dementia with Lewy bodies (DLB) patients) on both platforms. Passing bablok‐regression analysis showed that platforms were comparable and thus we selected the Ella platform for further validation in two independent cohorts (Amsterdam Dementia cohort (ADC) including 56 controls, 45 mild cognitive impairment with amyloid pathology (MCI‐Aβ+), 55 AD and 55 DLB patients; Sant Pau Initiative on Neurodegeneration (SPIN) cohort including 53 controls, 50 MCI‐Aβ+, 47 AD and 50 DLB patients). Differences in THOP1 levels between diagnostic groups and correlations to the AD CSF biomarkers were tested by ANCOVA (corrected for sex) and Spearman correlation analyses.ResultTHOP1 levels moderately correlated between proteomics analysis and the novel assays (Olink‐Ella: rho=0.720; Olink‐Simoa: rho=0.584; Ella‐Simoa rho=0.713; figure 1). Consistent with results obtained on the Olink proteomics platform, THOP1 levels were significantly increased in AD compared to DLB patients (Ella: 1.6‐fold; Simoa: 1.7‐fold) and controls (Ella: 1.8‐fold; Simoa: p>0.05). We next showed increased THOP1 levels in both MCI‐Aβ+ (ADC: 1.2‐fold, p<0.05; SPIN: 1.4‐fold, p<0.001) and AD patients (ADC: 1.03‐fold, p>0.05; SPIN: 1.4‐fold, p<0.001) compared to controls (Figure 2). THOP1 levels correlated moderately with levels of t‐Tau (ADC: rho=0.644; SPIN: rho=0.721), p‐Tau (ADC: rho=0.480; SPIN: rho=0.712) and Aβ40 (ADC: not available; SPIN: rho=0.625) but not with Aβ42 (ADC: rho=0.110; SPIN: rho=‐0.09).ConclusionWe show increased CSF THOP1 levels in amyloid positive AD and MCI patients and correlation with CSF Aβ40, which together with literature showing colocalization of THOP1 with Aβ plaques suggests a relationship with Aβ‐production. The association of THOP1 to tau pathologies highlights the potential of this protein as a disease monitoring biomarker.

Is Diminazene an Angiotensin-Converting Enzyme 2 (ACE2) Activator? Experimental Evidence and Implications.

Antiprotozoal veterinary drug diminazene aceturate (DIZE) has been proposed to be an angiotensin-converting enzyme 2 (ACE2) activator. Since then, DIZE was used in dozens of experimental studies, but its mechanism of action attributed to ACE2 activation and enhanced formation of angiontensin-(1-7) [Ang-(1-7)] from Ang II was not carefully verified. The aim of this study was to confirm the effect of DIZE on catalytic activity of ACE2 and extend it to other peptidases involved in formation and degradation of Ang-(1-7). Concentration-dependent effect of DIZE on the initial rate of a fluorogenic substrate hydrolysis by human and mouse recombinant ACE2 was measured at assay conditions imitating that of the original report, but no activation of ACE2 was documented. Similar results were obtained with a more physiologically relevant assay buffer. In addition, DIZE did not affect activity of recombinant neprilysin, neurolysin, thimet oligopeptidase, and ACE. Efficiency of the fluorogenic substrate hydrolysis (Vmax/Km value) by ACE2 in response to different concentrations of DIZE was also measured, but no substantial effects were documented. Likewise, DIZE failed to enhance the hydrolysis of ACE2 endogenous substrate Ang II. Identity of the commercial recombinant ACE2 variants used in these experiments was confirmed by inhibition with two well characterized inhibitors (DX600 and MLN4760), activation by NaCl, and Western Blotting using validated antibodies. These observations challenge the widely accepted notion about the molecular mechanism of DIZE action and call for not ascribing this molecule as an ACE2 activator. SIGNIFICANCE STATEMENT: DIZE has been proposed and widely used in experimental studies as an ACE2 activator. The detailed in vitro pharmacological studies failed to confirm that DIZE is an ACE2 activator. In addition, DIZE did not substantially affect the activity of other peptidases involved in formation and degradation of angiotensin-(1-7). Researchers should refrain from calling DIZE an ACE2 activator. Other mechanisms are responsible for the therapeutic benefits attributed to DIZE.

Probing the Conformational States of Thimet Oligopeptidase in Solution.

Thimet oligopeptidase (TOP) is a metallopeptidase involved in the metabolism of oligopeptides inside and outside cells of various tissues. It has been proposed that substrate or inhibitor binding in the TOP active site induces a large hinge-bending movement leading to a closed structure, in which the bound ligand is enclosed. The main goal of the present work was to study this conformational change, and fluorescence techniques were used. Four active TOP mutants were created, each equipped with a single-Trp residue (fluorescence donor) and a p-nitro-phenylalanine (pNF) residue as fluorescence acceptor at opposite sides of the active site. pNF was biosynthetically incorporated with high efficiency using the amber codon suppression technology. Inhibitor binding induced shorter Donor-Acceptor (D-A) distances in all mutants, supporting the view that a hinge-like movement is operative in TOP. The activity of TOP is known to be dependent on the ionic strength of the assay buffer and D-A distances were measured at different ionic strengths. Interestingly, a correlation between the D-A distance and the catalytic activity of TOP was observed: the highest activities corresponded to the shortest D-A distances. In this study for the first time the hinge-bending motion of a metallopeptidase in solution could be studied, yielding insight about the position of the equilibrium between the open and closed conformation. This information will contribute to a more detailed understanding of the mode of action of these enzymes, including therapeutic targets like neurolysin and angiotensin-converting enzyme 2 (ACE2).

Enhanced Antitumor Efficacy of PhAc-ALGP-Dox, an Enzyme-Activated Doxorubicin Prodrug, in a Panel of THOP1-Expressing Patient-Derived Xenografts of Soft Tissue Sarcoma.

Despite poor response rates and dose-limiting cardiotoxicity, doxorubicin (doxo) remains the standard-of-care for patients with advanced soft tissue sarcoma. We evaluated the efficacy of two tetrapeptidic doxo prodrugs (PhAc-ALGP-Dox or CBR-049 and CBR-050) that are locally activated by enzymes expressed in the tumor environment, in ten sarcoma patient-derived xenografts. Xenograft models were selected based on expression of the main activating enzyme, i.e., thimet oligopeptidase (THOP1). Mice were either randomized to vehicle, doxo, CBR-049 and CBR-050 or control, doxo, aldoxorubicin (aldoxo) and CBR-049. Treatment efficacy was assessed by tumor volume measurement and histological assessment of ex-mouse tumors. CBR-049 showed significant tumor growth delay compared to control in all xenografts investigated and was superior compared to doxo in all but one. At the same time, CBR-049 showed comparable efficacy to aldoxo but the latter was found to have a complex safety profile in mice. CBR-050 demonstrated tumor growth delay compared to control in one xenograft but was not superior to doxo. For both experimental prodrugs, strong immunostaining for THOP1 was found to predict better antitumor efficacy. The prodrugs were well tolerated without any adverse events, even though molar doses were 17-fold higher than those administered and tolerated for doxo.

Thimet Oligopeptidase—A Classical Enzyme with New Function and New Form

Peptidases generate bioactive peptides that can regulate cell signaling and mediate intercellular communication. While the processing of peptide precursors is initiated intracellularly, some modifications by peptidases may be conducted extracellularly. Thimet oligopeptidase (TOP) is a peptidase that processes neuroendocrine peptides with roles in mood, metabolism, and immune responses, among other functions. TOP also hydrolyzes angiotensin I to angiotensin 1–7, which may be involved in the pathophysiology of COVID-19 infection. Although TOP is primarily cytosolic, it can also be associated with the cell plasma membrane or secreted to the extracellular space. Recent work indicates that membrane-associated TOP can be released with extracellular vesicles (EVs) to the extracellular space. Here we briefly summarize the enzyme’s classical function in extracellular processing of neuroendocrine peptides, as well as its more recently understood role in intracellular processing of various peptides that impact human diseases. Finally, we discuss new findings of EV-associated TOP in the extracellular space.

Alterations in Gene Expression of Renin-Angiotensin System Components and Related Proteins in Colorectal Cancer.

Materials and Methods Quantitative expression of the RNA of these 17 genes in normal and cancerous tissues obtained using chip arrays from the public functional genomics data repository, Gene Expression Omnibus (GEO) application, was compared statistically. Results Expression of four genes, AGT (angiotensinogen), ENPEP (aminopeptidase A) MME (neprilysin), and PREP (prolyl endopeptidase), was significantly upregulated in CRC specimens. Expression of REN (renin), THOP (thimet oligopeptidase), NLN (neurolysin), PRCP (prolyl carboxypeptidase), ANPEP (aminopeptidase N), and MAS1 (Mas receptor) was downregulated in CRC specimens. Conclusions Presuming gene expression parallel protein expression, these results suggest that increased production of the angiotensinogen precursor of angiotensin (ANG) peptides, with the reduction of the enzymes that metabolize it to ANG II, can lead to accumulation of angiotensinogen in CRC tissues. Downregulation of THOP, NLN, PRCP, and MAS1 gene expression, whose proteins contribute to the ACE2/ANG 1-7/Mas axis, suggests that reduced activity of this RAS branch could be permissive for oncogenicity. Components of the RAS may be potential therapeutic targets for treatment of CRC.

Thimet Oligopeptidase Biochemical and Biological Significances: Past, Present, and Future Directions.

Thimet oligopeptidase (EC 3.4.24.15; EP24.15, THOP1) is a metallopeptidase ubiquitously distributed in mammalian tissues. Beyond its previously well characterized role in major histocompatibility class I (MHC-I) antigen presentation, the recent characterization of the THOP1 C57BL6/N null mice (THOP1−/−) phenotype suggests new key functions for THOP1 in hyperlipidic diet-induced obesity, insulin resistance and non-alcoholic liver steatosis. Distinctive levels of specific intracellular peptides (InPeps), genes and microRNAs were observed when comparing wild type C57BL6/N to THOP1−/− fed either standard or hyperlipidic diets. A possible novel mechanism of action was suggested for InPeps processed by THOP1, which could be modulating protein-protein interactions and microRNA processing, thus affecting the phenotype. Together, research into the biochemical and biomedical significance of THOP1 suggests that degradation by the proteasome is a step in the processing of various proteins, not merely for ending their existence. This allows many functional peptides to be generated by proteasomal degradation in order to, for example, control mRNA translation and the formation of protein complexes.

Renal Androgen and Renin Angiotensin System mRNA Expression in Polycystic Ovary Syndrome

BackgroundPolycystic Ovary Syndrome (PCOS), the most common endocrine disorder in women, is characterized by androgen‐excess and ovarian dysfunction. PCOS is associated with obesity, insulin resistance (IR) and increased blood pressure (BP). Currently therapeutic options to treat PCOS‐associated cardiovascular risk factors are limited. We have previously characterized a rat model of PCOS in which chronic androgen‐excess causes increased body weight (BW), fat mass (FM), IR, BP, and activation of some intrarenal Renin Angiotensin System (RAS) components. Still, the mechanisms responsible for increased BP in women with PCOS remain unclear. In the present study, we tested how chronic androgen‐excess changes the renal mRNA expression profile of the androgen receptor (AR) and RAS components in a model of PCOS.MethodsFour‐week old female SD rats were randomized to subcutaneously receive dihydrotestosterone (DHT, 7.5 mg/90 days) pellets (DHT) or sham surgeries (control) for 90 days (n= 6–10/grp). At the end of the experimental period, food intake (FI), BW, FM measured by Echo‐MRI, and BP using radiotelemetry were assessed in conscious, freely moving animals. Renal mRNA expression was assessed in cortical and medullary tissues separately by RT‐qPCR.ResultsAt the end of the experimental period, DHT rats exhibited higher FI, BW, FM and BP compared to control. In DHT rats, renal cortical mRNA expression of AR, angiotensinogen, AT1R, renin receptor, cathepsin A, aminoendopeptidase A, dipeptidyl peptidase 3, neprilysin, neurolysin, prolylendopeptidase, insulin‐like growth factor 2 receptor were significantly increased. In addition, renal medullary mRNA expression of angiotensin converting enzyme (ACE), angiotensin converting enzyme type 2 (ACE2), angiotensinogen, angiotensin 2 receptor type 1 (AT1R), membrane alanyl aminopeptidase, dipeptidyl peptidase 3, aminopeptidase A, kallikrein 1, neprilysin, neurolysin, prolylendopeptidase, and thimet oligopeptidase were significantly increased. Renal medullary mRNA expression of renin and aminopeptidase B were significantly decreased in DHT rats.Summary and ConclusionsIn summary, hyperandrogenemia leads to upregulation of AR in renal cortex and medulla. The mRNA expression of renal cortical and medullar ACE/AT1R axis components was upregulated in DHT rats. Additionally, mRNA expression of the ACE2 was upregulated mainly in the kidney medulla of DHT rats which could be a compensatory mechanism. This study highlights the effect of androgen‐excess modulating the intrarenal RAS and AR. Treatments focused on targeting RAS system components could be promising therapeutic tools to ameliorate the cardiovascular and renal abnormalities observes in women with PCOS.Support or Funding InformationAHA 0830239N (L.L.Y.C.) and 12SDG8980032 (D.G.R.), EFFERG (L.L.Y.C.), and NIH R21 DK‐113500 (D.G.R.) and P20 GM‐121334 (L.L.Y.C.)

The Relevance of Thimet Oligopeptidase in the Regulation of Energy Metabolism and Diet-Induced Obesity.

Thimet oligopeptidase (EC 3.4.24.15; EP24.15; THOP1) is a potential therapeutic target, as it plays key biological functions in processing biologically functional peptides. The structural conformation of THOP1 provides a unique restriction regarding substrate size, in that it only hydrolyzes peptides (optimally, those ranging from eight to 12 amino acids) and not proteins. The proteasome activity of hydrolyzing proteins releases a large number of intracellular peptides, providing THOP1 substrates within cells. The present study aimed to investigate the possible function of THOP1 in the development of diet-induced obesity (DIO) and insulin resistance by utilizing a murine model of hyperlipidic DIO with both C57BL6 wild-type (WT) and THOP1 null (THOP1−/−) mice. After 24 weeks of being fed a hyperlipidic diet (HD), THOP1−/− and WT mice ingested similar chow and calories; however, the THOP1−/− mice gained 75% less body weight and showed neither insulin resistance nor non-alcoholic fatty liver steatosis when compared to WT mice. THOP1−/− mice had increased adrenergic-stimulated adipose tissue lipolysis as well as a balanced level of expression of genes and microRNAs associated with energy metabolism, adipogenesis, or inflammation. Altogether, these differences converge to a healthy phenotype of THOP1−/− fed a HD. The molecular mechanism that links THOP1 to energy metabolism is suggested herein to involve intracellular peptides, of which the relative levels were identified to change in the adipose tissue of WT and THOP1−/− mice. Intracellular peptides were observed by molecular modeling to interact with both pre-miR-143 and pre-miR-222, suggesting a possible novel regulatory mechanism for gene expression. Therefore, we successfully demonstrated the previously anticipated relevance of THOP1 in energy metabolism regulation. It was suggested that intracellular peptides were responsible for mediating the phenotypic differences that are described herein by a yet unknown mechanism of action.

CPP-Ala-Ala-Tyr-PABA inhibitor analogs with improved selectivity for neurolysin or thimet oligopeptidase

Thimet oligopeptidase (TOP, EC 3.4.24.15) and neurolysin (NEL, EC 3.4.24.16) are closely related zinc-dependent metalo-oligopeptidases, which take part in the metabolism of oligopeptides (from 5 to 17 amino acid residues) inside and outside cells. Both peptidases are ubiquitously distributed in tissues. TOP is one of the main intracellular peptide-processing enzymes being important for the antigen selection in the MHC Class I presentation route, while NEL function has been more associated with the extracellular degradation of neurotensin. Despite efforts being made to develop specific inhibitors for these peptidases, the most used are: CPP-Ala-Ala-Tyr-PABA, described by Orlowski et al. in 1988, and CPP-Ala-Aib-Tyr-PABA (JA-2) that is an analog more resistant to proteolysis, which development was made by Shrimpton et al. in 2000. In the present work, we describe other analogs of these compounds but, with better discriminatory capacity to inhibit specifically NEL or TOP. The modifications introduced in these new analogs were based on a key difference existent in the extended binding sites of NEL and TOP: the negatively charged Glu469 residue of TOP corresponds to the positively charged Arg470 residue of NEL. These residues are in position to interact with the residue at the P1′ and/or P2′ of their substrates (mimicked by the Ala-Ala/P1′-P2′ residues of the CPP-Ala-Ala-Tyr-PABA). Therefore, exploring this single difference, the following compounds were synthesized: CPP-Asp-Ala-Tyr-PABA, CPP-Arg-Ala-Tyr-PABA, CPP-Ala-Asp-Tyr-PABA, CPP-Ala-Arg-Tyr-PABA. Confirming the predictions, the replacement of each non-charged residue of the internal portion Ala-Ala by a charged residue Asp or Arg resulted in compounds with higher selectivity for NEL or TOP, especially due to the electrostatic attraction or repulsion by the NEL Arg470 or TOP Glu469 residue. The CPP-Asp-Ala-Tyr-PABA and CPP-Ala-Asp-Tyr-PABA presented higher affinities for NEL, and, the CFP-Ala-Arg-Tyr-PABA showed higher affinity for TOP.

Sleep deprivation changes thimet oligopeptidase (THOP1) expression and activity in rat brain

The consequences of sleep deprivation on memory, cognition, nociception, stress, and endocrine function are related to the balance of neuropeptides, with peptidases being particularly essential. Thimet oligopeptidase (THOP1) is a metallopeptidase implicated in the metabolism of many sleep-related peptides, including angiotensin I, gonadotropin releasing hormone (GnRH), neurotensin, and opioid peptides. In the present study, we evaluated the effect of sleep deprivation and sleep recovery in male rats on THOP1 expression and specific activity in the central nervous system. In the striatum and hypothalamus, THOP1 activity decreased following sleep deprivation and a recovery period. Meanwhile, THOP1 activity and immunoexpression increased in the hippocampal dentate gyrus during the sleep recovery period. Changes in THOP1 expression after sleep deprivation and during sleep recovery can potentially alter the processing of neuropeptides. In particular, processing of opioid peptides may be related to the known increase in pain sensitivity in this model. These results suggest that THOP1 may be an important player in the effects of sleep deprivation.

Thimet Oligopeptidase (EC 3.4.24.15) Key Functions Suggested by Knockout Mice Phenotype Characterization.

Thimet oligopeptidase (THOP1) is thought to be involved in neuropeptide metabolism, antigen presentation, neurodegeneration, and cancer. Herein, the generation of THOP1 C57BL/6 knockout mice (THOP1−/−) is described showing that they are viable, have estrus cycle, fertility, and a number of puppies per litter similar to C57BL/6 wild type mice (WT). In specific brain regions, THOP1-/- exhibit altered mRNA expression of proteasome beta5, serotonin 5HT2a receptor and dopamine D2 receptor, but not of neurolysin (NLN). Peptidomic analysis identifies differences in intracellular peptide ratios between THOP1-/- and WT mice, which may affect normal cellular functioning. In an experimental model of multiple sclerosis THOP1-/- mice present worse clinical behavior scores compared to WT mice, corroborating its possible involvement in neurodegenerative diseases. THOP1-/- mice also exhibit better survival and improved behavior in a sepsis model, but also a greater peripheral pain sensitivity measured in the hot plate test after bradykinin administration in the paw. THOP1-/- mice show depressive-like behavior, as well as attention and memory retention deficits. Altogether, these results reveal a role of THOP1 on specific behaviors, immune-stimulated neurodegeneration, and infection-induced inflammation.

Separation-Based Enzymomics Assay for the Discovery of Altered Peptide-Metabolizing Enzymatic Activities in Biosamples.

We have developed a novel method to globally monitor the enzymatic activities of biological samples based on performing the global activity analysis on a proteome separated by native electrophoresis. The study of the alteration in peptide-metabolizing enzymatic activity in colorectal tumor specimens led us to the discovery of elevated thimet oligopeptidase activity, which contributed to the faster consumption of immune-stimulating peptide neurotensin.

SUN-478 The Role of Thimet Oligopeptidase (EP24.15) in Regulating Reproduction

For almost six decades, gonadotropin releasing hormone (GnRH) has been examined biochemically and physiologically in the hormonal control of reproduction. More recently, Kisspeptin (Kiss), neurokinin B (NKB) and dynorphin A (DynA) comprising KNDy neurons in the hypothalamus have been demonstrated to regulate gonadotropin releasing hormone (GnRH) release. To add to the complex circuitry of reproduction, and associated physiological processes, we sought to define the role of Phoenixin (Phx), a recently identified neuropeptide produced in the hypothalamus, that increases GnRH peptide, GnRH receptor and Kisspeptin mRNAs, increases GnRH secretion and mediates ovarian cyclicity. EP24.15 (EC 3.4.24.15, thimet oligopeptidase) is a neuropeptide processing metalloenzyme that is expressed throughout the hypothalamus and other brain regions. All neuropeptide hormones have their downstream signaling activity moderated enzymatically in the extracellular space though the specific enzymes remain unknown. EP24.15 is best known for regulating GnRH by degrading the hormone and thus, inactivating binding to the GnRH receptor. Likewise, we have proven that Kiss is likewise degraded by EP24.15 further pointing to a role in regulating reproduction. To validate both physiological and biochemical regulation of DynA and Phx, we determined if these neuropeptides and EP24.15 are co-expressed in the brain, (in addition to our previous studies of GnRH and Kiss) identifying a functional neuroendocrine circuit whereby EP24.15 could regulate reproduction. We have demonstrated Phx immunoreactivity in areas of the brain related to stress (amygdala and bed nucleus of the stria terminalis) and reproduction (hypothalamic arcuate nucleus). Biochemically, we used proteomics to identify new neuropeptide substrates, their binding to the active site and cleavage sites by high resolution 3D structural models, mass spectrometry, and enzyme kinetics. Two cleavage sites are present in Kiss and DynA whereas there is a single cleavage site in GnRH and Phx. Enzyme kinetic variables of DynA and Phx (Km and Vmax) are comparable to published values for GnRH and Kiss. The data demonstrates that EP24.15 cleaves DynA, and Phx in vivo as an additional layer to the elaborate mechanism of reproduction. Though NKB was co-expressed with EP24.1.5, it was not a substrate for the enzyme which allows for finer tuning of the reproductive axis. A better understanding of novel, regulated peptides that integrate stress and reproductive processes will provide insight into brain circuitry for moderating normal and aberrant reproductive function.

Differential Gene Expression Profile of Renin-Angiotensin System in the Left Atrium in Mitral Regurgitation Patients.

Background Left atrial enlargement is a mortality and heart failure risk factor in primary mitral regurgitation (MR) patients. Pig models of MR have shown differential expression of genes linked to the renin-angiotensin system. Therefore, the aim of this study was to investigate the key genes of the renin-angiotensin that are expressed differentially in the left atrial myocardium in MR patients. Methods Quantitative RT-PCR was used to compare gene expression in the renin-angiotensin system in the left atrium in MR patients, aortic valve disease patients, and normal subjects. Results Plasma angiotensin II concentrations did not significantly differ between MR patients and aortic valve disease patients (P = 0.582). Compared to normal controls, however, MR patients had significantly downregulated expressions of angiotensin-converting enzyme, angiotensin I converting enzyme 2, type 1 angiotensin II receptor, glutamyl aminopeptidase, angiotensinogen, cathepsin A (CTSA), thimet oligopeptidase 1, neurolysin, alanyl aminopeptidase, cathepsin G, leucyl/cystinyl aminopeptidase (LNPEP), neprilysin, and carboxypeptidase A3 in the left atrium. The MR patients also had significantly upregulated expressions of MAS1 oncogene (MAS1) and mineralocorticoid receptor compared to normal controls. Additionally, in comparison with aortic valve disease patients, MR patients had significantly downregulated CTSA and LNPEP expression and significantly upregulated MAS1 expression in the left atrium. Conclusions Expressions of genes in the renin-angiotensin system, especially CTSA, LNPEP, and MAS1, in the left atrium in MR patients significantly differed from expressions of these genes in aortic valve disease patients and normal controls. Notably, differences in expression were independent of circulating angiotensin II levels. The results of this study provide a rationale for pharmacological therapies or posttranslational regulation therapies targeting genes expressed differentially in the renin-angiotensin system to remedy structural remodeling associated with atrial enlargement and heart failure progression in patients with MR.

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.

Regulation of Gonadotropin-Releasing Hormone-(1-5) Signaling Genes by Estradiol Is Age Dependent.

Gonadotropin-releasing hormone (GnRH) is a key regulatory molecule of the hypothalamus–pituitary (PIT)–gonadal (HPG) axis that ultimately leads to the downstream release of estradiol (E2) and progesterone (P). These gonadal steroids feed back to the hypothalamus and PIT to regulate reproductive function and behavior. While GnRH is thought to be the master regulator of reproduction, its metabolic product GnRH-(1–5) is also biologically active. Thimet oligopeptidase 1 (also known as EP24.15) cleaves GnRH to form GnRH-(1–5). GnRH-(1–5) is involved in regulation of the HPG axis, exerting its actions through a pair of orphan G protein-coupled receptors, GPR101 and GPR173. The physiological importance of GnRH-(1–5) signaling has been studied in several contexts, but its potential role during reproductive senescence is poorly understood. We used an ovariectomized (OVX) rat model of reproductive senescence to assess whether and how GnRH-(1–5) signaling genes in hypothalamic subnuclei change in response to aging and/or different estradiol replacement regimens designed to model clinical hormone replacement in women. We found that Gpr101 and Gpr173 mRNA expression was increased with age in the arcuate nucleus, while expression of Gpr173 and EP24.15 increased with age in the medial preoptic area. Treatment with E2 in younger OVX animals increased expression of Gpr101, Gpr173, and EP24.15. However, older animals treated with E2 showed decreased expression of these GnRH-(1–5) signaling genes, displaying an age-related decline in responsiveness to E2. To our knowledge, this is the first study to systematically assess the effects of age and different clinically relevant regimens of E2 replacement on GnRH-(1–5) signaling genes.