Delineation of a Particulate Thyrotropin-Releasing Hormone-Degrading Enzyme in Rat Brain by the Use of Specific Inhibitors of Prolyl Endopeptidase and Pyroglutamyl Peptide Hydrolase

The degradation of thyrotropin-releasing hormone in rat brain homogenates was studied in the presence of N-benzyloxycarbonyl-prolyl-prolinal and pyroglutamyl diazomethyl ketone, specific and potent active-site-directed inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase, respectively. Substantial TRH degradation was observed, suggesting the presence of another thyrotropin-releasing hormone-degrading enzyme(s). Reports of a thyrotropin-releasing hormone-degrading enzyme with narrow specificity that cleaves the pGlu-His bond of this tripeptide led us to develop a coupled assay using pGlu-His-Pro-2NA as the substrate to measure this activity. Cleavage of the pGlu-His bond of this substrate under conditions in which pyroglutamyl peptide hydrolase is not expressed occurred in the particulate fraction of a rat brain homogenate. This particulate pyroglutamyl-peptide cleaving enzyme was not inhibited by pyroglutamyl diazomethyl ketone but was inhibited by metal chelators such as EDTA and o-phenanthroline. The particulate pyroglutamyl-peptide cleaving enzyme was found predominantly in the brain. Activity in brain regions varied widely with highest levels present in cortex and hippocampus and very low levels in pituitary. The data suggest that degradation of thyrotropin-releasing hormone by the particulate fraction of a brain homogenate is catalyzed mainly by an enzyme that cleaves the pGlu-His bond of thyrotropin-releasing hormone but is distinct from pyroglutamyl peptide hydrolase.     

A prolyl endopeptidase from murine macrophages, its assay and specific inactivation

The presence of a prolyl endopeptidase in the soluble fraction of murine peritoneal macrophages is reported. The prolyl endopeptidase is apparently highly specific for cleaving peptides after proline residues. A sensitive new fluorogenic assay substrate matching this specificity, benzyloxycarbonyl-Ala-Ala-Pro beta-methoxynaphthylamide, is described. The enzyme is rapidly inactivated by benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone, one of a class of reagents specific for cysteine proteinases, and by diisopropyl fluorophosphate, an inhibitor of serine proteinases. Culture of macrophages with the addition of low levels of benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone to the media allows the selective inhibition of the cytoplasmic enzyme as measured in lysates at the termination of culture. After exposure to inhibitor, macrophages resynthesize the enzyme over a period of days, a process which is inhibited by cycloheximide. Similar amounts of activity were found in both normal peritoneal macrophages and those elicited by prior injection of thioglycollate media. The enzyme from murine macrophages appears similar to that reported in bronchopulmonary lavage fluid and lung tissue and to those isolated from brain and pituitary tissues.     

Phosphorylation and dephosphorylation of the Ca2+ pump of human red cells in the presence of monovalent cations

A chloromethyl ketone derivative of pyroglutamic acid was newly synthesized and its reactivity with bacterial pyroglutamyl aminopeptidase (L-pyroglutamyl-peptide hydrolas, EC 3.4.11.8) as an affinity labelling reagent was examined. The compound was found to inactivate the enzyme markedly and rapidly at very low concentrations, though the enzyme was resistant to N-tosyl-phenylalanyl chloromethyl ketone. The rate of the enzyme inactivation by pyroglutamyl chloromethyl ketone was retarded in the presence of a poor substrate, pyroglutamyl valine. The enzyme inactivated by treating with p-chloromercuribenzoate failed to react with pyroglutamyl chloromethyl ketone. These results strongly suggest an active site-directed mechanism for the enzyme inactivation by pyroglutamyl chloromethyl ketone. This compound was shown to be useful as a titrant for the catalytically active protein of pyroglutamyl aminopeptidase.     

Regulation of Thyrotropin Releasing Hormone Degrading Enzymes in Rat Brain and Pituitary by L- 3,5,3''-Triiodothyronine

The effect of treatment with L-3,5,3'-triiodothyronine (T3) on the levels of pyroglutamyl peptidase I and pyroglutamyl peptidase II in rat brain regions, pituitary, and serum was studied. Pyroglutamyl peptidase I cleaves pyroglutamyl peptides such as thyrotropin releasing hormone (TRH), luteinizing hormone releasing hormone, neurotensin, and bombesin, whereas pyroglutamyl peptidase II appears to be specific for TRH. Acute administration of T3 did not affect pyroglutamyl peptidase I in any of the regions studied, whereas pyroglutamyl peptidase II was significantly elevated in frontal cortex and pituitary. Treatment with T3 for 10 or 14 days significantly elevated pyroglutamyl peptidase I in pituitary, hypothalamus, olfactory bulb, hippocampus, and thalamus. Chronic T3 treatment elevated pyroglutamyl peptidase II in frontal cortex and in serum. These studies demonstrate regulation of neuropeptide degrading enzymes by thyroid hormones in vivo. This regulation may play a role in the negative feedback control of thyroid status by T3.     

An evaluation of the role of a pyroglutamyl peptidase, a post-proline cleaving enzyme and a post-proline dipeptidyl amino peptidase, each purified from the soluble fraction of guinea-pig brain, in the degradation of thyroliberin in vitro

The degradation of thyroliberin (less than Glu-His-Pro-NH2) to its component amino acids by the soluble fraction of guinea pig brain is catalysed by four enzymes namely a pyroglutamate aminopeptidase, a post-proline cleaving enzyme, a post-proline dipeptidyl aminopeptidase and a proline dipeptidase. 1. The pyroglutamate aminopeptidase was purified to over 90% homogeneity with a purification factor of 2868-fold and a yield of 5.7%. In addition to catalysing the hydrolysis of thyroliberin, acid thyroliberin and pyroglutamate-7-amido-4-methylcoumarin the pyroglutamate aminopeptidase catalysed the hydrolysis of the peptide bond adjacent to the pyroglutamic acid residue in luliberin, neurotensin bombesin, bradykinin-potentiating peptide B, the anorexogenic peptide and the dipeptides pyroglutamyl alanine and pyroglutamyl valine. Pyroglutamyl proline and eledoisin were not hydrolysed. 2. The post-proline cleaving enzyme was purified to apparent electrophoretic homogeneity with a purification factor of 2298-fold and a yield of 10.6%. The post-proline cleaving enzyme catalysed the hydrolysis of thyroliberin and N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. It did not catalyse the hydrolysis of glycylproline-7-amido-4-methylcoumarin or His-Pro-NH2. 3. The post-proline dipeptidyl aminopeptidase was partially purified with a purification factor of 301-fold and a yield of 8.9%. The post-proline dipeptidyl aminopeptidase catalysed the hydrolysis of His-Pro-NH2 and glycylproline-7-amido-4-methylcoumarin but did not exhibit any post-proline cleaving endopeptidase activity against thyroliberin or N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. 4. Studies with various functional reagents indicated that the pyroglutamate aminopeptidase could be specifically inhibited by 2-iodoacetamide (100% inhibition at an inhibitor concentration of 5 microM), the post-proline cleaving enzyme by bacitracin (IC50 = 42 microM) and the post-proline dipeptidyl aminopeptidase by puromycin (IC50 = 46 microM). Because of their specific inhibitory effects these three reagents were key elements in the elucidation of the overall pathway for the metabolism of thyroliberin by guinea pig brain tissue enzymes.     

Comparative studies of thyrotropin releasing hormone diazomethyl ketone and chloromethyl ketone analogs

Diazomethyl ketone and chloromethyl ketone analogs of thyrotropin releasing hormones have been synthesized and studied for their inhibitory effects on thyrotropin releasing hormone-induced release of radioactive ¹²⁵I-labelled hormones from the thyroid gland of eight-week old male Long-Evans rats. When Long-Evans rats were pretreated with thyrotropin releasing hormone diazomethyl ketone (TRH-DMK) or the chloromethyl ketone derivative (TRH-CMK), a dose-related inhibition of thyrotropin releasing hormone-induced ¹²⁵I release was observed which could be partially reversed by thyrotropin stimulating hormone (TSH). The diazomethyl ketone was a more effective inhibitor than the chloromethyl ketone. These compounds may act as an active-site directed antagonists whose effects are unique to the hypothalamo-pituitary-thyroid system.     

N-alpha-carbobenzoxy pyroglutamyl diazomethyl ketone as active-site-directed inhibitor for pyroglutamyl peptidase

Pyroglutamyl-peptidase (L-pyroglutamyl-peptide hydrolase, EC 3.4.19.3) from Bacillus amyloliquefaciens was covalently labeled with a newly synthesized N-carbobenzoxy-L-pyroglutamyl diazomethyl ketone (Z-PGDK) and was completely inactivated. The inactivation reaction proceeded in pseudo-first order. The kinetic studies demonstrated a rate-limiting step in the inhibition reaction, resulting in the formation of a reversible (enzyme.reagent) complex. The calculated KI,app is 0.12 mM at pH 7.58. The rate of inactivation was pH dependent with an extrapolated pK value of approx. 8.6. The enzyme could be protected against inactivation by a poor substrate, pyroglutamyl-valine. The PCMB-inactivated enzyme, that could be reversibly reactivated by mercaptoethanol, failed to react with Z-PGDK. The enzyme was insensitive toward the D-isomer of Z-PGDK and other diazomethyl ketone derivatives of carbobenzoxy amino acids such as Z-L-proline and Z-L-phenylalanine. These results strongly suggest that the Z-PGDK reacts as an affinity label, presumably with a cysteine residue as the site of alkylation in pyroglutamyl-peptidase, as was reported for chloromethyl ketone derivatives of pyroglutamic acid and its N-carbobenzoxy derivative.     

Specificity of a serum peptidase hydrolyzing thyroliberin at pyroglutamyl-histidine bone

The substrate specificity of a serum enzyme which degrades thyroliberin (less than Glu-His-Pro-NH2) into pyroglutamic acid and His-Pro-NH2 has been investigated and compared with that of the pyroglutamyl aminopeptidase from calf liver. The latter enzyme has a broad specificity, causing rapid degradation of thyroliberin, pyroglutamyl beta-naphthylamide and luliberin. In contrast, the serum enzyme causes rapid stereospecific cleavage only of the pyroglutamyl-histidine bond of thyroliberin and closely related peptides. Compounds such as less than Glu-Ala, less than Glu-His and pyroglutamyl beta-naphthylamide, which are known substrates of the pyroglutamyl aminopeptidases (such as the liver enzyme), are not substrates of the serum enzyme, and inhibit it only poorly. Pyroglutamyl-containing peptides such as luliberin and neurotensin and thyroliberin analogues such as LLD-thyroliberin, less than Glu-His-Pro-NHCH3, less than Glu-His-Pro-Gly-NH2 and less than Glu-Phe-Pro-NH2 inhibit effectively the degradation of thyroliberin by the serum enzyme, but are not hydrolyzed by this enzyme. The high specificity of the serum enzyme implies a physiological function.     

Inactivation of cathepsin B1 by diazomethyl ketones

Benzyloxycarbonyl-phenylalanyl diazomethyl ketone and benzyloxy-carbonyl-phenylalanyl-phenylalanyl diazomethyl ketone, which have been shown to inactivate the thiol protease papain by a mechanism different from that of substrate chloromethyl ketone derivatives, have now been examined as inhibitors of cathepsin B₁ of beef spleen. The dipeptide derivative irreversibly inactivates this protease rapidly, apparently by affinity labeling.     

5-Oxoprolinal: transition-state aldehyde inhibitor of pyroglutamyl-peptide hydrolase

Pyroglutamyl-peptide hydrolase (EC 3.4.11.8) removes the N-terminal pyroglutamyl residue from pyroglutamyl-containing peptides such as thyrotropin-releasing hormone (TRH), luteinizing hormone-releasing hormone (LH-RH), neurotensin, and bombesin. The aldehyde analogue of pyroglutamate, 5-oxoprolinal, was synthesized as an active site directed transition-state inhibitor of the enzyme. 5-Oxoprolinal was found to be a potent (Ki = 26 nM) and specific competitive inhibitor of pyroglutamyl-peptide hydrolase. Other aldehydes tested inhibited the enzyme only weakly or not at all. 5-Oxoprolinal blocked the degradation of LH-RH by purified pyroglutamyl-peptide hydrolase. The inhibitor, when injected into mice, inhibited the enzyme after 10 and 30 min. 5-Oxoprolinal should be of value in studies probing the biological significance of pyroglutamyl-peptide hydrolase.     

Synthesis of histidine-containing dipeptide affinity-labelling agents. Relative inactivation rates of cathepsins B and L.

Peptidyl diazomethyl ketones and fluoromethyl ketones containing histidine in the C-terminal position were synthesized to determine their properties as proteinase inactivators. These were examined chiefly with derivatives of Z-Ala-His. The protection of histidine during conversion of the C-terminal residue to the diazomethyl ketone required unblocking conditions which avoid acid due to the lability of this function. This was achievable with a Cbz-imidazole derivative since aminolysis provided deblocking without disturbance of the diazomethyl ketone function. In the case of the fluoromethyl ketone synthesis using fluoroacetic anhydride (Dakin-West procedure), the desired product could be isolated without ring blocking. The Z-Ala-His products showed enhanced selectivity for inactivation of cathepsin B over L when compared to analogous dipeptide inhibitors.     

A comparison of the behavior of chymotrypsin and cathepsin B towards peptidyl diazomethyl ketones

Chymotrypsin is not inactivated by benzyloxycarbonyl-phenylalanyl diazomethyl ketone although disappearance of the diazo group can be followed spectroscopically. It is also inert to various dipeptide derivatives. Cathepsin B on the other hand is inactivated by this reagent, as described earlier as well as by other peptidyl diazomethyl ketones. It appears from initial studies that a phenylalanyl residue in the penultimate position of the inhibitor is favorable for effectiveness. Benzyloxycarbonyl-Phe-AlaCHN₂ emerges from this work as a powerful, relatively soluble inactivator of bovine spleen cathepsin B with K_i = 1.7 × 10^?6M.     

Specific inhibition of post proline cleaving enzyme by benzyloxycarbonyl-Gly-Pro-diazomethyl ketone

N-Benzyloxycarbonyl-Gly-Pro-diazomethyl ketone (Z-Gly-Pro-CHN2) was synthesized and tested as inhibitor of the post proline cleaving enzyme from bovine brain. The compound was found to inactivate the enzyme completely and irreversibly at low concentrations (0.3 microM) without affecting other proteolytic enzymes such as post proline dipeptidyl aminopeptidase, pyroglutamate aminopeptidase or trypsin. Substrates of post proline cleaving enzymes such as luliberin (LH-RH; pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) and Benzyloxycarbonyl-Gly-Pro-Ala protected the enzyme from the reaction with Z-Gly-Pro-CHN2. Thus, Z-Gly-Pro-CHN2 seems to be an active site directed, specific inhibitor of post proline cleaving enzyme. When administered intraperitoneally to rats, this inhibitor (8 mg/kg) completely inactivated the post proline cleaving enzyme in all tissues studied including brain. Therefore, Z-Gly-Pro-CHN2 should be a valuable tool for studies on the physiological function of this enzyme within the metabolism of neuropeptides.     

Evidence for tissue-specific forms of glutaminyl cyclase.

Glutaminyl cyclase (QC) is responsible for the presence of pyroglutamyl residues in many neuroendocrine peptides. An examination of the bovine tissue distribution of QC immunoreactivity, enzyme activity, and mRNA confirmed that QC was abundant in brain and pituitary by all three measures. However, enzymatic activity was considerably more widespread than either immunoreactivity or mRNA, suggesting multiple enzyme forms. Partially purified QC from bovine spleen differed significantly from the known bovine pituitary QC in physical and catalytic properties. We propose that this form of glutaminyl cyclase plays a role in the posttranslational processing of constitutively secreted pGlu-containing proteins.     

The selective inactivation of thiol proteasesin vitro andin vivo

Peptidyl diazomethyl ketone derivatives inactivate thiol proteases by affinity labeling. The peptide sequence permits the reagents to be targeted to individual proteases according to their specificity. Alkylation of the active center thiol group takes place. The reagents are not reactive to simple thiols such as mercaptoethanol and appear to inactivate thiol proteases by acting as suicide substrates. Other classes of proteases are not affected. Initial results indicate that peptidyl diazomethyl ketones can also be used to inactivate thiol proteases in vivo.     

The inhibition of macrophage protein turnover by a selective inhibitor of thiol proteinases.

1. A new inhibitor of thiol proteinases, benzyloxycarbonylphenylalanylalanine diazomethyl ketone (benzyloxycarbonylphenylalanylalanyldiazomethane, Z-Phe-Ala-CHN2) was added to cultured mouse peritoneal macrophages prelabelled with [14C]leucine. The degradation of protein was studied under conditions of basal proteolysis in the presence of 10% pig serum. After a lag of about 6 h a time- and dose-dependent inhibition of protein degradation was observed, up to a maximum of about 40%. 2. The inhibitor entered the cells with kinetics consistent with entry by pinocytosis, giving access to the lysosomal system. 3. Intracellular cathepsin B was almost completely inactivated after 90 min of exposure of the culture to 0.1 mm-inhibitor. 4. The inhibition of proteolysis and of cathepsin B was reversed virtually completely within 24 h, when the inhibitor was removed from the medium. Since the inhibitor forms a covalent bond with the enzyme, the recovery of cathepsin B activity presumably reflects production of new molecules of active enzyme. 5. The inhibitory effects of pepstatin, the carboxyl proteinase inhibitor, were under some circumstances additive with those Z-Phe-Ala-CHN2, and were also largely reversible. 6. It is concluded that thiol proteinases play a major role in lysosomal proteolysis in cultured macrophages.     

The properties of peptidyl diazoethanes and chloroethanes as protease inactivators

Earlier work has demonstrated the irreversible inactivation of serine and cysteine proteinases by peptides with a C-terminal chloromethyl ketone group. With a C-terminal diazomethyl ketone, on the other hand, peptides become reagents specific for cysteine proteinases. We have now synthesized and examined the properties of reagents with an additional methyl side chain near the reactive grouping with the goal of diminishing side reactions in a cellular environment. Derivatives of neutral amino acids as well as of lysine and arginine have been prepared. The chloroethyl ketones are about 60% less reactive to chemical nucleophiles than the chloromethyl ketones. However, the susceptibilities of the proteases examined varied remarkably. Cathepsins B and L of the papain family of cysteine proteinases were much less susceptible (about 2 orders of magnitude less) to both peptidyl diazoethyl and chloroethyl ketones. In marked contrast, clostripain, a cysteine proteinase of a separate family was decisively more susceptible to chloroethyl ketones. The serine proteinases showed a drop in susceptibility to the chloroethyl ketones generally, and this was similar to the drop in chemical reactivity in proceeding from the chloromethyl to the chloroethyl ketone.     

Neuropeptide-specific peptidases: does brain contain a specific TRH-degrading enzyme?

The particulate fraction of brain homogenates contains an enzyme that cleaves the pyroglutamyl-histidyl bond of thyrotropin-releasing hormone (TRH) but is clearly distinct from the more widely distributed pyroglutamyl peptidase (EC 3.4.19.3). This particulate enzyme is highly localized to brain where it is found on synaptosomal membranes. It exhibits an unusual degree of substrate specificity. For example, it does not cleave the pyroglutamyl-histidyl bond of luteinizing hormone-releasing hormone (LHRH) or the pyroglutamyl histidyl bond of the chromogenic substrate pyroglutamyl-histidyl-2-naphthylamide. Evidence is reviewed supporting the possibility that this enzyme, first detected in serum and originally referred to as "thyroliberinase", may be the first neuropeptide-specific peptidase to be characterized.     

Glutaminyl cyclases unfold glutamyl cyclase activity under mild acid conditions

N-terminal pyroglutamate (pGlu) formation from glutaminyl precursors is a posttranslational event in the processing of bioactive neuropeptides such as thyrotropin-releasing hormone and neurotensin during their maturation in the secretory pathway. The reaction is facilitated by glutaminyl cyclase (QC), an enzyme highly abundant in mammalian brain. Here, we describe for the first time that human and papaya QC also catalyze N-terminal glutamate cyclization. Surprisingly, the enzymatic Glu(1) conversion is favored at pH 6.0 while Gln(1) conversion occurs with an optimum at pH 8.0. This unexpected finding might be of importance for deciphering the events leading to deposition of highly toxic pyroglutamyl peptides in amyloidotic diseases.     

Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells

Mammalian cell lines were examined concerning their Glutaminyl Cyclase (QC) activity using a HPLC method. The enzyme activity was suppressed by a QC specific inhibitor in all homogenates. Aim of the study was to prove whether inhibition of QC modifies the posttranslational maturation of N-glutamine and N-glutamate peptide substrates. Therefore, the impact of QC-inhibition on amino-terminal pyroglutamate (pGlu) formation of the modified amyloid peptides Abeta(N3E-42) and Abeta(N3Q-42) was investigated. These amyloid-beta peptides were expressed as fusion proteins with either the pre-pro sequence of TRH, to be released by a prohormone convertase, or as engineered amyloid precursor protein for subsequent liberation of Abeta(N3Q-42) after beta- and gamma-secretase cleavage during posttranslational processing. Inhibition of QC leads in both expression systems to significantly reduced pGlu-formation of differently processed Abeta-peptides. This reveals the importance of QC-activity during cellular maturation of pGlu-containing peptides. Thus, QC-inhibition should impact bioactivity, stability or even toxicity of pyroglutamyl peptides preventing glutamine and glutamate cyclization.