Species Specificity of Thimet Oligopeptidase (EC 3.4.24.15)

Hayashi, Mirian A. F.; Gomes, Marcelo D.; Rebouças, Nancy Amaral; Fernandes, Beatriz Lieblich; Ferro, Emer S.; Camargo, Antônio Carlos Martins de

doi:10.1515/bchm3.1996.377.5.283

Cited by 12 publications

(6 citation statements)

References 39 publications

(22 reference statements)

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“…This activity was named Endo-A (EC 3.4.22. 19) and has been the subject of protracted debate as to whether it is identical to or distinct from EP24.15 (83)(84)(85). The consensus in the field in recent years has been that the two activities are attributable to the same enzyme.…”

Section: Endooligopeptidase Amentioning

confidence: 99%

Soluble Metalloendopeptidases and Neuroendocrine Signaling

2002

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Peptidases play a vital and often highly specific role in the physiological and pathological generation and termination of peptide hormone signals. The thermolysin-like family of metalloendopeptidases involved in the extracellular processing of neuroendocrine and cardiovascular peptides are of particular significance, reflecting both their specificity for particular peptide substrates and their utility as therapeutic targets. Although the functions of the membrane-bound members of this family, such as angiotensin-converting enzyme and neutral endopeptidase, are well established, a role for the predominantly soluble family members in peptide metabolism is only just emerging. This review will focus on the biochemistry, cell biology, and physiology of the soluble metalloendopeptidases EC 3.4.24.15 (thimet oligopeptidase) and EC 3.4.24.16 (neurolysin), as well as presenting evidence that both peptidases play an important role in such diverse functions as reproduction, nociception, and cardiovascular homeostasis.

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Section: Endooligopeptidase Amentioning

confidence: 99%

Soluble Metalloendopeptidases and Neuroendocrine Signaling

2002

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“…Rat testes recombinant TOP. Purified rat testes recombinant TOP was obtained as described [8] [5], and confirmed the homogeneity of the enzyme preparation. The specific activity of the recombinant enzyme used in this work was 2 U´mg 21 (one unit of enzyme activity is the amount of enzyme which hydrolyses 1 mmol of qf7 in 1 min).…”

Section: Preparation Of Enzymesmentioning

confidence: 75%

“…Rat testes recombinant TOP. Purified rat testes recombinant TOP was obtained as described [8]. The yield was 2.5 mg of pure protein per L of bacterial culture.…”

mentioning

confidence: 99%

Free ATP inhibits thimet oligopeptidase (EC 3.4.24.15) activity, induces autophosphorylation in vitro, and controls oligopeptide degradation in macrophage

Portaro

Hayashi

Silva

et al. 2001

European Journal of Biochemistry

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The fate of the proteasome-generated peptides depends upon the cytosolic peptidases whose activities ought to be regulated. One of the most important oligopeptide-degrading and -binding proteins in the cytosol is the thimet oligopeptidase (EC 3.4.24.15), ubiquitously found in mammalian tissues. To date, there is no indication whether thimet oligopeptidase activities are physiologically regulated. Here, we present evidences suggesting that the concentration of unbound ATP in the cytosol regulates the thimet oligopeptidase activities both, in vitro and ex vivo. To perform these studies two oligopeptides were used: a quenched fluorescent peptide, which is susceptible to thimet oligopeptidase degradation, and the ovalbumin 2572264 (MHC class I ovalbumin epitope), which displays high affinity to the thimet oligopeptidase without being degraded. We also showed that the thimet oligopeptidase undergoes autophosphorylation by ATP, a modification that does not affect the peptidase activity. The autophosphorylation is abolished in the presence of the thimet oligopeptidase substrates, as well as by the effect of a site directed inhibitor of this enzyme, and by the substitution of Glu474 for Asp at the metallo-peptidase motif.Altogether, the results presented here suggest that Zn 21 at the active center of the thimet oligopeptidase is the target for the ATP binding, leading to the inhibition of the enzyme activity, and inducing autophosphorylation. These effects, which depend upon the concentration of the unbound ATP, may help to explain the fate of the proteasomal-generated oligopeptides in the cytosol.Keywords: thimet oligopeptidase; ATP; autophosphorylation; chaperone.In eucaryotic cells most of the cytosolic peptides generated by the proteasome undergoes complete degradation by different classes of peptidases [1]. This process of intracellular protein degradation is part of the homeostatic mechanism, essential for the structure and function of the cells (reviewed in [2]) However, few peptides, which accomplish important biological tasks, are prevented from degradation by poorly understood mechanisms. This is the case of the MHC class I peptide epitopes which are presented at the cell surface in complex with the MHC class I molecule [3]. Our previous investigation on the susceptibility of a broad spectrum of randomly chosen MHC class I peptide epitopes to the thiol-activated metallo-oligopeptidase (TOP) indicated that in vitro few of them were degraded while the majority was resistant. Similar results were obtained using crude macrophage cytosol. Surprisingly, the majority of the epitopes, which were resistant to TOP degradation, displayed high affinity to the enzyme, thus suggesting a double role for TOP as a peptide degrading and also as peptide-binding protein [4].TOP is ubiquitously found in the cytosol of mammalian tissues [5], suggesting that it should perform a general function in the intracellular oligopeptide metabolism. Among other possibilities, the hydrolytic and the nonhydrolytic peptide binding activity of T...

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“…Northern blot analysis confirmed its preferential expression in the rabbit and rodent CNS (Hayashi et al, 1996;, although this could not be confirmed for humans . Later, the presence of Ndel1 in the adult brain was confirmed by in situ hybridization studies, with higher expression in some regions, such as the hippocampus, cerebellum, and basal nucleus of Meynert (Hayashi et al, 2001).…”

Section: Expression Of Ndel1 In the Developing Cns And Es Cellsmentioning

confidence: 80%