[32] Thimet oligopeptidase and oligopeptidase M or neurolysin

Barrett, Alan J.; Ma, Ben; Pm, Dando; Cg, Knight; McKie, Norman; Rawlings, Neil D.; Serizawa, Atsushi

doi:10.1016/0076-6879(95)48034-x

Cited by 98 publications

(104 citation statements)

References 60 publications

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“…Neurolysin (EC 3.4.24.16) is a ubiquitous enzyme very similar to TOP in structure, tissue distribution, specificity, and inhibitory profile (48, 56 -58), and is present in the cytosol, nucleus, and mitochondria (59). However, neurolysin has about a 100-fold lower affinity than TOP for the inhibitor Cpp-AAF-pAb (28), and the concentration that reduced degradation of proteasome products by 50% (10 M) does not markedly inhibit neurolysin. In addition, DTT (0.5 mM) did not affect the degradation of proteasome products in HeLa extracts, 2 though it inhibits neurolysin but not TOP (60).…”

Section: Discussionmentioning

confidence: 99%

“…These findings also indicate that the smaller peptides degraded in the absence of TOP are hydrolyzed by other peptidases in the cytosol. At the concentration used, Cpp-AAF-pAB can also partially inhibit neurolysin (28). However, neurolysin has a 100-fold lower affinity than TOP for this inhibitor, and only small amounts of this enzyme could be demonstrated in HeLa extracts, as assessed by immunoblotting and immunodepletion (see "Experimental Procedures").…”

Section: Degradation Of 9 -17 Residue Peptides In Hela Extractsmentioning

confidence: 99%

“…In addition to TPP-II, three other cytosolic endopeptidases have been proposed to function in this process: the insulindegrading enzyme, prolyl oligopeptidase, and neurolysin (23,27,28). However, no detailed studies of their roles have been undertaken.…”

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confidence: 99%

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Pathway for Degradation of Peptides Generated by Proteasomes

Šarić¹,

Graef²,

Goldberg

2004

Journal of Biological Chemistry

168

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The degradation of cellular proteins by proteasomes generates peptides 2-24 residues long, which are hydrolyzed rapidly to amino acids. To define the final steps in this pathway and the responsible peptidases, we fractionated by size the peptides generated by proteasomes from ␤-[ 14 C]casein and studied in HeLa cell extracts the degradation of the 9 -17 residue fraction and also of synthetic deca-and dodecapeptide libraries, because peptides of this size serve as precursors to MHC class I antigenic peptides. Their hydrolysis was followed by measuring the generation of smaller peptides or of new amino groups using fluorescamine. The 14 C-labeled peptides released by 20 S proteasomes could not be degraded further by proteasomes. However, their degradation in the extracts and that of the peptide libraries was completely blocked by o-phenanthroline and thus required metallopeptidases. One such endopeptidase, thimet oligopeptidase (TOP), which was recently shown to degrade many antigenic precursors in the cytosol, was found to play a major role in degrading proteasome products. Inhibition or immunodepletion of TOP decreased their degradation and that of the peptide libraries by 30 -50%. Pure TOP failed to degrade proteasome products 18 -24 residues long but degraded the 9 -17 residue fraction to peptides of 6 -9 residues. When aminopeptidases in the cell extract were inhibited with bestatin, the 9 -17 residue proteasome products were also converted to peptides of 6 -9 residues, instead of smaller products. Accordingly, the cytosolic aminopeptidase, leucine aminopeptidase, could not degrade the 9 -17 residue fraction but hydrolyzed the peptides generated by TOP to smaller products, recapitulating the process in cell extracts. Inactivation of both TOP and aminopeptidases blocked the degradation of proteasome products and peptide libraries nearly completely. Thus, degradation of most 9 -17 residue proteasome products is initiated by endoproteolytic cleavages, primarily by TOP, and the resulting 6 -9 residue fragments are further digested to amino acids by aminopeptidases.

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Section: Discussionmentioning

confidence: 99%

Section: Degradation Of 9 -17 Residue Peptides In Hela Extractsmentioning

confidence: 99%

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Pathway for Degradation of Peptides Generated by Proteasomes

Šarić¹,

Graef²,

Goldberg

2004

Journal of Biological Chemistry

168

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“…The member of this family most closely related to TOP, the peptidase neurolysin, has more than 60% sequence identity and a similar tissue distribution (1,40,41). Both TOP and neurolysin hydrolyze only short peptides, generally less than 20 residues in length, and they recognize cleavage sequences that vary widely, with no consistent preferences at any position relative to the site of hydrolysis (1,19,20,40,42). TOP and neurolysin cleave most bioactive or synthetic peptides at the same peptide bond, as might be expected from their high level of sequence similarity.…”

mentioning

confidence: 99%

“…TOP and neurolysin cleave most bioactive or synthetic peptides at the same peptide bond, as might be expected from their high level of sequence similarity. Interestingly though, the two peptidases hydrolyze some peptides at different sites (1,19,20,40), and systematic studies of sequence preferences show considerable differences at positions close to the cleavage site (43-48). For example, although both enzymes play an important role in metabolizing the 13-residue neuropeptide neurotensin, TOP cleaves between Arg-8 and Arg-9, whereas neurolysin cleaves between Pro-10 and Tyr-11.…”

mentioning

confidence: 99%

Crystal Structure of Human Thimet Oligopeptidase Provides Insight into Substrate Recognition, Regulation, and Localization

Ray¹,

Hines²,

Coll-Rodriguez

et al. 2004

Journal of Biological Chemistry

108

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Thimet oligopeptidase (TOP) is a zinc metallopeptidase that metabolizes a number of bioactive peptides and degrades peptides released by the proteasome, limiting antigenic presentation by MHC class I molecules. We present the crystal structure of human TOP at 2.0-Å resolution. The active site is located at the base of a deep channel that runs the length of the elongated molecule, an overall fold first seen in the closely related metallopeptidase neurolysin. Comparison of the two related structures indicates hinge-like flexibility and identifies elements near one end of the channel that adopt different conformations. Relatively few of the sequence differences between TOP and neurolysin map to the proposed substrate-binding site, and four of these variable residues may account for differences in substrate specificity. In addition, a loop segment (residues 599 -611) in TOP differs in conformation and degree of order from the corresponding neurolysin loop, suggesting it may also play a role in activity differences. Cysteines thought to mediate covalent oligomerization of rat TOP, which can inactivate the enzyme, are found to be surface-accessible in the human enzyme, and additional cysteines (residues 321,350, and 644) may also mediate multimerization in the human homolog. Disorder in the N terminus of TOP indicates it may be involved in subcellular localization, but a potential nuclear import element is found to be part of a helix and, therefore, unlikely to be involved in transport. A large acidic patch on the surface could potentially mediate a protein-protein interaction, possibly through formation of a covalent linkage.Thimet oligopeptidase (TOP, 1 3.4.24.15) is a 77-kDa zinc metalloendopeptidase that bears the His-Glu-Xaa-Xaa-His (HEXXH) active site sequence motif characteristic of a large superfamily of metallopeptidases (1-4). It is widely distributed in mammalian tissues with the highest expression levels in the brain, pituitary gland, and testis (5-8). TOP is present in different subcellular locations depending on cell type, with reports of secreted and cytosolic forms (5-16), membrane association (5-7, 17, 18), and nuclear localization (7,12,14). Consistent with its broad tissue and subcellular compartment distribution, TOP appears to play a variety of physiological roles. It has been implicated in the metabolism of a number of small peptides active in the central nervous system and the periphery including neurotensin, bradykinin, somatostatin, opioids, and angiotensin I (4, 6, 9, 16, 19 -26). In addition, recent reports demonstrate that TOP is primarily responsible for degrading peptides released from proteasomes, thereby limiting the extent of antigen presentation by MHC class I molecules (27-29). TOP has also been linked to amyloid precursor protein processing (30), and it promotes increased degradation of the A␤ peptide, a key component of amyloid plaques in Alzheimer's disease (31). Expression of TOP activity is regulated at the level of transcription (32-34), but activity may also be regulated by po...

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Characterization and localization of mitochondrial oligopeptidase (MOP) (EC 3.4.24.16) activity in the human cervical adenocarcinoma cell line HeLa

et al. 1997

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In this study we describe the partial purification and characterization of the HeLa cell oligopeptidase M or endopeptidase 3.4.24.16. The HeLa enzyme was isolated initially by its ability to hydrolyse a nonapeptide substrate (P9) which was cognate to the N-terminal cleavage site of preproTGF alpha. The enzyme was shown to be a metalloprotease as it was inhibited by Zn(2+)-chelating agents and DTT, and had an approximate molecular weight of 55-63 kD determined by gel filtration. Neurotensin, dynorphin A1-17 and GnRH1-9 were rapidly degraded by the enzyme while GnRH1-10 and somatostatin were not. Neurotensin was cleaved at the Pro10-Tyr11 bond, leading to the formation of neurotensin (1-10) and neurotensin (11-13). The K(m) for neurotensin cleavage was 7 microM and the Ki for the specific 24.16 dipeptide inhibitor (Pro-ile) was 140 microM which were similar to those observed from the human brain enzyme [Vincent et al. (1996): Brain Res 709:51-58]. Through the use of specific antibodies, the purified HeLa enzyme was shown to be oligopeptidase M. This enzyme and its closely related family member thimet oligopeptidase were shown to co-elute during the isolation procedure but were finally separated using a MonoQ column. Oligopeptidase M is located mainly in mitochondria though it was detected on the plasma membrane in an inactive form. The results obtained demonstrate the first recorded instance of this enzyme in human tissue cultured cells, and raise the issue of its function therein.

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[32] Thimet oligopeptidase and oligopeptidase M or neurolysin

Cited by 98 publications

References 60 publications

Pathway for Degradation of Peptides Generated by Proteasomes

Pathway for Degradation of Peptides Generated by Proteasomes

Crystal Structure of Human Thimet Oligopeptidase Provides Insight into Substrate Recognition, Regulation, and Localization

Characterization and localization of mitochondrial oligopeptidase (MOP) (EC 3.4.24.16) activity in the human cervical adenocarcinoma cell line HeLa

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