Human thimet oligopeptidase

170

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.

“…1C). These data indicate that TOP prefers substrates shorter than 17 residues in length, in accord with prior observations using specific peptides as substrates (29,33).…”

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

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Section: Products Of Proteasome Degradation Of ␤-[ 14 C]casein-inmentioning

confidence: 99%

See 1 more Smart Citation

Pathway for Degradation of Peptides Generated by Proteasomes

Šarić¹,

Graef²,

Goldberg

2004

170

“…Of the new ep24.15 and ep24.16 substrates identified here, at least seven are hemoglobin fragments. Interestingly, ep24.15 is present in large amounts in human erythrocytes, where hemoglobin also occurs in large quantities (46). Short hemoglobin fragments have been shown to be generated directly by the proteolytic action of the proteasome (47,48).…”

Section: Resultsmentioning

confidence: 99%

Novel Natural Peptide Substrates for Endopeptidase 24.15, Neurolysin, and Angiotensin-converting Enzyme

Rioli

Gozzo²,

Heimann

et al. 2003

159

165

The hemoglobin ␣-chain fragment PVNFKFLSH, which we have named hemopressin, produced dose-dependent hypotension in anesthetized rats, starting at 0.001 g/kg. The hypotensive effect of the peptide was potentiated by enalapril only at the lowest peptide dose. These results suggest a role for hemopressin as a vasoactive substance in vivo. The identification of these putative intracellular substrates for ep24.15 and ep24.16 is an important step toward the elucidation of the role of these enzymes within cells. Endopeptidase EC 3.4.24.15 (ep24.15; also referred to as thimet oligopeptidase) and endopeptidase EC 3. 4.24.16 (ep24.16; also referred to as neurolysin) were initially detected in and purified from rat brain homogenates (1, 2). The cloned rat brain ep24.16 (3) showed 80% similarity and 63% identity with the previously cloned rat testis ep24.15 (4). Both peptidases share most of their natural substrates, including bradykinin, neurotensin, opioids, angiotensin I, and gonadotrophinreleasing hormone (5, 6

“…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)(2)(3)(4). It is widely distributed in mammalian tissues with the highest expression levels in the brain, pituitary gland, and testis (5)(6)(7)(8).…”

mentioning

confidence: 99%

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

Ray¹,

Hines²,

Coll-Rodriguez

et al. 2004

108

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...