Functional characterization of the Mycobacterium tuberculosis zinc metallopeptidase Zmp1 and identification of potential substrates

Abstract

Zinc metallopeptidases of bacterial pathogens are widely distributed virulence factors and represent promising pharmacological targets. In this work, we have characterized Zmp1, a zinc metallopeptidase identifi ed as a virulence factor of Mycobacterium tuberculosis and belonging to the neprilysin (NEP; M13) family, whose X-ray structure has been recently solved. Interestingly, this enzyme shows an optimum activity toward a fl uorogenic substrate at moderately acidic pH values (i.e., 6.3), which corresponds to those reported for the Mtb phagosome where this enzyme should exert its pathological activity. Substrate specifi city of Zmp1 was investigated by screening a peptide library. Several sequences derived from biologically relevant proteins were identifi ed as possible substrates, including the neuropeptides bradykinin, neurotensin, and neuropeptide FF. Further, subsequences of other small bioactive peptides were found among most frequently cleaved sites, e.g., apelin-13 and substance P. We determined the specifi c cleavage site within neuropeptides by mass spectrometry, observing that hydrophobic amino acids, mainly phenylalanine and isoleucine, are overrepresented at position P1 ′ . In addition, the enzymatic mechanism of Zmp1 toward these neuropeptides has been characterized, displaying some differences with respect to the synthetic fl uorogenic substrate and indicating that the enzyme adapts its enzymatic action to different substrates.

The Endothelin System Has a Significant Role in the Pathogenesis and Progression of Mycobacterium tuberculosis Infection

“…Cleavage sites found in the ET-1 peptide (Fig. 3) were in accordance with the previously identified cleavage pattern for Zmp1 on other substrates (29), showing a preference for phenylalanine and isoleucine amino acid residues at the P1= position. In addition, the C-terminal as well as the N-terminal intramolecular loop structure is especially important for endothelin activity; thus, an enzymatic system might exist as a physiological mechanism for converting ET-1 to biologically inactive forms (36,37).…”

“…Zmp1 was previously described in M. tuberculosis (9,29) and in other bacteria, including Streptococcus parasanguis (30,31) and Porphyromonas gingivalis (32), as a protease with activity on small biologically active peptides, but no activation on endothelins was seen. In P. gingivalis, the protease showed activity against Big Endothelin (33), but the results were controversial, and ET-1 generation could not be confirmed by enzyme-linked immunosorbent assay (ELISA).…”

Metabolic relationship between diabetes and Alzheimer's Disease affected by Cyclo(His-Pro) plus zinc treatment

“…Others have shown that upregulation of Neprilysin (NEP) also reduces Aβ accumulation in the brain [81]. NEP, a zinc-dependent metalloprotease similar to IDE, cleaves and inactivates several peptide hormones including neurotensin, bradykinin, and neuropeptide FF [82]. IDE and NEP, both zinc enzymes, have comparable effects for clearing Aβ with similar catalytic action [83].…”

Characterization of the Prostate-Specific Antigen (PSA) Catalytic Mechanism: A Pre-Steady-State and Steady-State Study

“…The pH dependence of pre-steady-state and steady-state parameters was analyzed in the framework of the minimum reaction mechanism depicted in Figure 3 [21], [22], where two protonating residues are involved, according to Eqns. 7-12:…”