Fig 1: Scheme for the cleavage profiling of the proneuropeptide processing proteases cathepsin L, cathepsin V, PC1/3, ad PC2, by MSP-MS and analyses by fluorogenic peptide substrates. (a) Proneuropeptides undergo proteolytic processing at dibasic residue sites. Neuropeptides are generated from proneuropeptide precursors that require proteolytic processing at dibasic sites (K/R-K/R) to generate active neuropeptides. (b) Strategy for the cleavage profiling of cathepsin L, cathepsin V, PC1/3, and PC2 processing enzymes by MSP-MS and fluorogenic substrates. The cleavage profile properties of cathepsin L and cathepsin V cysteine proteases, combined with PC1/3 and PC2 serine proteases, were evaluated by global, unbiased multiplex substrate profiling by mass spectrometry (MSP-MS) and fluorogenic peptide-AMC substrates containing variant dibasic residue sequences. For MSP-MS, the 228 peptide library was incubated with each of the processing proteases (as described in the Methods section), and peptide cleavage products were subjected to nano-LC–MS/MS tandem mass spectrometry for identification and quantification. Peptide cleavage products were analyzed for the frequency of each of the different amino acid residues at positions P4–P4′ and at the cleaved P1↓P1′ cleavage site. Based on MSP-MS results, peptide-AMC substrates were designed to further assess the dibasic cleavage site preferences of these proteases.
Fig 2: PC1/3 and PC2 cleavage properties examined with variant dipeptide-AMC and tripeptide-AMC substrates containing dibasic residue sites. PC1/3 (panel a) and PC2 (panel b) were evaluated for the cleavage of dipeptide-AMC substrates containing the four dibasic variant cleavage sites KR, RK, KK, and RR and compared to the cleavage of tripeptide-AMC substrates containing the K-R with hydrophobic residues (Leu, Trp, Phe, Tyr, Val) or nonpolar residues (Gly, Ala) at the N-terminal side of the dibasic K-R site. After incubation of PC1/3 or PC2 with each of these substrates at 37 °C for 120 min, control buffer or the aminopeptidase cathepsin H was added and incubation at 37 °C continued for another 30 min (37 °C) to allow conversion of basic residue-extended AMC products to free AMC for fluorometric measurement. Comparison of fluorescence observed in the absence and presence of cathepsin H is illustrated and included evaluation of significant differences with p < 0.05 (Student’s t-test, n = 3).
Fig 3: Distinct dibasic cleavage properties of cathepsin L and cathepsin V cysteine proteases compared to PC1/3 and PC2 serine proteases involved in proneuropeptide processing. (a) Information is shown for the relative proteolytic activity by the MSP-MS analyses of peptide library substrates and relative proteolytic activity observed in peptide-AMC assays using standard substrates for cathepsin L and cathepsin V (Z-F-R-AMC), and PC1/3 and PC2 (pERTKR-AMC). (b) Locations of dibasic cleavage sites (#1, 2, and 3) for each of the proneuropeptide processing proteases cathepsin L, cathepsin V, PC1/3, and PC2 are indicated.
Fig 4: PC1/3 and PC2 cleavage profiling analyzed by MSP-MS. Volcano plots of PC1/3 (panel a) and PC2 (panel b) peptide cleavages from MSP-MS data show the log2 ratios of relative quantities of peptide products generated by PC1/3 and PC2 (60 min incubation at pH 5.5) compared to no enzyme activity controls, illustrated by −log10p values. Peptide products generated with at least a 5-fold change above controls and with p < 0.05 were analyzed for the frequencies of amino acid residues at the P4–P4′ positions of the P1–↓P1′ cleavage site.
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