Cathepsin B Dipeptidyl Carboxypeptidase and Endopeptidase Activities Demonstrated across a Broad pH Range

Abstract

Cathepsin B is a lysosomal protease that participates in protein degradation. However, cathepsin B is also active under neutral pH conditions of the cytosol, nuclei, and extracellular locations. The dipeptidyl carboxypeptidase (DPCP) activity of cathepsin B, assayed with the Abz-GIVR↓AK(Dnp)-OH substrate, has been reported to display an acidic pH optimum. In contrast, the endopeptidase activity, monitored with Z-RR-↓AMC, has a neutral pH optimum. These observations raise the question of whether other substrates can demonstrate cathepsin B DPCP activity at neutral pH and endopeptidase activity at acidic pH. To address this question, global cleavage profiling of cathepsin B with a diverse peptide library was conducted under acidic and neutral pH conditions. Results revealed that cathepsin B has (1) major DPCP activity and modest endopeptidase activity under both acidic and neutral pH conditions and (2) distinct pH-dependent amino acid preferences adjacent to cleavage sites for both DPCP and endopeptidase activities. The pH-dependent cleavage preferences were utilized to design a new Abz-G n VR↓AK(Dnp)-OH DPCP substrate, with norleucine (n) at the P3 position, having improved DPCP activity of cathepsin B at neutral pH compared to the original Abz-GIVR↓AK(Dnp)-OH substrate. The new Z-VR-AMC and Z-ER-AMC substrates displayed improved endopeptidase activity at acidic pH compared to the original Z-RR-AMC. These findings illustrate the new concept that cathepsin B possesses DPCP and endopeptidase activities at both acidic and neutral pH values. These results advance understanding of the pH-dependent cleavage properties of the dual DPCP and endopeptidase activities of cathepsin B that function under different cellular pH conditions.

Atg4B and Cathepsin B-Triggered in Situ Luciferin Formation for Precise Cancer Autophagy Bioluminescence Imaging

“…With the 3σ method (3σ/ s ), the limit of detection (LOD) of TFGC toward Atg4B was calculated to be 28.7 ng/mL (Table S1). We then investigated the kinetic parameters (i.e., turnover number k cat , Michaelis constant K m ) of TFGC toward Atg4B according to the reference . The k cat / K m value of TFGC was calculated to be 0.032 μM –1 s –1 , which was comparable to those of other reported Atg4B-catalyzed reactions (Figure S18, Table S3).…”

“…We then investigated the kinetic parameters (i.e., turnover number k cat , Michaelis constant K m ) of TFGC toward Atg4B according to the reference. 28 The k cat /K m value of TFGC was calculated to be 0.032 μM −1 s −1 , which was comparable to those of other reported Atg4B-catalyzed reactions (Figure S18, Table S3). Afterward, we tested the selectivity of TFGC toward Atg4B against other common substances in the model cell line MDA-MB-231 in this study, including cations (e.g., K + , Ca 2+ , Mg 2+ ), biomolecules (e.g., L-cysteine (L-Cys), glutathione (GSH)), and enzymes (e.g., alkaline phosphatase (ALP), carboxylesterase (CES), caspase 3 (Casp 3), and CTSB).…”

Cellular depletion of major cathepsin proteases reveals their concerted activities for lysosomal proteolysis

“…In SH-SY5Y cells, the CTSs with the highest expression are CTSA , CTSB , CTSC , CTSD , and CTSL . The endopeptidases CTSB, CTSD, and CTSL (SH-SY5Y), and in addition, CTSZ (HeLa) cleave their substrates within their peptide backbone, resulting in smaller peptides [ 12 , 30 , 31 ], initiating lysosomal proteolysis [ 32 , 33 ]. Given their high expression, CTSB, CTSD, CTSL, and CTSZ were selected for further knockout experiments in SH-SY5Y and HeLa cells.…”

Ion Mobility–Based Enrichment-Free N-Terminomics Analysis Reveals Novel Legumain Substrates in Murine Spleen

“…S17 E ) may hint at aminopeptidase activity following legumain cleavage. Cathepsin B has recently been characterized as a dipeptidyl carboxypeptidase with a preference to cleave substrates bearing asparagine in the P2′ position ( 104 ). We hypothesize that secondary cleavage by cathepsin B may obscure detection of legumain substrates in the lysosome.…”