Fluorogenic peptide substrates for carboxydipeptidase activity of cathepsin B.

Abstract

Cathepsin B is a lysosomal cysteine protease exhibiting mainly dipeptidyl carboxypeptidase activity, which decreases dramatically above pH 5.5, when the enzyme starts acting as an endopeptidase. Since the common cathepsin B assays are performed at pH 6 and do not distinguish between these activities, we synthesized a series of peptide substrates specifically designed for the carboxydipeptidase activity of cathepsin B. The amino-acid sequences of the P(5)-P(1) part of these substrates were based on the binding fragments of cystatin C and cystatin SA, the natural reversible inhibitors of papain-like cysteine protease. The sequences of the P'(1)-P'(2) dipeptide fragments of the substrates were chosen on the basis of the specificity of the S'(1)-S'(2) sites of the cathepsin B catalytic cleft. The rates of hydrolysis by cathepsin B and papain, the archetypal cysteine protease, were monitored by a continuous fluorescence assay based on internal resonance energy transfer from an Edans to a Dabcyl group. The fluorescence energy donor and acceptor were attached to the C- and the N-terminal amino-acid residues, respectively. The kinetics of hydrolysis followed the Michaelis-Menten model. Out of all the examined peptides Dabcyl-R-L-V-G-F- E(Edans) turned out to be a very good substrate for both papain and cathepsin B at both pH 6 and pH 5. The replacement of Glu by Asp turned this peptide into an exclusive substrate for cathepsin B not hydrolyzed by papain. The substitution of Phe by Nal in the original substrate caused an increase of the specificity constant for cathepsin B at pH 5, and a significant decrease at pH 6. The results of kinetic studies also suggest that Arg in position P(4) is not important for the exopeptidase activity of cathepsin B, and that introducing Glu in place of Val in position P(2) causes an increase of the substrate preference towards this activity.

OaAEP1 Ligase-Assisted Chemoenzymatic Synthesis of Full Cysteine-Rich Metal-Binding Cyanobacterial Metallothionein SmtA

“…Since the activation of this plant enzyme is not trivial, we examined its activity after this process using the FRET Dabcyl-YAKGNGL-Edans substrate. The nonfluorescent peptide turns into the fluorescent GL-Edans (λ ex = 340 nm, λ em = 490 nm) product only when the enzyme is active (Figure A,B) . Because this test is based on substrate hydrolysis, ligase activity has also been examined using model peptides containing recognition termini, YKLANGL and GVGKY-NH 2 .…”

Enzyme-Cleavable Polymeric Micelles for the Intracellular Delivery of Proapoptotic Peptides

“…Cathepsin B, a ubiquitous cysteine protease, is a demonstrated candidate for this application. Cathepsin B is primarily localized in endo/lysosomes, its cleavage substrates have been well-characterized, − and it is upregulated in a wide variety of cancers . While cathepsin B can be secreted in a variety of cancer cells, it has previously been employed for the selective intracellular degradation of a polymer conjugate of the angiogenesis inhibitor TNP-470 , and a nucleic acid delivery vehicle .…”

“…To address both the stability and conjugation limitations of the disulfide linkage, a synthetic strategy has been employed that creates a selectively cleavable BIM peptide macromonomer (Figure ). The macromonomer contains the BIM peptide sequence capped with the well-characterized cathepsin B substrate Phe-Lys-Phe-Leu (FKFL). − This substrate is flanked on either side by a six carbon spacer (6-aminohexanoic acid (Ahx)) to facilitate steric access of the cathepsin enzyme in the context of the surrounding PEG grafts. The peptide is functionalized on its N-terminus with methacrylamide and directly and stably polymerized into a multifunctional diblock copolymer.…”

Acidosis and proteolysis in the tumor microenvironment

“…In their studies, they found increases in expression and activity of cathepsin B in invadosomes and they propose that cathepsin B participates in invasion of the colon cancer cells by activating zymogens of MMPs. The pH optima for the two proteolytic activities of cathepsin B are acidic with that for the carboxydipeptidase activity more acidic than that for the endopeptidase activity [53]. The elegant studies by Busco et al [54] show that the peri-invadopodial space of breast cancer cells is acidified by NHE1, a Na+/H+ exchanger isoform, which would be consistent with a local environment that enhances cathepsin B activity.…”