The inactivation of the cysteinyl exopeptidases cathepsin H and C by affinity-labelling reagents

Abstract

An attempt has been made to extend to the cysteinyl exopeptidases cathepsins H and C affinity-labelling approaches shown to be effective with cysteinyl endopeptidases such as cathepsins B and L and the calcium-activated proteinase. This involved the preparation of amino acid and dipeptide derivatives with unblocked N-termini to satisfy the aminopeptidase and dipeptidyl aminopeptidase characteristics of cathepsins H and C respectively. For covalent reactivity, the possibilities examined included diazomethanes (-CHN2), fluoromethanes (-CH2F) and dimethylsulphonium salt [-CH2S+(CH3)2]. A dipeptidylfluoromethane with a free amino group could not be prepared, perhaps due to inherent instability. Cathepsin H was inactivated by 1 microM-H2N-Phe-CH2F (the 'H2N' indicates a free unblocked amino group) (k2 = 1878 M-1.s-1); this reagent was without effect on cathepsins C and B, even at 100-fold this concentration. Analogous selectivity was shown by H2N-Ser(OBzl)-CHN2 and H2N-Phe-CH2S+(CH3)2, members of other classes of covalently binding reagents. For cathepsin C the dipeptide derivatives H2N-Gly-Phe-CHN2 and H2N-Phe-Ala-CH2S+(CH3)2 caused rapid inactivation near 10(-7) M. Higher concentrations inactivated cathepsins H and B, but the rates were slower by two to three orders of magnitude than for cathepsin C.

Cysteinyl Proteinases and Their Selective Inactivation

“…An interesting observation was the fact that H2N-LeuCH2CI was more inhibitory to cathepsin H than to cathepsin L, in since cathepsin H has both exo-and endopeptidase properties (54, 55, 94). Other approaches to exopeptidase affinity labeling have been developed (95) and will be described later. This group of reagents can be used to provide radiolabeled derivatives by the inclusion of tyrosine either as a substitution for phenylalanine to promote binding in S2 or elsewhere in the peptide sequence, since conditions have been found for iodination without destruction of the chloromethyl ketone function (96).…”

“…H2NSer(O-Bzl)CHN2 readily inactivates cathepsin H with a kz = 2600 M -' s -l observed with a 0.5 p,M reagent at pH 6.8. This reagent at lop4 M has no effect on cathepsin C (pH 6) when incubated for 40-min; however, it is rapidly inactivated by HzNGly-PheCHNz in accordance with its specificity, with k2 = 6700 M -I s -' (95).…”

Cathepsin H Mediates the Processing of Talin and Regulates Migration of Prostate Cancer Cells

“…HepG2 cells were grown in DMEM supplemented with 10% FBS, 2 mM glutamine, and antibiotics. To inhibit the intracellular activity of CtsH, PC-3 cells were incubated for 24 h with the CtsH-specific inhibitor (CTSHi) H 2 N-Ser(benzyloxy)-CHN 2 (25), synthesized at the Faculty of Pharmacy of the University of Ljubljana (26). To knock down CtsH expression, cells were transiently transfected with CtsHspecific siRNA (HSS102489, Invitrogen) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.…”

Cathespin H Is an Fgf10 Target Involved in Bmp4 Degradation during Lung Branching Morphogenesis

“…Presently, there is only one inhibitor proven to be selective for Ctsh. H 2 N-Ser(O-Bzl)-CHN 2 (referred to hereafter as Ctshi) is a strong and irreversible inhibitor of Ctsh, which shows little or no activity toward two other lysosomal cysteine proteases with exopeptidase activity, Ctsb and Ctsc (23). In addition, Ctshi is a diazomethane derivative that penetrates easily across cell membranes and, thus, is able to block enzyme activity both intracellularly and extracellularly (see below) (37).…”