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ABSTRACT: Human β-tryptase is stored in secretory granules of human mast cells as a heparin-stabilized tetramer. β-Protryptase in solution can be directly processed to the mature enzyme by cathepsin (CTS) L and CTSB, and sequentially processed by autocatalysis at R(-3), followed by CTSC proteolysis. However, it is uncertain which CTS is involved in protryptase processing inside human mast cells, because murine bone marrow-derived mast cells from CTSC-deficient mice convert protryptase (pro-mouse mast cell protease-6) to mature mouse mast cell protease-6. This finding suggests that other proteases are important for processing human β-protryptase. In the current study, reduction of either CTSB or CTSL activity inside HMC-1 cells by short hairpin RNA silencing or CTS-specific pharmacologic inhibitors substantially reduced mature β-tryptase formation. Similar reductions of tryptase levels in primary skin-derived mast cells were observed with these pharmacologic inhibitors. In contrast, protryptase processing was minimally reduced by short hairpin RNA silencing of CTSC. A putative pharmacologic inhibitor of CTSC markedly reduced tryptase levels, suggesting an off-target effect. Skin mast cells contain substantially greater amounts of CTSL and CTSB than do HMC-1 cells, the opposite being found for CTSC. Both CTSL and CTSB colocalize to the secretory granule compartment of skin mast cells. Thus, CTSL and CTSB are central to the processing of protryptase(s) in human mast cells and are potential targets for attenuating production of mature tryptase in vivo.
The Journal of Immunology 08/2011; 187(4):1912-8. · 5.79 Impact Factor
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ABSTRACT: Human α- and β-protryptase zymogens are abundantly and selectively produced by mast cells, but the mechanism(s) by which they are processed is uncertain. β-Protryptase is sequentially processed in vitro by autocatalysis at R(-3) followed by cathepsin (CTS) C proteolysis to the mature enzyme. However, mast cells from CTSC-deficient mice successfully convert protryptase (pro-murine mast cell protease-6) to mature murine mast cell protease-6. α-Protryptase processing cannot occur by trypsin-like enzymes due to an R(-3)Q substitution. Thus, biological mechanisms for processing these zymogens are uncertain. β-Tryptase processing activity(ies) distinct from CTSC were partially purified from human HMC-1 cells and identified by mass spectroscopy to include CTSB and CTSL. Importantly, CTSB and CTSL also directly process α-protryptase (Q(-3)) and mutated β-protryptase (R(-3)Q) as well as wild-type β-protryptase to maturity, indicating no need for autocatalysis, unlike the CTSC pathway. Heparin promoted tryptase tetramer formation and protected tryptase from degradation by CTSB and CTSL. Thus, CTSL and CTSB are capable of directly processing both α- and β-protryptases from human mast cells to their mature enzymatically active products.
The Journal of Immunology 06/2011; 186(12):7136-43. · 5.79 Impact Factor
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ABSTRACT: A novel, sensitive method for detecting protease inhibitors by using fluorescent protease substrates in gels is described. The protease inhibitors were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels containing a copolymerized peptide substrate, namely 4-methyl-coumaryl-7-amide (MCA). As the incorporated substrates in the gel, Boc-Phe Ser-Arg-MCA was used for trypsin, Suc-Ala-Ala-Pro-Phe-MCA for alpha-chymotrypsin, and Z-Phe-Arg-MCA for papain. After electrophoresis, washing and incubating the gel with the target protease solutions allowed the substrate to be cleaved by the protease, and the release of the fluorescent 7 amino-4 methyl-coumarin (AMC), which was detected under a UV transilluminator. The uncleaved peptide-MCA substrate remained where the inhibitors were present, and was visualized as dark blue bands on the light-green fluorescent background gel. This new method offers several advantages over other previous methods including: (i) greatly increased sensitivity can be achieved in a shorter period of time, which may be useful for discovering new protease inhibitors in small amounts of crude material; (ii) the procedure is quite simple and quick since the incubation period is very short and no time is needed for staining and destaining steps; (iii) since these probes using substrate specificity/target proteases, they are excellent tools for detection and discrimination of unknown protease inhibitors for various target proteases.
Electrophoresis 04/2005; 26(6):1038-45. · 3.30 Impact Factor
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ABSTRACT: We found a novel procaspase-3 activating cascade mediated by lysosomal enzyme. The activating enzyme of procaspase-3, named lysoapoptase having the molecular weight of 78kDa was determined to be a lactoferrin located in the lysosome. Recombinant lactoferrin accelerated the processing of procaspase-3 to form active caspase-3 in vitro. D-Galactosamine is a well-known inducer of hepatocyte apoptosis. The caspase-3 which plays a common central role in the final step of various apoptosis cascades, was dramatically increased in the cytoplasm by the d-galactosamine administration in vivo. When D-galactosamine was administrated as a death signal in vivo, the lysosomal lactoferrin was released into the cytoplasm and procaspase-3 located in the cytoplasm was processed to form active caspase-3. The cotreatment of epigallo-catechin gallate resulted in the complete protection of the hepatocyte apoptosis suppressing the increases of caspase-3 in the cytoplasm. The caspase-3 activity was also inhibited directly by the epigallo-catechin gallate. Therefore, all apoptosis cascades mediated by caspase-3 should be suppressed by epigallo-catechin gallate. The caspase-3 activity was not only directly inhibited by epigallo-catechin gallate in vitro, but the release of lactoferrin from the lysosomes into the cytoplasm was also suppressed by epigallo-catechin gallate treatment in vivo. Therefore, the apoptosis induction was suppressed at the two successive steps by cotreatment of epigallo-catechin gallate in vivo.
Advances in Enzyme Regulation 02/2004; 44:1-10.
