Caspase-dependant activation of chymotrypsin-like proteases mediates nuclear events during Jurkat T cell apoptosis

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Abstract

Apoptosis involves a cascade of biochemical and morphological changes resulting in the systematic disintegration of the cell. Caspases are central mediators of this process. Supporting and primary roles for serine proteases as pro-apoptotic mediators have also been highlighted. Evidence for such roles comes largely from the use of pharmacological inhibitors; as a consequence information regarding their apoptotic function and biochemical properties has been limited. Here, we circumvented limitations associated with traditional serine protease inhibitors through use of a fluorescently labelled inhibitor of serine proteases (FLISP) that allowed for analysis of the specificity, regulation and positioning of apoptotic serine proteases within a classical apoptotic cascade. We demonstrate that staurosporine triggers a caspase-dependant induction of chymotrypsin-like activity in the nucleus of apoptotic Jurkat T cells. We show that serine protease activity is required for the generation of late stage nuclear events including condensation, fragmentation and DNA degradation. Furthermore, we reveal caspase-dependant activation of two chymotrypsin-like protein species that we hypothesize mediate cell death-associated nuclear events.

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Methods and materials

Cell culture and treatments. Jurkat T cells were obtained from CATCC and maintained in RPMI 1640 medium (Sigma) supplemented with 10% heat-inactivated foetal calf serum, 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere of 5% CO2 in air at 37 °C. Cells were maintained in logarithmic growth phase by routine passage every 2–3 days. For induction of apoptosis, cells were seeded at a density of 106/ml in fresh cell culture medium and staurosporine was added to

Staurosporine-induced apoptosis in Jurkat T cells is accompanied by an increase in FFCK-binding proteins

Activation of non-caspase proteases has been implicated in a number of types of cell death including apoptosis, necrosis and autophagy [16], [39]. Therefore, in order to characterise the potential apoptotic roles of serine proteases, it is important to define the type of cell death in which they are active. In our initial studies we demonstrated that treatment of Jurkat T cells with 1 μM staurosporine (sts) for 2 h led to the development of features characteristic of apoptosis (Fig. 1A–C).

Acknowledgments

This work was funded by Science Foundation Ireland, Irish Council for Science and Engineering Technology, National Development Plan, Higher Education Authority, National University of Ireland, NUIG Millennium Research Fund, and Enterprise Ireland.

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