Caspases: their intracellular localization and translocation during apoptosis.

The activation of the caspase family of proteases has been detected in numerous cell systems and appears to function as a common pathway through which apoptotic mechanisms may operate. Caspases are synthesized as precursors (pro-caspases) and are converted into mature enzymes by apoptotic signals. The effects of caspases in apoptosis are accomplished by the cleavage of numerous proteins located in different intracellular compartments.

Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis.

The release of two mitochondrial proteins, cytochrome c and apoptosis-inducing factor (AIF), into the soluble cytoplasm of cells undergoing apoptosis is well established. Using spectrophotometric determination of enzyme activity, the accumulation of adenylate kinase (AK) activity in the cytosolic fraction of apoptotic cells has also been observed recently. However, three isozymes, AK1, AK2 and AK3, have been characterized in mammalian cells and shown to be localized in the cytosol, mitochondrial intermembrane space and mitochondrial matrix, respectively, and it is unknown which one of these isozymes accumulates in the cytosol during apoptosis.

Two different proteases are involved in the proteolysis of lamin during apoptosis.

To investigate the involvement of different proteases in the execution step of apoptosis and to determine their intracellular location, isolated rat thymocyte nuclei were incubated either in the presence of Ca2+ and Mg2+ or with cytosolic extract from Jurkat T lymphocytes treated with anti-Fas (APO-1, CD-95) antibody. Inhibitors of caspases, VADcmk and DEVDcho, were not effective in hindering Ca2+-induced apoptotic changes in isolated nuclei, but did prevent similar changes in nuclei treated with the cytosolic extract from apoptotic Jurkat cells. In contrast, the inhibitor of the Ca2+-regulated, nuclear scaffold-associated serine protease, AAPFcmk, was able to inhibit lamin B1 breakdown, as well as chromatin cleavage in nuclei incubated in the presence of Ca2+ and Mg2+, but only partially prevented the same changes induced with cytosolic extract.

The role of proteolysis in T cell apoptosis triggered by chelation of intracellular Zn2+.

Our previous work showed that chelation of intracellular Zn2+ with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) induces apoptosis in rat thymocytes. The molecular mechanism involved in TPEN-triggered apoptosis remains unknown, except that it is a Ca2+-independent process. In the present study, we show that TPEN is unable to induce DNA fragmentation when added to isolated thymocyte nuclei, indicating that activation of a cytoplasmic component is essential for TPEN-induced apoptosis.

Multiple proteases are involved in thymocyte apoptosis.

To investigate the involvement of proteases in apoptosis, rat thymocytes were treated with the glucocorticoid hormone methylprednisolone or the topoisomerase II inhibitor etoposide in the presence of selective substrate inhibitors of either interleukin-1 beta-converting enzyme (ICE), (Z-Val-Ala-Asp-chloromethylketone, VADcmk) or Ca(2+)-regulated serine protease (Suc-Ala-Ala-Pro-Phe-chloromethylketone, AAPFcmk). VADcmk protected from lamin proteolysis, chromatin fragmentation, cell shrinkage, and formation of apoptotic nuclei in both methylprednisolone- and etoposide-treated thymocytes when present during the initiation of the apoptotic process. AAPFcmk prevented lamin breakdown, chromatin fragmentation, and apoptotic morphological changes in thymocytes treated with methylprednisolone, but not with etoposide.