Caspase Substrate

TABLE 35-1 Examples of caspase substrates Cytoskeletal and associated proteins Nuclear and DNA-associated proteins... 

A large number of caspase substrates have been identified, some of which have a direct relationship to survival of the cell. The following examples illustrate important cellular processes and substrates that are targeted for proteolysis during apoptosis Inactivation of proteins that protect from apoptosis ... 

Kamada, S., H. Kusano, H. Fujita, M. Ohtsu, R.C. Koya, N. Kuzumaki, and Y. Tsujimoto. 1998. A cloning method for caspase substrates that uses the yeast two-hybrid system cloning of the antiapoptotic gene gelsolin. Proc Natl Acad Sci USA. 95 8532—7. 

Some caspase substrates, such as catenins, cadherins, plakoglobin, and des-moglein, maintain cell adhesion and cell-cell communications in adherens and gap junctions, or in desmosomes. Cleavage of these proteins causes loss of contact between cells [78, 79]. 

Both techniques can be applied to most experimental models of apoptosis, including cells in culture and biopsies. In addition, techniques based on the cleavage of synthetic caspase substrates can be applied to caspases, activated in vitro and in vivo [82, 83], Whereas Western blotting is time consuming and mainly a qualitative assay, the detection of caspase activity by cleavage of synthetic substrates is a quantitative, relatively fast and sensitive method [82]. However, both techniques do not provide information about the type and distribution of the cells with activated caspases in the tissue examined. 

Fischer U, Janicke RU, Schulze-Osthoff K. Many cuts to ruin A comprehensive update of caspase substrates. Cell Death Differ 2003 10 76-100. 

Figure 2 Small-molecule inhibitors of caspases. (a) Schematic of a typical caspase inhibitor (b) caspases substrate specificities preferred amino acids in PI -P4 positions (c) chemical structure of PAC-1, procaspase-3 activator (EC50 of 0.22 uM) (d) low-nanomolar inhibitors of caspases-3 and -7 (compound 1) and caspases-3, -7 and -9 (compound 2) (e) caspase-1 peptidomimetic inhibitor VX-740 or pralnacasan from Vertex Pharmaceutical (Cambridge, MA) (f) peptidomimetic irreversible oxamyl dipeptide pan-caspase inhibitor IDN-6556 from IDUN Pharmaceuticals and (g) caspase-3 inhibitor.

Most caspase-inhibitor design strategies target their active sites and are based on caspase substrate preferences. Tetrapep-tide inhibitors, which are based on identihed sequences of the four amino acid recognition motifs, were shown to inhibit caspase family members selectively (24) (Fig. 2a). Overall, a typical peptide-based caspase inhibitor consists of three major structural components ... 

Stroh, C. and K. Schulze-Osthoff. Death by a thousand cuts an ever increasing list of caspase substrates. Cell Death Differ. 5 997-1000, 1998. 

XIAP BIR1-3 domains recognize and bind caspase substrate for ubiquitination... 

Assay of cleavage of known intracellular caspases substrates (i.e., Poly ADP Ribose Polymerase)... 

Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates. J. Exp. Med. 191(11), 1819-1828. 

The aforementioned results demonstrate that in Jurkat cells treated with STS, the natural caspase substrate PARP is cleaved, DEVD-afc cleavage activity is activated, caspase-7 is proteolytically processed, and bzVKD-... 

Caspase activity in the cell lysate was determined by a fluorogenic assay. 50 pg of total protein was pre-incubated in buffer containing 25 mM HEPES pH7.5, 5 mM MgClz, 5 mM EDTA, 0.1 M NaCl and 5 mM DTT at 30 C for 30 min. Then 25 pM of various caspase substrates, such as Ac-YVAD-AMC for caspase-... 

Mack, A. Furmann, C. Hacker, G. Detection of caspase-activation in intact lymphoid ceUs using standard caspase substrates and inhibitors. J. Immunol. Methods 2000,241,19-31. 

Zhang, H. Kasibhatla, S. Guastella, J. Tseng, B. Drewe, L Cai, S. X. N-Ac-DEVD-N -(Polyfluorobenzoyl)-Rl 10 Novel cell-permeable fluorogenic caspase substrates for the detection of caspase activity and apoptosis. Bioconjugate Chem. 2003,14,458 63. 

Tawa, R, Tam, J., Cassady, R., Nicholson, D.W., and Xanthoudakis, S., Quantitative analysis of fluorescent caspase substrate cleavage in intact cells and identification of novel inhibitors of apoptosis, Cell Death Differ., 8, 1, 30, 2001. 

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