Coupling cellular mitogenesis to apoptosis by designed biomolecules.
ABSTRACT Cellular signal transduction pathways transduce input signals to produce corresponding output effects, ensuring correct response to extracellular signals. Manipulation of components in signaling pathways will alter correlation of input signals to output effects. Here we report that by reconstructing the components in mitogenic and apoptotic signaling pathways, Ras, Raf, and caspase-3, we manipulated the cells to couple mitogenic signal input to apoptotic output. The reconstructed biomolecules that couple mitogenesis to apoptosis are designated as "mitogenesis coupled-apoptosis molecular device" (MCAMD). As mitogenesis in cancer cells is constitutively active, MCAMD may have potential applications for cancer gene therapy.
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ABSTRACT: Caspases are cysteine proteases that play a critical role in the initiation and regulation of apoptosis. These enzymes act in a cascade to promote cell death through proteolytic cleavage of intracellular proteins. Since activation of apoptosis is implicated in human diseases such as cancer and neurodegenerative disorders, caspases are targets for drugs designed to modulate their action. Active caspases are heterodimeric enzymes with two symmetrically arranged active sites at opposite ends of the molecule. A number of crystal structures of caspases with peptides or proteins bound at the active sites have defined the mechanism of action of these enzymes, but molecular information about the active sites before substrate engagement has been lacking. As part of a study of peptidyl inhibitors of caspase-3, we crystallized a complex where the inhibitor did not bind in the active site. Here we present the crystal structure of the unoccupied substrate-binding site of caspase-3. No large conformational differences were apparent when this site was compared with that in enzyme-inhibitor complexes. Instead, the 1.9 A structure reveals critical side chain movements in a hydrophobic pocket in the active site. Notably, the side chain of tyrosine204 is rotated by approximately 90 degrees so that the phenol group occupies the S2 subsite in the active site. Thus, binding of substrate or inhibitors is impeded unless rotation of this side chain opens the area. The positions of these side chains may have important implications for the directed design of inhibitors of caspase-3 or caspase-7.Journal of Molecular Recognition 01/2003; 16(3):121-4. · 3.01 Impact Factor