Tetramer formation by the caspase-activated fragment of the Par-4 tumor suppressor.

The prostate apoptosis response-4 (Par-4) tumor suppressor can selectively kill cancer cells via apoptosis while leaving healthy cells unharmed. Full length Par-4 has been shown to be predominantly intrinsically disordered in vitro under neutral conditions. As part of the apoptotic process, cellular Par-4 is cleaved at D131 by caspase-3, which generates a 24 kDa C-terminal activated fragment (cl-Par-4) that enters the nucleus and inhibits pro-survival genes, thereby preventing cancer cell proliferation. Here, the structure of cl-Par-4 was investigated using CD spectroscopy, dynamic light scattering, intrinsic tyrosine fluorescence, and size exclusion chromatography with mutli-angle light scattering. Biophysical characterization shows that cl-Par-4 aggregates and is disordered at low ionic strength. However, with increasing ionic strength, cl-Par-4 becomes progressively more helical and less aggregated, ultimately forming largely ordered tetramers at high NaCl concentration. These results, together with previous results showing induced folding at acidic pH, suggest that the in vivo structure and self-association state of cl-Par-4 may be strongly dependent upon cellular environment.

pH-Induced Folding of the Caspase-Cleaved Par-4 Tumor Suppressor: Evidence of Structure Outside of the Coiled Coil Domain.

Prostate apoptosis response-4 (Par-4) is a 38 kDa largely intrinsically disordered tumor suppressor protein that functions in cancer cell apoptosis. Par-4 down-regulation is often observed in cancer while up-regulation is characteristic of neurodegenerative conditions such as Alzheimer's disease. Cleavage of Par-4 by caspase-3 activates tumor suppression via formation of an approximately 25 kDa fragment (cl-Par-4) that enters the nucleus and inhibits Bcl-2 and NF-ƙB, which function in pro-survival pathways. Here, we have investigated the structure of cl-Par-4 using biophysical techniques including circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and intrinsic tyrosine fluorescence. The results demonstrate pH-dependent folding of cl-Par-4, with high disorder and aggregation at neutral pH, but a largely folded, non-aggregated conformation at acidic pH.