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.

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.

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Intrinsically disordered proteins play important roles in cell signaling, and dysregulation of these proteins is associated with several diseases. Prostate apoptosis response-4 (Par-4), an approximately 40 kilodalton proapoptotic tumor suppressor, is a predominantly intrinsically disordered protein whose downregulation has been observed in various cancers. The caspase-cleaved fragment of Par-4 (cl-Par-4) is active and plays a role in tumor suppression by inhibiting cell survival pathways. Here, we employed site-directed mutagenesis to create a cl-Par-4 point mutant (D313K). The expressed and purified D313K protein was characterized using biophysical techniques, and the results were compared to that of the wild-type (WT). We have previously demonstrated that WT cl-Par-4 attains a stable, compact, and helical conformation in the presence of a high level of salt at physiological pH. Here, we show that the D313K protein attains a similar conformation as the WT in the presence of salt, but at an approximately two times lower salt concentration. This establishes that the substitution of a basic residue for an acidic residue at position 313 alleviates inter-helical charge repulsion between dimer partners and helps to stabilize the structural conformation.

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