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

Abstract

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.

Keywords: cancer; circular dichroism; coiled coil (CC), leucine zipper (LZ), apoptosis; intrinsically disordered protein (IDP); prostate apoptosis response-4 (Par-4).

Figure 1

(a) Block diagram of caspase-3-cleaved Par-4 (cl-Par-4) domain structure. (b) DisEMBL disorder prediction. (c) GOR4 (Garnier-Osguthorpe-Robson) alpha helix prediction.

Figure 2

(a) Circular dichroism (CD) analysis of cl-Par-4 at pH 4, 7, 10, and in SDS (sodium dodecyl sulfate). (b) Secondary structure content at pH 4, 7 and 10. (c) CD analysis at pH 5, 5.5, and 6.5, near the pI. (d) Secondary structure content near the pI.

Figure 3

(a) Circular dichroism (CD) versus temperature at pH 7. (b) CD versus temperature at pH 4.(c) Secondary structure versus temperature at pH 7. (d) Secondary structure versus temperature at pH 4.

Figure 4

(a) Measured R_s of cl-Par-4 under native and denaturing conditions by Dynamic Light Scattering (DLS). (b) Relationship of zeta potential (dashed) to pH and R_s (solid line). (c) Measured R_s over seven days at pH 4, 7, and 10.

Figure 5

(a) Intrinsic tyrosine fluorescence at pH 4 (solid) and 7 (dashed) over 250–400 nm at 25 °C (b) Thermal denaturation at pH 4 (solid) and 7 (dashed) monitored by fluorescence emission at 310 nm.

Figure 6

Helical wheel representation of Par-4 leucine zipper parallel dimer (DrawCoil 1.0, Dartmouth College, Hanover, NH, USA). Basic residues are blue and acidic residues are red. The red dashed line represents inter-helical charge repulsion and the blue dashed line represents an inter-helical salt bridge.

Figure 7

GalaxyWEB model of cl-Par-4 at acidic pH. The N-terminal residues are yellow, the selective for apoptosis induction in cancer cells (SAC) domain with nuclear localization signal 2 (NLS2) is red, the linker region is gray, and coiled coil (CC) is blue. Darker blue is the leucine zipper (LZ) region of the CC. The position of NLS2 is also indicated.