Structural Analysis of the cl-Par-4 Tumor Suppressor as a Function of Ionic Environment

Abstract

Prostate apoptosis response-4 (Par-4) is a proapoptotic tumor suppressor protein that has been linked to a large number of cancers. This 38 kilodalton (kDa) protein has been shown to be predominantly intrinsically disordered in vitro. In vivo, Par-4 is cleaved by caspase-3 at Asp-131 to generate the 25 kDa functionally active cleaved Par-4 protein (cl-Par-4) that inhibits NF-κB-mediated cell survival pathways and causes selective apoptosis in tumor cells. Here, we have employed circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) to assess the effects of various monovalent and divalent salts upon the conformation of cl-Par-4 in vitro. We have previously shown that high levels of sodium can induce the cl-Par-4 fragment to form highly compact, highly helical tetramers in vitro. Spectral characteristics suggest that most or at least much of the helical content in these tetramers are non-coiled coils. Here, we have shown that potassium produces a similar effect as was previously reported for sodium and that magnesium salts also produce a similar conformation effect, but at an approximately five times lower ionic concentration. We have also shown that anion identity has far less influence than does cation identity. The degree of helicity induced by each of these salts suggests that the “Selective for Apoptosis in Cancer cells” (SAC) domain—the region of Par-4 that is most indispensable for its apoptotic function—is likely to be helical in cl-Par-4 under the studied high salt conditions. Furthermore, we have shown that under medium-strength ionic conditions, a combination of high molecular weight aggregates and smaller particles form and that the smaller particles are also highly helical, resembling at least in secondary structure, the tetramers found at high salt.

Enhancing the Conformational Stability of the cl-Par-4 Tumor Suppressor via Site-Directed Mutagenesis

“…Expression and purification of D313K were conducted following the established procedure for WT cl-Par-4 [ 37 , 39 ]. In brief, BL21 (DE3) codon plus E. coli cells, transformed with the codon-optimized mutant construct D313K, were cultured in Luria-Bertani (LB) medium supplemented with ampicillin for selective growth at 37 °C.…”

“…We previously found that cl-Par-4 at pH 7.0 requires a high salt concentration to form compact helical tetramers [ 38 , 39 ]. For NMR analysis, high salt and tetramer formation are problematic in terms of the signal-to-noise ratio and line width.…”

“…However, the analysis shown in Figure 1 indicates that the cl-Par-4 fragment has higher order propensity than the PAF fragment. Most notably, the C-terminal 80 residues of cl-Par-4 are known to form a coiled coil (CC), at least under certain conditions [ 14 , 37 , 38 , 39 ]. Even in the CC, the order propensity is not strong.…”

“…Our previous studies have shown that cl-Par-4 achieves a compact, highly helical (approximately 80% helix), and stable conformation in the presence of either high salt or acidic pH [ 37 , 38 ]. We have also demonstrated that cl-Par-4 shows a similar conformation in the presence of divalent cations as it does in the presence of monovalent cations, but at a five times lower cation concentration [ 39 ]. This requirement of high salt or low pH could be due to the existence of inter-helical electrostatic repulsion between charged residues at the leucine zipper dimer interface near the C-terminus [ 41 , 42 ].…”

The Amazing World of IDPs in Human Diseases II

“…The work presented by Raut and colleagues [15] investigated Par-4 (Prostate apoptosis response-4), a predominantly intrinsically disordered protein, acting as a tumoural suppressor that is capable of selectively inducing apoptosis in cancer cells while leaving healthy cells unaffected. The authors performed experimental studies, employing circular dichroism spectroscopy and dynamic light scattering to assess the effects of various monovalent and divalent salts upon the conformation of cl-Par-4 in vitro.…”