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.
Highlights
Prostate apoptosis response-4 (Par-4) is a tumor suppressor protein that is capable of selectively inducing apoptosis in cancer cells while leaving healthy cells unaffected [1]
The harvested cells were lysed by a combination of mechanical and enzymatic methods and extracted protein was purified with immobilized metal affinity chromatography (IMAC)
Full-length Par-4 (FL-Par-4) has been shown to be a largely intrinsically disordered protein (IDP) in vitro: circular dichroism (CD) spectroscopy and other techniques have previously been used to show that the C-terminal coiled-coil folds in FL-Par-4, while a large majority of the remainder of the protein maintains a disordered state under neutral conditions [34]
Summary
Par-4 was first identified through its down-regulation in therapy-resistant prostate cancer cells and has since been shown to be ubiquitously expressed, localized in the cytoplasm, the nucleus and extracellularly, and linked to a variety of cancers [2,3,4,5]. While Par-4 has been shown to be largely intrinsically disordered in vitro, it does contain several identifiable domains and features. The first identified domain was the C-terminal coiled-coil, which is highly conserved across humans, mice, and rats [7]. This region contains a small nuclear export signal (NES). The coiled-coil is responsible for Par-4 dimerization, and for most known interactions with cellular proteins
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
