Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA 23529, USA.
Biomolecules. 2021 Mar 5;11(3):386. doi: 10.3390/biom11030386.
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.
前列腺凋亡反应蛋白-4（Par-4）是一种促凋亡的肿瘤抑制蛋白，与许多癌症有关。这种 38 千道尔顿（kDa）的蛋白质在体外主要表现为无序结构。在体内，Par-4 被半胱天冬酶-3 在天冬氨酸 131 位切割，生成具有 25 kDa 功能活性的切割 Par-4 蛋白（cl-Par-4），该蛋白抑制 NF-κB 介导的细胞存活途径，并导致肿瘤细胞选择性凋亡。在这里，我们采用圆二色性（CD）光谱和动态光散射（DLS）技术评估了各种单价和二价盐对 cl-Par-4 体外构象的影响。我们之前已经表明，高水平的钠可以诱导 cl-Par-4 片段在体外形成高度紧凑、高度螺旋的四聚体。光谱特征表明，这些四聚体中的大部分或至少大部分螺旋含量是非卷曲螺旋。在这里，我们已经表明，钾产生与以前报道的钠类似的效果，并且镁盐也产生类似的构象效果，但在大约五倍低的离子浓度下。我们还表明，阴离子的身份比阳离子的身份影响小得多。这些盐诱导的螺旋度表明，“选择性杀伤癌细胞”（SAC）域——对 Par-4 凋亡功能最不可或缺的区域——在研究的高盐条件下，cl-Par-4 可能是螺旋的。此外，我们已经表明，在中等强度的离子条件下，会形成高分子量聚集体和较小颗粒的混合物，并且这些较小的颗粒也具有高度的螺旋性，至少在二级结构上类似于在高盐条件下发现的四聚体。
