Department of Molecular Biology, Rowan University, School of Osteopathic Medicine and Graduate School of Biomedical Sciences, New Jersey, 08084, Stratford.
Department of Cell Biology, Rowan University, School of Osteopathic Medicine and Graduate School of Biomedical Sciences, Stratford, New Jersey, 08084.
Protein Sci. 2018 Oct;27(10):1797-1809. doi: 10.1002/pro.3481. Epub 2018 Sep 24.
dUTPase is an enzyme found in all organisms that have thymine as a constituent of DNA. Through evolution, humans have two major isoforms of dUTPase: a mitochondrial (mDut) and a nuclear (nDut) isoform. The nuclear isoform of dUTPase is a 164-amino-acids-long protein containing three cysteine residues. nDut's starting methionine is post-translationally cleaved, leaving four unique amino acids on its amino-terminus including one cysteine residue (C3). These are not present in the mitochondrial isoform (mDut). Using mass spectrometry analyses of recombinant dUTPase constructs, we have discovered an intermolecular disulfide bridge between cysteine-3 of each nDut monomer. We have found that these two residues stabilize a dimer configuration that is unique to the nDut isoform. We have also uncovered an intramolecular disulfide linkage between cysteine residues C78 and C134, stabilizing the monomeric state of the protein. Of note, both disulfide linkages are essential for nDut's enzymatic activity and dimeric formation can be augmented by the addition of the oxidizing agent, hydrogen peroxide to cells. Analyses of endogenous cellular dUTPase proteins confirm these differences between the two isoforms. We observed that mDut appears to be a mixture of monomer, dimer, and trimer conformations, as well as higher-order subunit interactions. In contrast, nDut appeared to exist only in monomeric and dimeric forms. Cysteine-based redox "switches" have recently emerged as a distinct class of post-translational modification. In light of this and our results, we propose that nDut possesses a redox switch whereby cysteine interactions regulate nDut's dUTP-hydrolyzing activity.
dUTP 酶是一种存在于所有含有胸腺嘧啶作为 DNA 成分的生物体中的酶。通过进化，人类有两种主要的 dUTP 酶同工型：线粒体（mDut）和核（nDut）同工型。核 dUTP 酶同工型是一种 164 个氨基酸长的蛋白质，含有三个半胱氨酸残基。nDut 的起始甲硫氨酸在翻译后被切割，其氨基末端留下四个独特的氨基酸，包括一个半胱氨酸残基（C3）。这些残基不存在于线粒体同工型（mDut）中。使用重组 dUTP 酶构建体的质谱分析，我们发现每个 nDut 单体的半胱氨酸-3 之间存在一个分子间二硫键。我们发现这两个残基稳定了一个独特的 nDut 同工型二聚体构象。我们还发现了半胱氨酸残基 C78 和 C134 之间的分子内二硫键连接，稳定了蛋白质的单体状态。值得注意的是，这两个二硫键连接对于 nDut 的酶活性都是必不可少的，并且可以通过向细胞中添加氧化剂过氧化氢来增强二聚体的形成。对内源性细胞 dUTP 酶蛋白的分析证实了这两种同工型之间的差异。我们观察到 mDut 似乎是单体、二聚体和三聚体构象的混合物，以及更高阶的亚基相互作用。相比之下，nDut 似乎只以单体和二聚体形式存在。基于半胱氨酸的氧化还原“开关”最近作为一类独特的翻译后修饰出现。鉴于此和我们的结果，我们提出 nDut 具有氧化还原开关，其中半胱氨酸相互作用调节 nDut 的 dUTP 水解活性。
