Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland.
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland.
Biochim Biophys Acta Gen Subj. 2021 Nov;1865(11):129968. doi: 10.1016/j.bbagen.2021.129968. Epub 2021 Jul 27.
Human HINT2 is an important mitochondrial enzyme involved in many processes such as apoptosis and bioenergetics, but its endogenous substrates and the three-dimensional structure of the full-length protein have not been identified yet.
An HPLC assay was used to test the hydrolytic activity of HINT2 against various adenosine, guanosine, and 2'-deoxyguanosine derivatives containing phosphate bonds of different types and different leaving groups. Data on binding affinity were obtained by microscale thermophoresis (MST). Crystal structures of HINT2, in its apo form and with a dGMP ligand, were resolved to atomic resolution.
HINT2 substrate specificity was similar to that of HINT1, but with the major exception of remarkable discrimination against substrates lacking the 2'-hydroxyl group. The biochemical results were consistent with binding affinity measurements. They showed a similar binding strength of AMP and GMP to HINT2, and much weaker binding of dGMP, in contrast to HINT1. A non-hydrolyzable analog of Ap4A (JB419) interacted with both proteins with similar K and Ap4A is the signaling molecule that can interact with hHINT1 and regulate the activity of some transcription factors.
Several forms of homo- and heterodimers of different lengths of N-terminally truncated polypeptides resulting from degradation of the full-length protein were described. Ser144 in HINT2 appeared to be functionally equivalent to Ser107 in HINT1 by supporting the protonation of the leaving group in the hydrolytic mechanism of HINT2.
Our results should be considered in future studies on the natural function of HINT2 and its role in nucleotide prodrug processing.
人类 HINT2 是一种重要的线粒体酶，参与许多过程，如细胞凋亡和生物能量学，但它的内源性底物和全长蛋白的三维结构尚未确定。
使用 HPLC 测定法测试 HINT2 对各种含有不同类型和不同离去基团的磷酸键的腺苷、鸟苷和 2'-脱氧鸟苷衍生物的水解活性。通过微尺度热泳动（MST）获得结合亲和力数据。解析 HINT2 的apo 形式和 dGMP 配体的晶体结构，分辨率达到原子分辨率。
HINT2 的底物特异性与 HINT1 相似，但有一个主要的例外，即对缺乏 2'-羟基的底物有明显的区分。生化结果与结合亲和力测量结果一致。它们表明 AMP 和 GMP 与 HINT2 的结合强度相似，而与 HINT1 相比，dGMP 的结合强度较弱。Ap4A 的非水解类似物（JB419）与两种蛋白质的相互作用相似，K 值相近，Ap4A 是一种信号分子，可以与 hHINT1 相互作用并调节一些转录因子的活性。
描述了全长蛋白降解产生的不同长度 N 端截断多肽的几种同源和异源二聚体形式。 HINT2 中的 Ser144 似乎通过支持水解机制中离去基团的质子化，在 HINT2 中与 HINT1 中的 Ser107 具有相同的功能。
我们的研究结果应在未来关于 HINT2 的自然功能及其在核苷酸前药处理中的作用的研究中加以考虑。
