Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, 247 667, India.
CSIR-Institute of Microbial Technology, Chandigarh, 160 036, India.
Protein J. 2022 Jun;41(3):381-393. doi: 10.1007/s10930-022-10060-x. Epub 2022 Jun 8.
A class of plant defense and storage proteins, including Putranjiva roxburghii PNP protein (PRpnp), belongs to PNP-UDP family. The PRpnp and related plant proteins contain a disrupted PNP-UDP domain as revealed in previous studies. In PRpnp, the insert disrupting the domain contains the trypsin inhibitory site. In the present work, we analyzed native PRpnp (nPRpnp) complex formation with trypsin and inosine using SAXS experiments and established its dual functionality. Results indicated a relatively compact nPRpnp:Inosine structure, whereas trypsin complex showed conformational changes/flexibility. nPRpnp also exhibited a strong anti-cancer activity toward breast cancer (MCF-7), prostate cancer (DU-145) and hepatocellular carcinoma (HepG2) cell lines. MCF-7 and DU-145 were more sensitive to nPRpnp treatment as compared to HepG2. However, nPRpnp treatment showed no effect on the viability of HEK293 cells indicating that nPRpnp is specific for targeting the viability of only cancer cells. Further, acridine orange, DAPI and DNA fragmentation studies showed that cytotoxic effect of nPRpnp is mediated through induction of apoptosis as evident from the apoptosis-associated morphological changes and nuclear fragmentation observed after PRpnp treatment of cancer cells. These results suggest that PRpnp has the potential to be used as an anticancer agent. This is first report of anticancer activity as well as SAXS-based analysis for a PNP enzyme with trypsin inhibitory activity.
一类植物防御和储存蛋白，包括印度獐牙菜 PN 水解酶蛋白（PRpnp），属于 PN- UDP 家族。以前的研究表明，PRpnp 和相关植物蛋白含有一个破坏的 PN-UDP 结构域。在 PRpnp 中，破坏该结构域的插入序列包含胰蛋白酶抑制位点。在本工作中，我们使用 SAXS 实验分析了天然 PRpnp（nPRpnp）与胰蛋白酶和肌苷的复合物形成，并确定了其双重功能。结果表明，nPRpnp：肌苷结构相对紧凑，而胰蛋白酶复合物显示出构象变化/灵活性。nPRpnp 还对乳腺癌（MCF-7）、前列腺癌（DU-145）和肝癌（HepG2）细胞系表现出强烈的抗癌活性。与 HepG2 相比，MCF-7 和 DU-145 对 nPRpnp 处理更为敏感。然而，nPRpnp 处理对 HEK293 细胞的活力没有影响，表明 nPRpnp 特异性针对仅癌细胞的活力。此外，吖啶橙、DAPI 和 DNA 片段化研究表明，nPRpnp 的细胞毒性作用是通过诱导细胞凋亡介导的，这从 PRpnp 处理癌细胞后观察到的凋亡相关形态变化和核片段化中可以明显看出。这些结果表明 PRpnp 有可能被用作抗癌剂。这是首例报道具有胰蛋白酶抑制活性的 PN 酶的抗癌活性和基于 SAXS 的分析。
