Villa-Medina María Cristina, Díaz-Gaxiola Cecilia, Rosales-Reyes Roberto, Durán-Pérez Sergio Alonso, Vega-Castillo Ulises, Rodríguez-Rochín Jesús Alberto, León-Sicairos Claudia Del Rosario, Beltrán-López Evangelina, López-Moreno Héctor Samuel
Lab. Biomedicina Molecular, Posgrado en Ciencias Biomédicas, CAC-UAS-264, Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, Mexico.
Unidad de Medicina Experimental, Hospital General de México, Universidad Nacional Autónoma de México, Mexico City, Mexico.
Biomed Res Int. 2025 Mar 6;2025:3420875. doi: 10.1155/bmri/3420875. eCollection 2025.
In eukaryotic cells, mitochondria play a key role in apoptosis; however, ancestral eukaryotic cells such as only possess a mitochondrial remnant, the mitosome. Interestingly, this protozoan still undergoes an apoptosis-like process; therefore, we focused primarily on the search for the mitochondria-independent executor DNase. Here, we identified, cloned, expressed, and characterized the caspase-activated DNase (CAD) from . Using a commercial polyclonal antibody that recognizes mouse, rat, and human caspase-activated DNase (hCAD), we developed an immunoproteomic analysis using a crude extract of curcumin-treated trophozoites (CEGl) and detected a spot of 42 kDa and pI 9.4, similar to hCAD and sequenced by LC-MS. The proteomic profile matched a novel protein of 383 residues, with a predicted 42 kDa, pI 9.4, a CIDE-N domain, and putative H-K-H catalytic motif. Afterward, we cloned the full-length gene (GenBank: ON707040), expressed it, and purified it as a 6-His tag-recombinant protein in , which was also recognized by commercial anti-CAD. In conclusion, genetic, proteomic, and structural analyses showed that the identified gCAD is an orthologous protein of hCAD, and its DNase role in the apoptosis-like signaling pathway of can be further analyzed.
在真核细胞中，线粒体在细胞凋亡中起关键作用；然而，诸如仅拥有线粒体残余物——微小体的原始真核细胞。有趣的是，这种原生动物仍会经历类似细胞凋亡的过程；因此，我们主要专注于寻找不依赖线粒体的执行性脱氧核糖核酸酶。在此，我们从 中鉴定、克隆、表达并表征了半胱天冬酶激活的脱氧核糖核酸酶（CAD）。使用一种可识别小鼠、大鼠和人半胱天冬酶激活的脱氧核糖核酸酶（hCAD）的商业多克隆抗体，我们利用姜黄素处理的滋养体粗提物（CEGl）开展了免疫蛋白质组学分析，并检测到一个42 kDa、pI 9.4的斑点，与hCAD相似，并通过液相色谱-质谱联用仪进行了测序。蛋白质组图谱与一种由383个残基组成的新蛋白质匹配，预测分子量为42 kDa，pI 9.4，有一个CIDE-N结构域和假定的H-K-H催化基序。之后，我们克隆了全长基因（GenBank：ON707040），进行了表达，并在 中作为6-组氨酸标签重组蛋白进行了纯化，其也能被商业抗CAD抗体识别。总之，遗传学、蛋白质组学和结构分析表明，所鉴定的gCAD是hCAD的直系同源蛋白，其在 类似细胞凋亡信号通路中的脱氧核糖核酸酶作用可进一步分析。
