Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics
Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics
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DOI：
10.3390/toxins6051644
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发表时间：
2014-05-01
期刊：
影响因子：
4.2
通讯作者：
Weng, Alexander
中科院分区：
文献类型：
--
作者：
Gilabert-Oriol, Roger;Thakur, Mayank;Weng, Alexander
以胞浆为靶点的基于蛋白质的治疗药物一旦从内小体或溶酶体中逃脱，就能够显示出它们的治疗效果。本研究考察了辣根过氧化物酶(HRP)、Alexa Fluor 488(Alexa)和蓖麻毒素A链(RTA)对核糖体失活蛋白Saporin内切/溶酶体逃逸的监控能力。构建了Saporin-HRP、(Alexa)Saporin和Saporin-KQ-RTA结合物，并对这些结合物的内切/溶酶体逃逸(缺乏内切/溶酶体释放)或与某些结构特异性的三萜皂苷(有效的内切/溶酶体逃逸)进行了表征。HRP未能报告Saporin的内切/溶酶体逃逸。相比之下，Alexa Fluor 488成功地在毒素浓度为1000 NM的情况下报告了这一过程。此外，单一内切/溶酶体分析有助于确定从每个囊泡释放的(Alexa)皂苷的量。RTA也成功地报道了酶失活突变体Saporin-KQ的内切/溶酶体逃逸，但在这种情况下，该方法的灵敏度达到了10 NM的毒素浓度。总之，同时使用Alexa Fluor 488和RTA作为报告可能提供在10-1000 nM的浓度范围内监测基于蛋白质的治疗药物的内切/溶酶体逃逸的可能性。
Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters-horseradish peroxidase (HRP), Alexa Fluor 488 ((Alexa)) and ricin A-chain (RTA)-were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates-saporin-HRP, (Alexa)saporin and saporin-KQ-RTA-were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of (Alexa)saporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10-1000 nM.