Lei Kunjian, Li Jingying, Tu Zewei, Gong Chuandong, Liu Junzhe, Luo Min, Ai Wenqian, Wu Lei, Li Yishuang, Zhou Zhihong, Chen Zhihao, Lv Shigang, Ye Minhua, Wu Miaojing, Long Xiaoyan, Zhu Xingen, Huang Kai
Department of Neurosurgery, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi 330006, P. R. China.
Key Laboratory of Neurological Diseases in Jiangxi Province, Nanchang, Jiangxi 330031, P. R. China.
Theranostics. 2024 Jul 22;14(11):4481-4498. doi: 10.7150/thno.98574. eCollection 2024.
Since oncogene expression products often exhibit upregulation or abnormally activated activity, developing a technique to regulate abnormal protein levels represent a viable approach for treating tumors and protein abnormality-related diseases. We first screened out eMIATAC components with high targeted degradation efficiency and explored the mechanism by which eMIATAC induced target protein degradation, and verified the degradation efficiency of the target protein by protein imprinting and flow cytometry. Next, we recombined eMIATAC with some controllable elements to verify the regulatable degradation performance of the target protein. Subsequently, we constructed eMIATAC that can express targeted degradation of AKT1 and verified its effect on GBM cell development in vitro and in vivo. Finally, we concatenated eMIATAC with CAR sequences to construct CAR-T cells with low BATF protein levels and verified the changes in their anti-tumor efficacy. we developed a system based on the endosome-microautophagy-lysosome pathway for degrading endogenous proteins: endosome-MicroAutophagy TArgeting Chimera (eMIATAC), dependent on Vps4A instead of lysosomal-associated membrane protein 2A (LAMP2A) to bind to the chaperone Hsc70 and the protein of interest (POI). The complex was then transported to the lysosome by late endosomes, where degradation occurred similarly to microautophagy. The eMIATACs demonstrated accuracy, efficiency, reversibility, and controllability in degrading the target protein EGFP. Moreover, eMIATAC exhibited excellent performance in knocking down POI when targeting endogenous proteins in vivo and in vitro. The eMIATACs could not only directly knock down abnormal proteins for glioma treatment but also enhance the therapeutic effect of CAR-T cell therapy for tumors by knocking down T cell exhaustion-related proteins. The newly developed eMIATAC system holds promise as a novel tool for protein knockdown strategies. By enabling direct control over endogenous protein levels, eMIATAC has the potential to revolutionize treatment for cancer and genetic diseases.
由于癌基因表达产物常常表现出上调或异常激活的活性，开发一种调节异常蛋白质水平的技术是治疗肿瘤和蛋白质异常相关疾病的可行方法。我们首先筛选出具有高靶向降解效率的内体-微自噬-溶酶体途径靶向嵌合体（eMIATAC）成分，并探索eMIATAC诱导靶蛋白降解的机制，通过蛋白质印迹和流式细胞术验证靶蛋白的降解效率。接下来，我们将eMIATAC与一些可控元件重组，以验证靶蛋白的可调节降解性能。随后，我们构建了能够表达对AKT1进行靶向降解的eMIATAC，并在体外和体内验证了其对胶质母细胞瘤细胞发育的影响。最后，我们将eMIATAC与嵌合抗原受体（CAR）序列连接，构建出BATF蛋白水平较低的CAR-T细胞，并验证了其抗肿瘤疗效的变化。我们开发了一种基于内体-微自噬-溶酶体途径降解内源性蛋白质的系统：内体-微自噬靶向嵌合体（eMIATAC），它依赖Vps4A而非溶酶体相关膜蛋白2A（LAMP2A）来结合伴侣蛋白Hsc70和目的蛋白（POI）。然后该复合物通过晚期内体被转运到溶酶体，在那里发生与微自噬类似的降解。eMIATAC在降解靶蛋白绿色荧光蛋白（EGFP）方面表现出准确性、高效性、可逆性和可控性。此外，当在体内和体外靶向内源性蛋白质时，eMIATAC在敲低目的蛋白方面表现出色。eMIATAC不仅可以直接敲低异常蛋白用于治疗胶质瘤，还可以通过敲低T细胞耗竭相关蛋白来增强CAR-T细胞疗法对肿瘤的治疗效果。新开发的eMIATAC系统有望成为蛋白质敲低策略的一种新型工具。通过直接控制内源性蛋白质水平，eMIATAC有潜力彻底改变癌症和遗传疾病治疗方法。
