Yu Ting-Ting, Zhou Xun, Vakifahmetoglu-Norberg Helin
Department of Physiology and Pharmacology, Solnavägen 9, Biomedicum, Karolinska Institutet, Stockholm, Sweden.
Department of Medical Genetics, School of Basic Medical Science, Longmian Avenue 101, Nanjing Medical University, Nanjing, China.
Methods Mol Biol. 2022;2445:39-50. doi: 10.1007/978-1-0716-2071-7_3.
Chaperone-mediated autophagy (CMA) is a highly specific lysosomal-dependent protein degradation pathway. A critical molecular component of CMA is the lysosome-associated membrane protein (LAMP) type 2A, which is required for substrate uptake by the lysosome. Defects in the CMA pathway have been associated with various human pathologies, including malignancies, increasing the overall interest in methods to monitor this selective autophagy process. Yet isogenic LAMP-2A knockout cancer cell models are still lacking. This is likely to depend on challenges related to that human LAMP-2 gene undergoes alternative splicing of its pre-mRNA, generating three isoform variants, LAMP-2A, LAMP-2B, and LAMP-2C. However, without assessment of the impact of LAMP-2A loss of function specifically in human cells, the involvement of CMA in human pathologies, including carcinogenesis remains speculative. Here, we describe the generation of isoform-specific CRISPR-Cas9 genomic editing of LAMP-2A in human cancer cells, without affecting the other two isoforms, allowing for experimental evaluation of LAMP-2A, thus CMA in human cancer models.
伴侣介导的自噬（CMA）是一种高度特异性的溶酶体依赖性蛋白质降解途径。CMA的一个关键分子成分是溶酶体相关膜蛋白（LAMP）2A，它是溶酶体摄取底物所必需的。CMA途径的缺陷与包括恶性肿瘤在内的各种人类疾病相关，这增加了人们对监测这种选择性自噬过程方法的总体兴趣。然而，同基因LAMP-2A敲除癌细胞模型仍然缺乏。这可能取决于与人类LAMP-2基因前体mRNA发生可变剪接有关的挑战，产生三种异构体变体，即LAMP-2A、LAMP-2B和LAMP-2C。然而，在没有专门评估LAMP-2A功能丧失对人类细胞的影响的情况下，CMA在包括致癌作用在内的人类疾病中的参与仍然是推测性的。在这里，我们描述了在人类癌细胞中对LAMP-2A进行异构体特异性CRISPR-Cas9基因组编辑的方法，而不影响其他两种异构体，从而能够对LAMP-2A进行实验评估，进而在人类癌症模型中评估CMA。
