Abstract
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.
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References
Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147(4):728–741
Kaushik S, Cuervo AM (2018) The coming of age of chaperone-mediated autophagy. Nat Rev Mol Cell Biol 19(6):365–381
Dice JF (2007) Chaperone-mediated autophagy. Autophagy 3(4):295–299
Cuervo AM, Dice JF (1997) A receptor for the selective uptake and degradation of proteins by lysosomes. Science 273(5274):501–503
Gough NR, Hatem CL, Fambrough DM (1995) The family of LAMP-2 proteins arises by alternative splicing from a single gene: characterization of the avian LAMP-2 gene and identification of mammalian homologs of LAMP-2b and LAMP-2c. DNA Cell Biol 14(10):863–867
Andrade-Tomaz M, de Souza I, Rocha CRR, Gomes LR (2020) The role of chaperone-mediated autophagy in cell cycle control and its implications in cancer. Cell 9:2140
Schneider JL, Villarroya J, Diaz-Carretero A, Patel B, Urbanska AM, Thi MM, Villarroya F, Santambrogio L, Cuervo AM (2015) Loss of hepatic chaperone-mediated autophagy accelerates proteostasis failure in aging. Aging Cell 14(2):249–264
Kon M, Kiffin R, Koga H, Chapochnick J, Macian F, Varticovski L, Cuervo AM (2011) Chaperone-mediated autophagy is required for tumor growth. Sci Transl Med 3(109):109ra17
Galan-Acosta L, Xia H, Yuan J, Vakifahmetoglu-Norberg H (2015) Activation of chaperone-mediated autophagy as a potential anticancer therapy. Autophagy 11(12):2370–2371
Arias E, Cuervo AM (2020) Pros and cons of chaperone-mediated autophagy in cancer biology. Trends Endocrinol Metab 31(1):53–66
Vakifahmetoglu-Norberg H, Kim M, Xia HG, Iwanicki MP, Ofengeim D, Coloff JL, Pan L, Ince TA, Kroemer G, Brugge JS, Yuan J (2013) Chaperone-mediated autophagy degrades mutant p53. Genes Dev 27(15):1718–1730
Xia H-G, Najafov A, Geng J, Galan-Acosta L, Han X, Guo Y, Shan B, Zhang Y, Norberg E, Zhang T, Pan L, Liu J, Coloff JL, Ofengeim D, Zhu H, Wu K, Cai Y, Yates JR, Zhu Z, Yuan J, Vakifahmetoglu-Norberg H (2015) Degradation of HK2 by chaperone-mediated autophagy promotes metabolic catastrophe and cell death. J Cell Biol 210(5):705–716
Hao Y, Kacal M, Ouchida AT, Zhang B, Norberg E, Vakifahmetoglu-Norberg H (2019) Targetome analysis of chaperone-mediated autophagy in cancer cells. Autophagy 15(9):1558–1571
Kacal M, Zhang B, Hao Y, Norberg E, Vakifahmetoglu-Norberg H (2021) Quantitative proteomic analysis of temporal lysosomal proteome and the impact of the KFERQ-like motif and LAMP2A in lysosomal targeting. Autophagy 26:1–10
Wang H, La Russa M, Qi LS (2016) CRISPR Cas9 in genome editing and beyond. Annu Rev Biochem 85(1):227–264
Musunuru K (2017) The hope and hype of CRISPR-Cas9 genome editing: a review. JAMA Cardiol 2(8):914–919
Xu CF, Chen GJ, Luo YL, Zhang Y, Zhao G, Lu ZD, Czarna A, Gu Z, Wang J (2019) Rational designs of in vivo CRISPR-Cas delivery systems. Adv Drug Deliv Rev 168:3–29
Acknowledgments
This work was supported by grants from Karolinska Institutet, the Swedish Research Council (VR), Ragnar Söderberg Foundation, and Swedish Cancer Society (Cancerfonden). Special thanks to MSc. Merve Kacal for her help with technical and practical details for optimizing the procedure.
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Yu, Tt., Zhou, X., Vakifahmetoglu-Norberg, H. (2022). CRISPR-Cas9 Gene Editing to Generate Isoform-Specific LAMP-2A Knockout in Human Cancer Cells. In: Norberg, H., Norberg, E. (eds) Autophagy and Cancer. Methods in Molecular Biology, vol 2445. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2071-7_3
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DOI: https://doi.org/10.1007/978-1-0716-2071-7_3
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