title: Development of a novel method for quantification of autophagic protein degradation by AHA labeling.
abstract: Autophagy is a catabolic process during which cellular components including protein aggregates and organelles are degraded via a lysosome-dependent process to sustain metabolic homeostasis during nutrient or energy deprivation. Measuring the rate of proteolysis of long-lived proteins is a classical assay for measurement of autophagic flux. However, traditional methods, such as a radioisotope labeling assay, are technically tedious and have low sensitivity. Here, we report a novel method for quantification of long-lived protein degradation based on L-azidohomoalanine (AHA) labeling in mouse embryonic fibroblasts (MEFs) and in human cancer cells. AHA is a surrogate for L-methionine, containing a bio-orthogonalazide moiety. When added to cultured cells, AHA is incorporated into proteins during active protein synthesis. After a click reaction between an azide and an alkyne, the azide-containing proteins can be detected with an alkyne-tagged fluorescent dye, coupled with flow cytometry. Induction of autophagy by starvation or mechanistic target of rapamycin (MTOR) inhibitors was able to induce a significant reduction of the fluorescence intensity, consistent with other autophagic markers. Coincidently, inhibition of autophagy by pharmacological agents or by Atg gene deletion abolished the reduction of the fluorescence intensity. Compared with the classical radioisotope pulse-labeling method, we think that our method is sensitive, quantitative, nonradioactive, and easy to perform, and can be applied to both human and animal cell culture systems.
authors.affiliation: Department of Physiology; Yong Loo Lin School of Medicine; National University of Singapore; Singapore.
authors.forename: Jianbin
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Biological Sciences; Faculty of Science; National University of Singapore; Singapore.
authors.forename: Jigang
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Physiology; Yong Loo Lin School of Medicine; National University of Singapore; Singapore.
authors.forename: Shukie
authors.initials: S
authors.lastname: Ng
authors.affiliation: Department of Biological Sciences; Faculty of Science; National University of Singapore; Singapore.
authors.forename: Qingsong
authors.initials: Q
authors.lastname: Lin
authors.affiliation: Department of Physiology; Yong Loo Lin School of Medicine; National University of Singapore; Singapore.
authors.forename: Han-Ming
authors.initials: HM
authors.lastname: Shen
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D001343:Autophagy; D001681:Biological Assay / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D051379:Mice; D059748:Proteolysis; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; C506098:azidohomoalanine; D000409:Alanine
keywords: AHA; autophagy; click-chemistry; flow cytometry; proteolysis