Cancer-associated mutations in autophagy-related proteins analyzed in yeast and human cells.

Abstract

Macroautophagy/autophagy is a conserved process among eukaryotes and is essential to maintain cell homeostasis; the dysregulation of autophagy has been linked with multiple human diseases, including cancer. However, not many studies have focused on the cancer-related mutations in ATG (autophagy related) proteins, which are likely to affect the protein function, influence autophagy activity and further contribute to the progression of the disease. In this study, we focused on the four ATG4 isoforms, which have a higher mutation frequency compared with the other core ATG proteins (i.e. those involved in autophagosome formation). We first studied the mutations in conserved residues and characterized one cancer-associated mutation that significantly impairs protein function and autophagy activity. Extending the study, we determined a region around the mutant residue to be essential for protein function, which had yet to be examined in previous studies. In addition, we created a yeast system expressing the human ATG4B protein to study mutations in the residues that are not conserved from human to yeast. Using this yeast model, we identified six cancer-associated mutations affecting autophagy. The effects of these mutations were further tested in mammalian cells using a quadruple ATG4 gene knockout cell line. Our study proves the principle of using human disease-associated mutations to study Atg proteins in yeast and generates a yeast tool that is helpful for a rapid screen of mutations to determine the autophagy phenotype, providing a new perspective in studying autophagy and its relation with cancer.Abbreviations: 4KO: ATG4 tetra knockout; ATG: autophagy related; BafA1: bafilomycin A₁; GFP: green fluorescent protein; LC3-II: PE-conjugated form of LC3B; ORF: open reading frame; PE: phosphatidylethanolamine; RFP: red fluorescent protein; SEP: superecliptic pHluorin; Ubl: ubiquitin-like; UCEC: uterine corpus endometrial carcinoma.