Arginine 205 Mutation in PHAF1/MYTHO: Implications for Autophagy Regulation and Cancer Biology

Autophagy is a vital cellular process essential for maintaining homeostasis, regulating the protein quality control, and facilitating organelle turnover. Dysregulation of autophagy is implicated in various diseases, including neurodegeneration, muscular dystrophies, and cancer. Among the emerging regulators of autophagy, the PHAF1/MYTHO gene has recently been identified as a key player in skeletal muscle integrity and autophagic flux. Unpublished data have indicated that zebrafish MYTHO knockout models develop pancreatic and testis tumors. Thus, the study of MYTHO mutations and their potential role in cancer biology may represent a novel research avenue. Analysis of publicly available databases identified specific MYTHO mutations at 90 distinct sites in 3% of tumors, including colon, pancreas, uterus, and testis tumors. This thesis aimed to investigate the Arginine 205 mutation, prevalent in multiple cancers such as pancreatic cancer, providing the first steps toward understanding the molecular mechanisms by which MYTHO might contribute to oncogenesis. Arginine 205 MYTHO mutants were generated using a plasmid vector containing WT MYTHO fused to green fluorescent protein (GFP) and transfected into human embryonic kidney (HEK) cells, resulting in the expression of MYTHO-GFP proteins. Confocal fluorescence microscopy revealed that certain MYTHO mutations altered the protein subcellular localization and distribution. In addition, immunoprecipitation assays identified differential interactions between mutant MYTHO proteins and key autophagy regulators, BCAS3 and WIPI2, compared to the WT MYTHO. Therefore, the mutations appear to disrupt MYTHO binding affinity and interaction dynamics, which might impact autophagic processes. These findings provide initial insights into the potential involvement of MYTHO in cancer biology, highlighting MYTHO as a putative therapeutic target for autophagy-related cancers.