Changes in the Lysosomal Proteome Under Stressed Conditions

Abstract

Autophagy is a critical cellular process that is implicated in a vast array of human diseases, including amyotrophic lateral sclerosis (ALS). The targets of this degradative mechanism are frequently altered depending upon the inducing condition, however exactly how these targets change has rarely been explored on a proteomic level. To assess the targets of autophagy, this project analyzed the proteome of lysosomes collected by immunoprecipitation from cells undergoing starvation or recovering from oxidative stress. Oxidative stress induces stress granule formation and is frequently used as a cellular model for ALS. Our results show that while lysosomes from these two conditions retain a similar array of traditionally lysosomal proteins, they nevertheless display substantial differences indicating that their autophagic targets differ. Using Ingenuity Pathway Analysis (IPA) as well as gene ontology (GO) term analysis, the purine biosynthesis pathway was identified as being significantly enriched in lysosomes collected from sodium arsenite treated cells. The enrichment of the purine biosynthesis pathway was heavily dependent on ATG7, indicating these proteins localized to the lysosome primarily through macroautophagy. IMPDH2, the rate-limiting enzyme for guanylate production in the purine biosynthesis pathway drew particular attention due to its significant enrichment in sodium arsenite lysosomes, and its partial dependence on ATG7. Due to this discovery, and the presence of KFERQ-like sequences within the protein, our data suggests that IMPDH2 may localize to the lysosome through both macroautophagy and chaperone-mediated autophagy. Furthermore, numerous ALS-linked proteins were identified to be enriched in sodium arsenite lysosomes including SOD1, DCTN1, PFN1, TUBA4A, SQSTM1, VCP, CHMP2B, TDP-43, VAPB and TMEM106B. Their presence within the lysosome strengthens arguments that autophagy plays a key role in ALS. Overall, this project confirms the changing substrates of autophagy depending on the environmental condition and highlights the purine biosynthesis pathway as being involved in the cellular response to oxidative stress.