Negative Modulation of Macroautophagy by Stabilized HERPUD1 is Counteracted by an Increased ER-Lysosomal Network With Impact in Drug-Induced Stress Cell Survival.

Gabriela Vargas,Jorge Cancino,Ronald T Hay,Patricia V Burgos,Sergio Hernández,Claudio Retamal,Manuel Varas-Godoy,Alexis E González,Michael H Tatham,Eloisa Arias-Muñoz,Alejandro Rojas-Fernández,Omar Cortés,Hianara A Bustamante,Cristobal Cerda-Troncoso,Laura Hernández,Viviana A Cavieres

doi:10.3389/fcell.2022.743287

Abstract

Macroautophagy and the ubiquitin proteasome system work as an interconnected network in the maintenance of cellular homeostasis. Indeed, efficient activation of macroautophagy upon nutritional deprivation is sustained by degradation of preexisting proteins by the proteasome. However, the specific substrates that are degraded by the proteasome in order to activate macroautophagy are currently unknown. By quantitative proteomic analysis we identified several proteins downregulated in response to starvation independently of ATG5 expression. Among them, the most significant was HERPUD1, an ER membrane protein with low expression and known to be degraded by the proteasome under normal conditions. Contrary, under ER stress, levels of HERPUD1 increased rapidly due to a blockage in its proteasomal degradation. Thus, we explored whether HERPUD1 stability could work as a negative regulator of autophagy. In this work, we expressed a version of HERPUD1 with its ubiquitin-like domain (UBL) deleted, which is known to be crucial for its proteasome degradation. In comparison to HERPUD1-WT, we found the UBL-deleted version caused a negative role on basal and induced macroautophagy. Unexpectedly, we found stabilized HERPUD1 promotes ER remodeling independent of unfolded protein response activation observing an increase in stacked-tubular structures resembling previously described tubular ER rearrangements. Importantly, a phosphomimetic S59D mutation within the UBL mimics the phenotype observed with the UBL-deleted version including an increase in HERPUD1 stability and ER remodeling together with a negative role on autophagy. Moreover, we found UBL-deleted version and HERPUD1-S59D trigger an increase in cellular size, whereas HERPUD1-S59D also causes an increased in nuclear size. Interestingly, ER remodeling by the deletion of the UBL and the phosphomimetic S59D version led to an increase in the number and function of lysosomes. In addition, the UBL-deleted version and phosphomimetic S59D version established a tight ER-lysosomal network with the presence of extended patches of ER-lysosomal membrane-contact sites condition that reveals an increase of cell survival under stress conditions. Altogether, we propose stabilized HERPUD1 downregulates macroautophagy favoring instead a closed interplay between the ER and lysosomes with consequences in drug-cell stress survival.

Highlights

Macroautophagy is a catabolic pathway that mediates the engulfment of aberrant or damaged cytoplasmic constituents into double-membrane autophagosomes that subsequently fuse with lysosomes to form a hybrid organelle called the autolysosome that mediates the degradation of the cargo by acid hydrolases (Mizushima et al, 2008; Khaminets et al, 2016)
Previous reports have demonstrated a close interplay between autophagy and the Ubiquitin-Proteasome System (Bustamante et al, 2018), to date, few proteasomal substrates are known as modulators of autophagy (Jia and Bonifacino, 2019; Guarascio et al, 2020; Thayer et al, 2020)
Among all the proteins downregulated by EBSS starvation, we found that in both H4 cell lines, shRNA against the luciferase gene (shLuc) (Figure 1B) and shATG5 (Figure 1C) the most significantly downregulated protein was HERPUD1, a protein originally identified as a homocysteine-inducible gene, that is upregulated by endoplasmic reticulum (ER) stress (Kokame et al, 2000; Kokame et al, 2001)

Summary

Introduction

Macroautophagy (from here referred to as autophagy) is a catabolic pathway that mediates the engulfment of aberrant or damaged cytoplasmic constituents into double-membrane autophagosomes that subsequently fuse with lysosomes to form a hybrid organelle called the autolysosome that mediates the degradation of the cargo by acid hydrolases (Mizushima et al, 2008; Khaminets et al, 2016). Autophagy is implicated in the degradation of cellular constituents under basal conditions, playing an essential role in the maintenance of cellular homeostasis upon a variety of environmental conditions such as nutrient restriction or other stressors (Murrow and Debnath, 2013). Autophagy is highly inducible by environmental changes being a very dynamic process that resolves a variety of cellular demands (Murrow and Debnath, 2013). Increased autophagy is protective in different cells and organisms, playing a crucial role in cell maintenance and survival under different insults (Moreau et al, 2010). Initially autophagy was thought to work independently of the ubiquitin proteasome system (UPS), increasing evidence shows many layers of both negative and positive regulation (Bustamante et al, 2018), revealing an interconnected network with important roles in cellular homeostasis and maintenance (Korolchuk et al, 2010). To date a limited number of substrates of the UPS system are known to play a regulatory role in autophagy (Jia and Bonifacino, 2019; Thayer et al, 2020) and many aspects about the functional role of this interconnected network between autophagy and UPS remain elusive

Methods

Results

Conclusion