Catalase inhibition induces pexophagy through ROS accumulation

Highlights

• Catalase deficiency or its inhibition by 3 A T during serum starvation results in pexophagy.

• Pexophagy mediated by catalase inhibition is accompanied by PEX5 ubiquitination.

• ROS accumulation in peroxisome is the key factor to induce pexophagy.

• N-acetyl-l-cysteine prevents ROS-mediated pexophagy.

Abstract

Peroxisomes are dynamic and multifunctional organelles involved in various cellular metabolic processes, and their numbers are tightly regulated by pexophagy, a selective degradation of peroxisomes through autophagy to maintain peroxisome homeostasis in cells. Catalase, a major peroxisome protein, plays a critical role in removing peroxisome-generated reactive oxygen species (ROS) produced by peroxisome enzymes, but the contribution of catalase to pexophagy has not been reported. Here, we investigated the role of catalase in peroxisome degradation during nutrient deprivation. Both short interfering RNA-mediated silencing of catalase and pharmacological inhibition by 3-aminotriazole (3AT) decreased the number of peroxisomes and resulted in the downregulation of peroxisomal proteins, such as PMP70 and PEX14 under serum starvation. In addition, treatment with 3AT induced NBR1-dependent autophagy and PEX5 ubiquitination in the absence of serum, which was accompanied by accumulation of ROS. Co-treatment with antioxidant agent N-acetyl-l-cysteine (NAC) prevented ROS accumulation and pexophagy by modulating peroxisome protein levels and the association of NBR1, a pexophagy receptor with peroxisomes. Taken together, these findings demonstrate that catalase plays an important role in pexophagy during nutrient deprivation.

Introduction

The peroxisome is a cellular organelle that plays an important role in various metabolic processes including β-oxidation of very long chain and branched chain fatty acids, synthesis of bile acids and ether lipids, and decomposition of hydrogen peroxide (H₂O₂) [[1], [2], [3]]. Peroxisome number is tightly controlled according to cellular needs. Under conditions of metabolic or cellular stresses, the number of peroxisomes is reduced through pexophagy [[4], [5], [6]]. Pexophagy is a specialized form of autophagy that degrades peroxisomes that is critical for maintaining cellular homeostasis [7,8]. Ubiquitination on the cytosolic face of peroxisomes is required for recruitment of the autophagy receptor proteins sequestosome (SQSTM)1/p62 and neighbor of BRCA1 gene (NBR)1, which bind to microtubule-associated proteins 1 A/1 B light chain (LC)3-associated autophagosomes for degradation [[9], [10], [11], [12]]. Among peroxisome proteins, PMP70 and PEX5 are known to be ubiquitinated during pexophagy [13,14]. Furthermore, it has been reported that peroxisome E3 ligases PEX2, PEX10, and PEX12 are related to PEX5 ubiquitination for peroxisome degradation [14,15]. Since oxygen is consumed in various metabolic reactions in peroxisomes, oxygen availability able to regulate peroxisome number. Under hypoxic conditions, the peroxisome fails to function properly and pexophagy is induced to remove the damaged peroxisomes through hypoxia-inducible factor 2 alpha (Hif-2α) activation [16]. Also, it has been suggested that ROS triggers pexophagy through ATM activation [17].

Catalase, a porphyrin heme-containing enzyme is a classical marker protein of peroxisomes. Catalase decomposes H₂O₂ generated by various peroxisomal oxidases including acyl-CoA oxidase, urate oxidase, and xanthine oxidase [18,19]. Even though peroxisome contains several antioxidant enzymes, catalase plays a major role for protecting the adverse effects of accumulating peroxides [20,21]. Catalase inhibition deteriorated peroxisomal fatty acyl-CoA beta-oxidation by accumulated H₂O₂ in rodent liver [22]. Also, catalase protects the kidney under diabetic condition through maintaining peroxisome function [23]. In addition, the overexpression of catalase in mice extends lifespan of animals by reducing H₂O₂ production [24].

While the role of catalase on antioxidant effects has been intensively investigated, the relationship between catalase and pexophagy remains elusive. Here, we examined the role of catalase on pexophagy during serum starvation. Our data show that an inhibition of catalase activity increases peroxisome degradation through pexophagy during serum starvation. Also, catalase inhibition triggers the accumulation of ROS during starvation, which induces peroxisome loss through activation of selective autophagy.