The crosstalk of NAD, ROS and autophagy in cellular health and ageing

Abstract

Cellular adaptation to various types of stress requires a complex network of steps that altogether lead to reconstitution of redox balance, degradation of damaged macromolecules and restoration of cellular metabolism. Advances in our understanding of the interplay between cellular signalling and signal translation paint a complex picture of multi-layered paths of regulation. In this review we explore the link between cellular adaptation to metabolic and oxidative stresses by activation of autophagy, a crucial cellular catabolic pathway. Metabolic stress can lead to changes in the redox state of nicotinamide adenine dinucleotide (NAD), a co-factor in a variety of enzymatic reactions and thus trigger autophagy that acts to sequester intracellular components for recycling to support cellular growth. Likewise, autophagy is activated by oxidative stress to selectively recycle damaged macromolecules and organelles and thus maintain cellular viability. Multiple proteins that help regulate or execute autophagy are targets of post-translational modifications (PTMs) that have an effect on their localization, binding affinity or enzymatic activity. These PTMs include acetylation, a reversible enzymatic modification of a protein's lysine residues, and oxidation, a set of reversible and irreversible modifications by free radicals. Here we highlight the latest findings and outstanding questions on the interplay of autophagy with metabolic stress, presenting as changes in NAD levels, and oxidative stress, with a focus on autophagy proteins that are regulated by both, oxidation and acetylation. We further explore the relevance of this multi-layered signalling to healthy human ageing and their potential role in human disease.

Keywords: Acetylation; Ageing; Autophagy; NAD; ROS; Sirtuins.

Fig. 1

Molecular outcomes of NAD⁺ cleavage. The three major groups of NAD⁺-consuming enzymes include sirtuins (SIRT), poly(ADP-ribose) polymerases (PARPs) and cyclic ADP-ribose synthases (cADPRs, CD38, CD157). a SIRT1-3 are NAD + -dependent deacetylases that bind an acetylated (Ac) protein substrates and transfer the Ac moiety onto ADP-ribose (ADPR) to give rise to O-acetyl-ADP-ribose, a deacetylated protein substrate and a by-product of the reaction, nicotinamide (NAM). b PARPs are indiscriminate NAD⁺ consumers that use NAD⁺ as a co-substrate to generate poly(ADP)-ribose (PAR) chains on protein substrates, and generate NAM as a by-product. c cADPRs consume NAD⁺ to generate cyclic ADP-ribose (cADPR), a second messenger, and a by-product, NAM

Fig. 2

Autophagy targets of acetylation and oxidation. Nutrient and oxidative stresses affect proteins that participate in autophagy by lysine (K) acetylation or cysteine (C) oxidation. a Localization of transcriptional factor EB (TFEB), a master regulator of autophagy and lysosomal gene expression, is regulated by oxidative stress. Indirectly, oxidative modification of mucolipin 1 (MCOLN1) leads to TFEB dephosphorylation by Ca²⁺-sensitive phosphatase, calcineurin and its translocation to the nucleus. Directly, oxidation of TFEBs redox-sensitive residue, C²¹², promotes rapid nuclear localization. In addition, inhibitory lysine acetylation of K²⁷⁴ and K²⁷⁹ that is regulated by the general control non-repressed protein 5 (GCN5) prevents TFEB dimerization. The molecular and functional outcomes of K¹¹⁶ are not known, but are opposed by nutrient sensitive, NAD + -dependent lysine deacetylase (KDAC), SIRT1. b Acetylation-sensitive lysine residues were detected within members of the ULK1 complex, the class III PI(3)K complex and both ubiquitin-like conjugation systems. Unc-51-like kinase 1 (ULK1, ULK1 complex) contains two lysine residues, K¹⁶² and K⁶⁰⁶ (* in mouse) that are acetylated by TIP60 in response to serum starvation. Vacuolar protein sorting 34 (VPS34, class III PI(3)K complex) contains two acetylation sensitive lysine residues (K²⁹ and K⁷⁷¹) that are subject to inhibitory acetylation in fed conditions. Inhibitory acetylation of residues K⁴³⁰ and K⁴³⁷ in Beclin 1 (class III PI(3)K complex) is opposed by nutrient-sensitive SIRT1 KDAC. Within the ubiquitin-like conjugation systems, LC3 (K⁴⁹, K⁵¹), ATG5 (unknown), ATG7 (unknown) and ATG12 (unknown) are subject to inhibitory acetylation by p300 (not shown) in fed conditions. Acetylation of LC3, ATG5 and ATG7 residues is opposed by SIRT1 deacetylase. ATG3 is subject to activating acetylation by TIP60 in starved conditions. Acetylation of lysine residues K¹⁹, K⁴⁸ and K¹⁸³ (** in yeast) is necessary for ATG3 enzymatic activity and LC3 binding affinity. ATG3 and ATG7 are also subject to inactivation by oxidative stress due to oxidation of their catalytic thiols, C²⁶⁴ and C⁵⁷², respectively. c Selective autophagy receptor p62 is a target of both, acetylation and oxidation. TIP60-dependent activating acetylation of K⁴²⁰ and K⁴³⁵ residues within the ubiquitin-associated (UBA) domain prevent UBA dimerization and enhance ubiquitin (Ub) binding affinity. Oxidation of C¹⁰⁵ and C¹¹³ promotes p62 oligomerization and stimulates autophagy by intermolecular disulphide bond formation