Nutrient deprivation or cellular stress leads to the activation of a catabolic pathway that is conserved across species, known as autophagy. This process is considered to be adaptive and plays an important role in a number of cellular processes, including metabolism, immunity and development. Autophagy has also been linked to diseases, such as cancer and neurodegeneration, highlighting the importance of a better insight into its regulation. In the present chapter, we discuss how PTMs (post-translational modifications) of lysine residues by acetylation and ubiquitination alter the function of key proteins involved in the activation, maturation and substrate selectivity of autophagy. We also discuss the clinical potential of targeting these modifications to modulate autophagic activities.
Autophagy is a conserved catabolic process whereby, in response to nutrient deprivation or stress, a cell degrades proteins and/or organelles via the lysosomal pathway. There are three forms of autophagy: macroautophagy, microautophagy and CMA (chaperone-mediated autophagy). Although they are all activated in response to nutrient deprivation, they differ in their substrate-specificity, membrane source and mode of delivery to the lysosome (Table 1). Among the three forms of autophagy, macroautophagy, which encloses and delivers substrates to the lysosome via a double-membrane vesicle called the autophagosome, is best characterized and will be the main focus of this chapter.
Autophagy probably evolved in unicellular organisms to provide essential nutrients by digesting cellular constituents during periods of nutrient deprivation – a function that is conserved through mammals. However, the function of autophagy has grown beyond nutrient recycling, which is considered non-selective degradation. It has become clear that autophagy is also a dominant mechanism for organelle QC (quality-control), protein aggregate clearance and cellular defence against pathogens . Autophagy, in this context, demonstrates exquisite substrate selectivity, which is in contrast with the bulk degradation seen in starvation-induced autophagy. These findings not only greatly expand the functional repertoire of autophagy, but also reveal unexpected molecular diversity of this highly conserved degradation machinery.
Autophagy is a stress response that enhances cellular and organismal fitness; however, it can also contribute to disease development. For instance, autophagy can sustain cancerous cells under low nutrient conditions by providing essential nutrients and macromolecules. Autophagy can also limit oxidative stress by degrading mitochondria that are damaged by cancer drugs, which might cause resistance to anti-cancer therapies. On the other hand, loss of autophagy through mutations of Beclin-1 (Atg6) can predispose individuals to cancer (reviewed in ). The complex biology of autophagy is also observed in heart disease, where autophagy can function as a protective mechanism during ischaemic injury, yet it can be harmful during reperfusion . These complicated effects highlight the importance of maintaining an adequate level of autophagy. As such, understanding how autophagy is regulated could help to develop therapies targeting autophagy in different diseases. In this chapter, we will discuss how PTMs (post-translational modifications) of lysine residues, including acetylation and ubiquitination, modulate autophagy, as well as the implications in disease.
Ubiquitination in autophagy
Protein ubiquitination involves conjugation of Ub (ubiquitin) molecules on to protein substrates sequentially catalysed by E1 (Ub-activating), E2 (Ub-conjugating) and E3 (Ub ligase) enzymes (Figure 1). In macroautophagy, a group of autophagy proteins (ATG proteins) catalyse biochemical reactions analogous to the Ub-conjugation system (Figure 1). Different from the protein Ub system, Atg8 (also known as MAP1-LC3), which adopts an Ub-like fold, is conjugated to lipids [PE (phosphatidylethanolamine)] instead of proteins, like the Atg5–Atg12 complex, to stimulate auophagosomal membrane formation. The Atg5–Atg12 complex is also formed through a Ub-like conjugation system (Figure 1). Other than these intriguing similarities, ubiquitination also regulates autophagy by several different mechanisms discussed below.
Ubiquitination of Atg proteins
Atg1, which is known as Ulk1/2 in mammalian cells, is a serine/threonine kinase essential for induction of autophagy as it regulates localization of other Atg proteins at the phagophore assembly site. Nutrient deprivation triggers the autophagic response in part through AMPK (AMP-activated protein kinase), which associates with and phosphorylates Ulk1 only when nutrients are unavailable. This AMPK-mediated phosphorylation activates Ulk1 kinase activity to drive autophagy. On the other hand, mTOR (mammalian target of rapamycin), which blocks autophagy, induces inhibitory phosphorylation of Ulk1, which prevents activation by AMPK. The AMPK-mTOR pathway will be discussed in more detail in a later section. Interestingly, in response to NGF (nerve growth factor) signalling, Ulk1 becomes modified by Lys63-linked Ub chains, which typically are not associated with proteasome-mediated degradation. This modification leads to Ulk1 binding by a Ub-binding protein, p62, which results in attenuation of the NGF receptor, TrkA, signalling in sensory neurons . This is of interest in autophagy, as p62 is also a key component of selective autophagy, as discussed in more detail in the following section. Furthermore, PI3K (phosphoinositide 3-kinase) and Akt, which are positive regulators of mTOR, are downstream targets of NGF/TrkA signalling . Thus NGF-induced Lys63-polyubiquitination of Ulk1 could potentially link NGF signalling to the autophagy machinery.
Beclin-1 (Atg6) is a key component of the PI3K complex responsible for autophagy activation. Beclin-1 can be inhibited by binding to Bcl-2 or Bcl-xL . Beclin 1 is also subject to Lys63-specific ubiquitination, which is catalysed by the E3 ligase TRAF6 [TNF (tumour-necrosis-factor)-receptor-associated factor 6]; interestingly, TRAF6 is a p62-interactive protein. In macrophages activated by LPS (lipopolysaccharide), TRAF6-mediated ubiquitination blocks the Bcl-2/Beclin-1 interaction, thereby permitting TLR4 (Toll-like receptor 4)-mediated autophagy, which is thought to enhance the clearance of ingested microbes. Recently, a deubiquitinase, A20, was found to inhibit TLR4-induced autophagy by removing the Ub from Beclin-1, which restores the interaction between Beclin-1 and Bcl-2  (Figure 2). Thus TRAF6 and A20 regulate Beclin-1 ubiquitination in order to control the induction of autophagy in response to inflammation.
Ub in selective and QC autophagy
Autophagy has been traditionally considered a non-selective degradation process, where cytosolic contents are digested to recycle macromolecules as part of the emergency response to starvation. However, since autophagy is also responsible for the clearance of protein aggregates and damaged or supernumerary organelles, it must also possess substrate selectivity. Protein aggregates derived from misfolded proteins are almost always ubiquitinated. This Ub modification, in turn, is recognized by Ub-binding p62/sequestersome 1 and the related protein NBR1 [neighbour of BRCA1 (breast cancer early-onset 1) gene 1]. p62 was initially characterized as a protein that binds and coalesces ubiquitinated proteins into large aggregates and inclusion bodies . Subsequently, it was discovered that p62 also binds LC3, the central component of the autophagosome. p62 and NBR1 can simultaneously bind both LC3 and ubiquitinated substrates via their LIR (LC3-interacting region) and UBA (Ub-association) domains respectively  (Figure 2). This dual binding activity led to the proposal that p62 and NBR1 act as adaptor proteins that recruit autophagosomes to ubiquitinated protein aggregates and other substrates (reviewed in ). In addition to p62, aggregates are also associated with HDAC6 (histone deacetylase 6), a protein deacetylase with intrinsic Ub-binding activity. As we will discuss later in this chapter, HDAC6 stimulates autophagy maturation. In essence, by recruiting Ub-binding p62 and HDAC6, specific Ub modification provides both specificity and efficiency for autophagy to dispose of protein aggregates.
Ubiquitination in the clearance of damaged mitochondria
Mitophagy refers to the removal of mitochondria by autophagy. By selectively eliminating damaged mitochondria, mitophagy serves as an important QC mechanism, which ensures a healthy population of mitochondria within a cell. The molecular mechanism of mitophagy has recently come to light from studies of two proteins encoded by genes mutated in familial forms of PD (Parkinson's disease: an E3 ligase, parkin, and the mitochondria-localized serine-threonine kinase, PINK1 [PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1]. In mammalian cell-based models, PINK1 becomes stabilized on the outer mitochondrial membrane in response to mitochondrial depolarization, and this recruitment is thought to label the damaged organelles (reviewed in ). PINK1 then recruits parkin from the cytosol to the depolarized mitochondria, where parkin ubiquitinates many mitochondrial proteins, which are subsequently degraded by proteasomes . In addition, parkin-mediated ubiquitination is required for mitophagy, as parkin mutants deficient in Ub E3 ligase activity do not ubiquitinate mitochondria and do not support mitophagy (reviewed in ). In fact, many parkin mutants associated with familial PD are defective in mitophagy. In principle, a defect in mitophagy would certainly lead to deterioration in mitochondrial function, which is indeed a prominent phenotype in PD patients and animals deficient in parkin or PINK1.
Autophagy-mediated removal of protein aggregates and impaired mitochondria share many features. Both entities are transiently concentrated to the perinuclear region, forming Ub-positive inclusion body-like structures . These structures, termed the aggresome or mito-aggresome, are enriched for p62, HDAC6 and proteasomes. In mitophagy, parkin-mediated ubiquitination of mitochondria is required for recruiting both HDAC6 and p62, which probably work in tandem to clear the Ub-tagged mitochondria  (Figure 2). Although the absolute requirement of these factors for mitophagy remains controversial (reviewed in ), these findings support a critical role for ubiquitination in the execution of mitophagy and provide a molecular mechanism for the prevalence of mitochondrial dysfunction and inclusion bodies in PD.
Polyubiquitin chains can form via different lysine residues in Ub. While Lys48-linked Ub chains are best characterized as the recognition signal for proteasome-mediated degradation, it has become apparent that different lysine-linked Ub chains, such as Lys63 or Lys27, could have different functions. Whether a specific type of Ub modification serves as a targeting signal for autophagy remains to be determined. Earlier studies have indicated that p62 and HDAC6 both preferentially bind Lys63-linked Ub chains [4,10]. Interestingly, parkin-mediated ubiquitination of mito-aggregates has been shown to predominantly involve an increase in Lys63-specific ubiquitination . These findings suggest that Lys63-linked Ub conjugation could be part of the signal for selective QC autophagy.
The involvement of Ub in mitophagy, however, is more complicated. In addition to recruiting the autophagy machinery, parkin also recruits the proteasome to depolarized mitochondria, where it degrades outer mitochondrial membrane proteins [8,12,13]. In this context, these outer membrane proteins are probably modified with Lys48-linked and Lys27-linked Ub species, which would target them to the proteasome . Paradoxically, proteasome-dependent degradation of mitochondrial proteins operates independently of autophagy, but proteasomal activity was shown to be required for mitophagy [8,12]. These findings reveal a complex mode of protein ubiquitination events required for mitophagy.
Role of ubiquitination in xenophagy
Xenophagy, or the autophagy of foreign micro-organisms, has been implicated in the host defence mechanism against invasion by bacteria. Listeria, Shigella and Salmonella typhimurium have been shown to be ubiquitinated upon entrance into host cells. This ubiquitination is followed by recruitment of p62 and LC3 and clearance of the organisms (reviewed in ). This observation follows the recurrent theme of Ub-dependent autophagy. In response to this host cell defence system, many bacteria have evolved mechanisms to subvert ubiquitination in order to achieve successful infection. For instance, Listeria encodes ActA, which recruits host proteins into an actin-binding scaffold that acts as a shield, disguising the bacteria from recognition by host ubiquitination machinery (reviewed in ). Insight into the mechanisms through which bacteria can subvert the autophagic response to infection could lead to novel therapeutics to treat these diseases.
Acetylation in autophagy
Lysine acetylation is best known for its key role in the histone code, wherein it dictates the dynamic output of gene transcription (reviewed in ). However, studies have revealed that a large number of non-histone proteins are also subject to acetylation, suggesting a broad regulatory role for acetylation. Reversible lysine acetylation is catalysed by acetyltransferases, including CBP [CREB (cAMP-response-element-binding protein)-binding protein]/p300, TIP60 [Tat (transactivator of transcription)-interactive protein 60 kDa] and PCAF [p300 (E1A-associated protein of 300 kDa)/CBP-associated factor] family members, and removed by HDACs, which are divided into classes I, II and III. Class I and II HDACs are structurally related, whereas class III HDACs, also known as sirtuins, constitute a distinct deacetylase group. Both HDACs and sirtuins, as well as acetyltransferases such as p300, play important regulatory roles in autophagy.
Acetylation of Atg proteins in nutrient-regulated autophagy
One prominent feature of the sirtuin family is their unique dependence on NAD+ for enzymatic activity. The central role of NAD+ in metabolism has therefore implicated sirtuins in various aspects of nutrient and metabolic regulation, which are intimately linked to autophagy. Yeast Sir2 (silent information regulator 2), the founding member, has been extensively studied for its role in aging (reviewed in ). Sirtuins, especially SIRT1, have also being implicated in lifespan extension induced by CR (caloric restriction) in other organisms. Although the role of ectopic SIRT1 in lifespan extension in multicellular organisms has recently been called into question , CR has been shown to activate SIRT1 and induce autophagy. Indeed, overexpression of SIRT1 can also induce autophagy in vitro. These findings provide a plausible link between nutrients, sirtuins and autophagy in lifespan regulation (reviewed in ).
Atg proteins are essential regulators of macroautophagy from yeast to mammals. Interestingly, a number of these proteins are reversibly acetylated. Atg5, Atg7, Atg8 and Atg12, which are required for effective autophagosome formation, can all be acetylated and inhibited by the acetyltransferase p300, which interacts with Atg proteins when nutrients are abundant (Figure 2) . As nutrients are depleted, sirtuins become activated, which leads to deacetylation of these Atg proteins. This relieves the inhibitory acetylation and allows autophagy to progress. Ectopic SIRT1 has also been shown to form a complex with Atg5, Atg7 and Atg8, increasing basal levels of autophagy (reviewed in ). In support of a role for SIRT1 as the deacetylase of these Atg proteins, SIRT1-knockout mice show an increase in basal acetylation of these proteins and fail to fully activate autophagy upon starvation (reviewed in ).
In addition to SIRT1, class I HDAC members are also involved in activation of autophagy. In HDAC1/HDAC2 double-knockout muscle, fasting-induced autophagy was impaired, and progressive myopathy occurred . Importantly, the myopathy and autophagy defects can be effectively suppressed by a high-fat diet. Although the specific molecular mechanism remains to be established, these findings support a critical role for HDAC1 and HDAC2 in nutrient supply and metabolic adaptation in skeletal muscle, which is probably linked to autophagy.
Acetylation in AMPK-mTOR signalling
Metabolism and autophagy are intimately linked. Changes in environmental nutrient availability and internal energy status can dominantly influence autophagy. This regulation is mainly controlled by mTOR, a kinase that integrates both nutrient and energy information to co-ordinate key cellular activities, including ribosome biogenesis, cell growth and autophagy . When nutrients are abundant, active mTOR inhibits autophagy. Conversely, nutrient deprivation inactivates mTOR, which results in activation of autophagy. A decrease in intracellular ATP levels also inactivates mTOR. This is mediated by AMPK, which is activated by the tumour suppressor LKB (liver kinase B) kinase and its cofactors, MO25 and STRAD. LKB1 is also subject to acetylation, which can be reversed by SIRT1 (Figure 2). LKB1 deacetylation enhances its interaction with its cofactors, thereby increasing its kinase activity  towards AMPK. Acetylation of LKB, therefore, provides an additional mechanism by which sirtuins regulate autophagy.
Recently, AMPK activity in yeast was shown to be associated with a decrease in replicative lifespan extension . An activating mutation of the AMPK repressor (Sip2) that mimics acetylation enhanced lifespan. Also, it was shown that Sip2 acetylation levels decreased during aging. Yeast replicative lifespan is thought to require autophagy, which can be driven by AMPK activation. However, these paradoxical findings indicate that repression of AMPK results in lifespan extension. This indicates that AMPK may play a role in intrinsic aging, independent of a CR-mediated nutrient-sensing pathway.
Acetylation in clearance of protein aggregates
Protein aggregates are normally cleared through autophagy. Failure of this process can result in the build-up of toxic protein aggregates, such as the aggregates of mHtt [mutant Htt (huntingtin)] in HD (Huntington's disease). Recently, Htt was shown to be acetylated. Increasing Htt acetylation by expressing the acetyltransferase CBP or inhibition of the deacetylase HDAC1 accelerated Htt aggregate clearance by autophagy and conferred neuroprotection . This mode of regulation highlights the exciting therapeutic possibility of targeting Htt acetylation in HD. Indeed, HDAC inhibitors can ameliorate the HD phenotype in animal models . Whether this acetylation-dependent clearance is applicable to other disease-associated protein aggregates remains to be established.
Acetylation regulates maturation of autophagy
The acetylation status of microtubules has been linked to autophagy. Tubulin acetylation, which is regulated by the class II deacetylase HDAC6, affects motor-dependent transport . During starvation, the Atg12–Atg5 conjugate is transported along microtubules, and this recruitment depends upon tubulin acetylation (reviewed in ). On the other hand, in QC autophagy, HDAC6 deacetylates tubulin to promote the enrichment of lysosomes and autophagosomes at the MTOC (microtubule-organizing centre) via retrograde transport . This concentration of autophagosomes and lysosomes at the MTOC should increase the efficiency of autophagy by increasing the probability of autophagosome–lysosome fusion.
HDAC6 is dispensable for starvation-induced autophagy. In contrast, HDAC6 is required for efficient autophagosome–lysosome fusion associated with clearance of protein aggregates and damaged mitochondria. This finding clearly demonstrates that autophagy consists of distinct forms . In HDAC6-dependent autophagy, termed QC autophagy, HDAC6 stimulates autophagosome–lysosome fusion by deacetylation and activation of cortactin, a key regulator of F-actin (filamentous actin) assembly (Figure 2). F-actin network assembly potentially provides a platform for vesicle fusion. Thus acetylation of both cortactin and microtubules is regulated by HDAC6 to modulate the efficiency of QC autophagy.
Autophagy is not only a physiological response, it also contributes to various diseases, including PD and HD. Thus it is highly desirable to develop pharmacological agents that modulate autophagy. Evidence strongly supports an important and complex regulatory role for lysine-targeted PTMs in autophagy. Importantly, enzymes that catalyse reversible acetylation are amenable to pharmacological inhibition. Inhibitors for DUBs (deubiquitinating enzymes) are also being developed . These PTM-modulating compounds could be useful in manipulating and fine-tuning the activity of autophagy activity, thereby providing therapeutic effects in a number of diseases.
• Lysine residues can be modified by acetylation or ubiquitination to regulate autophagy.
• The ubiquitination status of proteins is regulated by the opposing activities of ubiquitin ligases and deubiquitinases.
• Lys63-linked ubiquitination of Beclin-1 blocks its interaction with Bcl-2/Bcl-xL to allow for activation of autophagy; Beclin-1 is ubiquitinated by TRAF6 and deubiquitinated by A20.
• Adaptor proteins (p62, NBR1) can target ubiquitinated substrates for degradation through binding to LC3 (Atg8).
• Lys63-linked ubiquitination of mitochondria is increased during parkin-mediated mitophagy.
• During parkin-mediated mitophagy, mitochondrial proteins undergo both Lys63-linked ubiquitination and degradation by the proteasome, although proteasomal degradation is independent of mitophagy.
• The acetylation status of proteins is regulated by the opposing activities of acetyltransferases and deacetylases.
• Sirtuins are activated by nutrient deprivation and can deacetylate Atg proteins, as well as AMPK, LKB1 and mTOR.
• In mammals, LKB1 deacetylation increases phosphorylation and activation of AMPK, which inhibits mTOR to promote autophagy.
• Acetylation of mHtt facilitates its clearance by autophagy.
• HDAC6 regulates deacetylation of tubulin and cortactin to mediate QC autophagy and promotes concentration of ubiquitinated substrates at the MTOC through its BUZ (Ub-binding zinc finger) domain.
• Direct targeting of PTMs that regulate autophagy (ubiquitination in PD, acetylation in HD etc.) may allow for more specific therapeutics in disease.
- © The Authors Journal compilation © 2012 Biochemical Society