New links between protein N-terminal acetylation, dauer diapause, and the insulin/IGF-1 signaling pathway in Caenorhabditis elegans

Abstract

Protein N-terminal acetylation is a widespread posttranslational modification in eukaryotes that is catalyzed by N-terminal acetyltransferases (NATs). The biochemical activity of NATs has been characterized extensively, whereas the biological function of NATs is only beginning to be defined. Here we comment on recent progress in understanding the function of NAT activity in C. elegans based on the characterization of natc-1 by Warnhoff et al. (2014) and daf-31 by Chen et al. (2014).^1,2 natc-1 encodes an auxiliary subunit of the NatC complex and modulates stress tolerance, dauer entry, and adult lifespan. daf-31 encodes the catalytic subunit of the NatA complex and affects dauer entry, dauer formation, and adult lifespan. The analysis of these genes and genetic studies of NATs in other organisms suggests protein N-terminal acetylation plays an evolutionarily conserved role in promoting growth and development and inhibiting stress resistance. Furthermore, we propose that NATs may regulate growth and development in response to external cues such as nutrient deprivation and other physiologic stresses.

Background

Organisms have evolved to cope with a wide variety of stressful conditions in their quest to survive, grow, develop, and ultimately reproduce. Multiple strategies are employed, including avoiding stressful environments, adopting forms that are protective against stress-related damage, and repairing damage that does occur. The analysis of these strategies has revealed important information about mechanisms of repair and turnover of macromolecules, environmental sensing, and development. These topics have important implications for human health, since people are exposed to a wide variety of toxic compounds and stressful environments.

Caenorhabditis elegans is an important animal model system for studies of stress tolerance. When challenged with high temperature, low nutrient availability, and high population density, C. elegans larvae enter dauer diapause, an alternate third larval stage that is stress resistant. Genetic studies of dauer formation led to the discovery of an evolutionarily conserved insulin/insulin-like growth factor (IGF-1) pathway (reviewed by Hu (2007)³). The C. elegans insulin-like receptor tyrosine kinase, DAF-2, signals through a protein kinase cascade to inhibit the function of the FOXO transcription factor DAF-16. DAF-16 and the target genes it regulates have been analyzed extensively because of their functions in stress tolerance, dauer formation, and adult longevity.^4,5 Here we discuss 2 recent publications in PLOS Genetics, Warnhoff et al. (2014) and Chen et al. (2014), that advance the understanding of stress resistance and the insulin/IGF-1 pathway by describing new roles for protein N-terminal acetylation.^1,2 These results are exciting because they contribute to a growing body of evidence from yeast, plants, and animals implicating this post-translational protein modification in specific biological processes.

N-terminal acetyltransferases (NATs) are multi-subunit enzymes that catalyze the transfer of the acetyl group of acetyl coenzyme A to the α-amino group of the first amino acid of a target protein (Fig. 1). N-terminal acetylation is a widespread posttranslational modification affecting the majority of eukaryotic proteins; for example, ∼80–85% of human proteins are N-terminally acetylated.⁶ Eukaryotes possess multiple NAT complexes that vary in subunit composition and substrate specificity, named NatA, NatB, NatC, etc. (reviewed by Starheim et al. (2012)⁷). While the biochemical activity of NATs is well characterized, the functional consequences of N-terminal acetylation of specific proteins and the biological function of these enzymes is only beginning to be determined.

Figure 1.

Protein N-terminal acetylation is a posttranslational modification catalyzed by NAT complexes. NAT complexes are composed of a catalytic subunit (shaded) and usually one or more auxiliary subunits (open). NAT complexes catalyze the transfer of the acetyl group of acetyl-CoA to the α-amino group of the first amino acid of the target protein. For the NatA complex, the amino acid following Met (Xaa) is typically Ser, Ala, Thr, Gly, Val, or Cys. For the NatC complex, Xaa is typically Ile, Leu, Trp, or Phe.

natc-1 encodes an auxiliary subunit of the NatC complex that influences stress tolerance, dauer formation, and lifespan and is regulated by the insulin/IGF-1 pathway

Zinc is an essential nutrient for C. elegans and all forms of life; however, excess zinc is toxic, and the ability to tolerate high levels of zinc is a type of stress resistance.⁸ To identify genes involved in this type of stress resistance, Bruinsma et al. (2008) performed a forward genetic screen for worms that are resistant to the toxicity caused by high levels of dietary zinc and isolated 2 mutations in natc-1.⁹ Warnhoff et al. (2014) identified the affected gene by positional cloning and demonstrated that the natc-1(am138) and natc-1(am134) mutations cause a strong loss-of-function. natc-1 is predicted to encode a protein homologous to human Naa35, an auxiliary subunit of the NatC complex that acetylates translating proteins that begin with Met-Ile, Met-Leu, Met-Trp, or Met-Phe.¹⁰ The natc-1 expression pattern was inferred from transgenic animals expressing NATC-1::GFP fusion protein. NATC-1 is expressed throughout development in multiple tissues including the pharynx, intestine, vulva, somatic gonad, and body wall muscles. These observations suggest that natc-1(lf) mutations disrupt the function of the NatC complex, resulting in altered N-terminal acetylation of multiple proteins in a variety of tissues. Consistent with this interpretation, RNAi against natc-2, which encodes the predicted catalytic subunit, causes overlapping defects.¹ However, changes in protein acetylation have not been analyzed biochemically in mutant animals.

A detailed analysis revealed that natc-1(lf) mutations have multiple effects. In addition to increasing tolerance to high dietary zinc, natc-1(lf) mutations also increase tolerance to high levels of other transition metals, high heat, and excess oxidation. These findings indicate that natc-1 function is necessary for wild-type levels of sensitivity to a wide range of stressful conditions. The formation of dauer larvae is an important developmental response to unfavorable growth conditions during larval development. Although natc-1(lf) mutants do not display an independent dauer-constitutive (Daf-c) phenotype, the natc-1(lf) mutations strongly enhance the Daf-c phenotype of daf-2(lf) mutant animals. Thus, natc-1 is necessary to inhibit dauer formation in a sensitive genetic background. Furthermore, natc-1(lf) animals display a reduced lifespan, indicating that natc-1 is necessary for longevity under standard C. elegans growth conditions.

Although mutations of natc-1 had not been previously characterized, the natc-1 promoter had been noted to contain an evolutionarily conserved DAF-16 binding site.⁴ This binding site is likely to be physiologically relevant, since genome-wide studies of DAF-16 binding in vivo demonstrated an interaction with the natc-1 promoter.^11,12 Warnhoff et al. (2014) showed that transcription of natc-1 is repressed by DAF-16 activity. These findings indicate that natc-1 is a target of the insulin/IGF-1 pathway that is directly regulated by DAF-16. The genetic findings support these molecular conclusions, since the stress resistance caused by natc-1(lf) is epistatic to daf-16(lf). This genetic relationship is strikingly different from the upstream regulator daf-2, since the stress resistance cause by daf-2(lf) is not epistatic to daf-16(lf). Taken together, these findings demonstrate that natc-1 is an important effector of the insulin/IGF-1 pathway that functions downstream of DAF-16 to inhibit stress resistance and dauer formation.

The interpretation of mutations that increase stress resistance

Stress resistance is frequently interpreted as an adaptive trait; if animals live in environments with fluctuating levels of stress, then the ability to survive stressful extremes would appear to be an advantage during evolution. In light of this reasoning, it is perplexing that loss-of-function mutations can be identified that increase stress resistance. The conclusion from this observation is that the mutated gene is necessary to promote wild-type levels of stress sensitivity. In other words, the function of the gene causes the animals to be more vulnerable to stress. natc-1(lf) mutations increase resistance to a broad range of environmental stresses, suggesting that natc-1 function increases vulnerability to stress. Mutations in other genes cause a similar phenotype, such as daf-2(lf) mutations.^13,14 These observations raise 2 important issues: (1) What is the mechanism by which these mutations increase stress resistance and (2) If stress resistance is adaptive, then why aren't such mutations selected for during evolution?

Here we consider 2 general models for the mechanism of increased stress resistance displayed by mutant animals. The first model is that the mutation causes increased expression of proteins that promote stress resistance, such as heat shock proteins. An example of a direct mechanism would be a gene that inhibits transcription of heat shock genes; a loss-of-function mutation would cause increased transcription of heat shock genes and thereby increased stress resistance. An example of an indirect mechanism would be a gene that promotes protein folding. A loss-of-function mutation would cause proteins to be misfolded, which would stimulate the unfolded protein response and thereby increase stress resistance. This type of mechanism has been referred to as hormesis. The general concept of hormesis is that a perturbation that causes a minor dysfunction will stimulate a response that equips the organism to withstand a major stress in the future. For example, prior exposure to a mild heat shock causes increased resistance to a major heat shock, and animals that experienced the mild heat shock survive better than control animals kept in benign conditions.¹⁵

The second model is that the gene promotes an activity that causes vulnerability to stress. An example of this is histidine ammonia lyase (haly-1), a gene that encodes an enzyme that metabolizes histidine.¹⁶ haly-1(lf) mutants display accumulation of histidine, because it cannot be broken down. haly-1(lf) mutants display increased resistance to high zinc toxicity, because histidine functions as a chelator that detoxifies zinc, and haly-1(lf) mutant animals have more histidine than wild-type animals. Histidine ammonia lyase is a highly conserved enzyme that is present in humans and worms; it is likely to be conserved because under standard growth conditions the metabolites of histidine serve important functions and accumulation of histidine is not beneficial. However, in conditions of high zinc stress, haly-1 mutant animals survive whereas wild-type animals perish. haly-1 is a specific example of this general model, but many other examples can be imagined.

If stress resistance is adaptive, then why aren't mutations that increase stress resistance selected for during evolution? Because this is an evolutionary question, and we are reasoning from molecular genetic observations, our models are speculative. The goal of animal life is reproduction, and one possibility is that mechanisms that promote stress resistance also inhibit reproduction. For example, if accumulation of histidine promotes resistance to high zinc toxicity and metabolites of histidine promote reproduction, then animals face a tradeoff. If animals frequently encounter toxic levels of zinc in the environment, then it might be adaptive to reduce haly-1 activity and survive at the expense of generating fewer histidine metabolites that enhance reproduction. If animals rarely encounter toxic levels of zinc in the environment, then it might be adaptive to metabolize histidine and run the risk of vulnerability to high zinc toxicity. The concept is that in environments with fluctuating levels of stress, animals have evolved stress resistance mechanisms that are inducible and thus tuned to the environment. Mutations that constitutively activate these mechanisms are clearly adaptive in laboratory settings where animals are challenged with severe stresses, but are unlikely to be adaptive in native environments.

daf-31 encodes the catalytic subunit of the NatA complex that affects dauer entry, dauer formation, and adult lifespan

The daf-31 mutant was isolated in genetic screens for dauer-like mutants.¹⁷ Dauer mutants were originally classified as either dauer constitutive (Daf-c) or dauer defective (Daf-d). Dauer-like mutants define a third class characterized by animals that are incapable of executing either complete dauer or non-dauer development. Chen et al. (2014) used positional cloning to identify the affected gene and showed that the daf-31(m655) deletion mutation causes a strong loss-of-function.² daf-31 is predicted to encode the C. elegans homolog of human Naa10, the catalytic subunit of the NatA complex. The NatA complex acetylates translating proteins that begin with Met-Ser, Met-Ala, Met-Thr, Met-Gly, Met-Val, or Met-Cys.¹⁰ The daf-31 expression pattern was inferred from a daf-31 promoter fused to GFP and expressed in transgenic worms. The daf-31 gene is expressed in multiple tissues including the pharynx, intestine, hypodermis, and some neurons, indicating DAF-31 functions in many tissues. These observations suggest that daf-31(lf) mutant animals have multiple proteins with altered N-terminal acetylation, although this prediction was not confirmed biochemically.

The daf-31(lf) mutation causes lethality; homozygous mutant animals that are derived from heterozygous hermaphrodites arrest development at the larval stage. The lethality seems to occur because mutant animals form abnormal dauer larvae under standard lab conditions. daf-31 mutant animals have some characteristics of wild-type dauer larvae, such as a dark body, fully constricted pharynx, and a cuticle with dauer alae. Fat accumulation is an important characteristic of dauer larvae, and daf-31(lf) mutant animals accumulate fat droplets similar to known Daf-c mutants such as daf-2(e1370). However, these mutant animals are abnormal, since they are not SDS-resistant and cannot resume development when food is provided. Thus, daf-31 is necessary to inhibit dauer formation and promote reproductive development when food is available. Furthermore, daf-31 is necessary to form specific features of dauer larvae, such as SDS resistance.

To examine the role of daf-31 in adults, Chen et al. (2014) used feeding RNA interference (RNAi). Reducing the activity of daf-31 shortens adult lifespan in the RNAi sensitive rrf-3(pk1426) genetic background, although this effect was not observed in a wild-type genetic background (Bristol N2). Thus, daf-31 is necessary for wild-type longevity in a specific genetic background. The effect of increasing daf-31 activity was analyzed by introducing multiple copies of the wild-type gene into transgenic animals. Overexpression in a wild-type genetic background slightly decreased lifespan, whereas overexpression in daf-2(lf) animals enhanced the daf-2(lf) lifespan extension. Thus, daf-31 is sufficient to increase longevity in a specific genetic background. The effect of daf-31 overexpression on the insulin/IGF pathway is not fully defined, since it was not sufficient to influence DAF-16 nuclear localization, although it was sufficient to increase transcript levels of 2 DAF-16 target genes.

The analyses of natc-1 and daf-31 link protein N-terminal acetylation to reproductive development and adult longevity

natc-1 encodes an auxiliary subunit of the NatC complex, suggesting that natc-1(lf) mutations reduce the N-terminal acetylation of multiple proteins that begin with Met-Ile, Met-Leu, Met-Trp, or Met-Phe. daf-31 encodes the catalytic subunit of the NatA complex, suggesting that daf-31(lf) mutations reduce or abrogate the N-terminal acetylation of multiple proteins that begin with Met-Ser, Met-Ala, Met-Thr, Met-Gly, Met-Val, or Met-Cys. natc-1 and daf-31 are both expressed throughout development in many tissues, suggesting NatC and NatA mediate protein N-terminal acetylation in many cell types. Thus, while mutations in both genes are likely to cause widespread defects in protein N-terminal acetylation, there is probably no overlap in the specific proteins that are affected due to the distinct specificities of the enzyme complexes.

natc-1(lf) mutations cause a dauer constitutive (Daf-c) phenotype in a sensitized background, whereas daf-31(lf) mutations cause constitutive formation of dauer-like larva. Thus, natc-1 and daf-31 both promote the reproductive fate during larval development and inhibit dauer diapause. natc-1(lf) mutants display reduced adult lifespan, whereas overexpression of daf-31 is sufficient to extend adult lifespan in a specific genetic background. Thus, both genes appear to promote adult longevity.

natc-1 appears to be a downstream effector of the insulin/IGF-1 pathway. DAF-16 binds and regulates the natc-1 promoter, and genetic interactions with daf-2 and daf-16 support the model that natc-1 is a physiologically significant target of the insulin/IGF pathway. Genetic studies indicate daf-31 also interacts with daf-2 and daf-16, but the relationship of these genes remains to be clarified. Overall, these findings indicate that protein N-terminal acetylation is necessary for reproductive development during larval stages and longevity in adults. Disruption of protein N-terminal acetylation promotes entry into dauer diapause and shortens adult lifespan.

NATs appear to play evolutionarily conserved roles in balancing reproductive growth and development with stress resistance

The functions of specific NATs have been characterized using genetic analysis in organisms as diverse as yeast, plants, worms, flies, and humans (reviewed in^7,18). Table 1 shows genes encoding NatA and NatC subunits from multiple species and the phenotypes caused by mutations or knock down of these genes. These observations reveal intriguing similarities that suggest protein N-terminal acetylation modulates growth, development, and reproduction in multiple organisms. Furthermore, several of these genes also modulate stress tolerance and entry into stress tolerant developmental stages. Thus, the activity of NATs may balance growth and development with survival and stress tolerance. Here we highlight the analysis of genes from yeast, worms, and plants that are consistent with this model.

Table 1.

Genetic analyses of NatA and NatC

NatA Complex
Organism	Gene	Subunit Type	Mutant Phenotypes1	Reference1
S. cerevisiae	ARD1	Catalytic	Inability to enter stationary phase, sporulate, and respond to α-factor	Whiteway et al. (1985)
C. elegans	daf-31	Catalytic	Larval lethal and dauer-like	Chen et al. (2014)
D. melanogaster	Ard1	Catalytic	Lethal, female sterility, oogenesis defects	Wang et al. (2010)
H. sapiens	hNaa10p	Catalytic	Ogden syndrome: infant lethal, global developmental delays, aged appearance, craniofacial anomalies, cardiac arrhythmia	Rope et al. (2011)
NatC Complex
S. cerevisiae	MAK10	Auxiliary	Sub-optimal growth on nonfermentable carbon sources	Lee et al. (1992)
C. elegans	natc-1	Auxiliary	Stress resistance, synthetic dauer constitutive, and decreased lifespan	Warnhoff et al. (2014)
A. thaliana	atmak3–1	Catalytic	Reduced photosynthesis and slow growth	Pesaresi et al. (2003)
D. rerio	egap	Auxiliary	Embryonic lethal and slow growth	Wenzlau et al. (2006)
H. sapiens	hMak3	Catalytic	Increased apoptosis and reduced cell growth	Starheim et al. (2009)

¹Mutant phenotypes are caused by reducing gene activity. Studies of ARD1, daf-31, Ard1, hNaa10p, MAK10, natc-1, and atmak3–1 were performed with chromosomal loss-of-function mutations. Studies of egap were conducted with morpholinos, and studies of hMak3 were conducted with small interfering RNAs.

²Citations are: ARD1,²⁴ daf-31,² Ard1,²⁷ hNaa10p,²⁸ MAK10,²⁶ natc-1¹ atmak3–1,¹⁹ egap,²⁹ and hMak3.³⁰

Mutations in genes encoding NatC subunits have been analyzed in yeast (MAK10), worms (natc-1), plants (atmak3–1), fish (egap), and humans (hMak3). In the plant Arabidopsis thaliana, a mutation that affects the catalytic subunit of the NatC complex was identified because it dramatically reduces photosynthetic efficiency. atmak3–1 mutants also displayed a reduction in growth rate and overall size.¹⁹ Plants that evolve in low-resource, stressful environments share a common set of traits including low rates of growth, photosynthesis, tissue turnover, and nutrient absorption. This phenomenon is referred to as “stress resistance syndrome” (SRS), and SRS plants resist a wide range of physiological stresses.^20-23 atmak3–1 loss of function causes reduced photosynthetic activity and growth rates, which are traits of SRS. SRS has interesting analogies with dauer diapause in C. elegans, since both strategies allow organisms to balance growth and reproduction with stress tolerance and survival. Loss of function in natc-1 promotes dauer diapause and stress tolerance, traits reminiscent of SRS. These observations suggest there may be a conserved role for the NatC complex in balancing organismal growth and reproduction with the ability to tolerate stressful environments.

Mutations in genes encoding NatA subunits have been analyzed in yeast (ARD1), worms (daf-31), flies (Ard1) and humans (hNaa10p). In the yeast S. cerevisiae, ARD1 is necessary for entry into stationary phase and for sporulation during nitrogen deprivation.²⁴ Yeast in stationary phase display a thickened cell wall, accumulation of reserve carbohydrates, and resistance to environmental stresses such as high heat.²⁵ Stationary phase has similarities to dauer formation in C. elegans, since both are stress resistant. Both dauer and sporulation are developmental stages that promote survival during nutrient deprivation, a physiologic stress. daf-31 is necessary to promote reproductive development and inhibit entry into the dauer stage of development; daf-31 is also necessary for normal dauer larvae development. These observations suggest a role for the NatA complex and protein N-terminal acetylation in developmental switches that respond to nutrient limitation.

Regulation of NAT activity

An important topic that is just beginning to be explored is the regulation of NAT activity. The prevalence of N-terminal acetylation among eukaryotes raises a major question: is N-terminal acetylation a constitutive modification or are levels of N-terminal acetylation regulated in response to intrinsic developmental signals or extrinsic environmental cues?

We postulate that NAT activity has the capacity to shift the balance between reproductive growth and stress resistance. Therefore, in theory it is possible that organisms alter NAT activity to control these shifts, thus allowing for more flexibility in response to environmental stresses. Warnhoff et al. (2014) showed that natc-1 is transcriptionally regulated by the insulin/IGF-1 pathway, a pathway known to respond to environmental cues such as nutrient deprivation and other physiologic stresses. In addition, the yeast NatC auxiliary subunit MAK10 is repressed by glucose, a critical nutrient.²⁶ These are important demonstrations of NAT regulation that suggest NAT activity is altered by environmental factors such as nutrient availability. The mechanism by which DAF-16-mediated reduction of natc-1 expression influences stress resistance remains an important question. A detailed understanding is likely to require the identification of specific target proteins, the characterization of how reduced acetylation affects individual protein function, and elucidation of the consequences for the whole animal.

The function and regulation of NAT complexes is only beginning to be characterized and the discovery of roles for natc-1 and daf-31 are important advances for the field. The powerful genetic and molecular techniques available in C. elegans will be valuable for addressing key questions about the role of protein N-terminal acetylation in animal physiology.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.