N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity.

Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility.

The Final Maturation State of ß-actin Involves N-terminal Acetylation by NAA80, not N-terminal Arginylation by ATE1.

Actin is a hallmark protein of the cytoskeleton in eukaryotic cells, affecting a range of cellular functions. Actin dynamics is regulated through a myriad of actin-binding proteins and post-translational modifications. The mammalian actin family consists of six different isoforms, which vary slightly in their N-terminal (Nt) sequences. During and after synthesis, actins undergo an intricate Nt-processing that yields mature actin isoforms. The ubiquitously expressed cytoplasmic ß-actin is Nt-acetylated by N-alpha acetyltransferase 80 (NAA80) yielding the Nt-sequence Ac-DDDI-. In addition, ß-actin was also reported to be Nt-arginylated by arginyltransferase 1 (ATE1) after further peptidase-mediated processing, yielding RDDI-. To characterize in detail the Nt-processing of actin, we used state-of-the-art proteomics. To estimate the relative cellular levels of Nt-modified proteoforms of actin, we employed NAA80-lacking cells, in which actin was not Nt-acetylated. We found that targeted proteomics is superior to a commercially available antibody previously used to analyze Nt-arginylation of ß-actin. Significantly, despite the use of sensitive mass spectrometry-based techniques, we could not confirm the existence of the previously claimed Nt-arginylated ß-actin (RDDI-) in either wildtype or NAA80-lacking cells. A very minor level of Nt-arginylation of the initially cleaved ß-actin (DDDI-) could be identified, but only in NAA80-lacking cells, not in wildtype cells. We also identified small fractions of cleaved and unmodified ß-actin (DDI-) as well as cleaved and Nt-acetylated ß-actin (Ac-DDI-). In sum, we show that the multi-step Nt-maturation of ß-actin is terminated by NAA80, which Nt-acetylates the exposed Nt-Asp residues, in the virtual absence of previously claimed Nt-arginylation.

Human NAA30 can rescue yeast mak3∆ mutant growth phenotypes.

N-terminal acetylation is an irreversible protein modification that primarily occurs co-translationally, and is catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). The NatC complex (NAA30-NAA35-NAA38) is a major NAT enzyme, which was first described in yeast and estimated to N-terminally acetylate ∼20% of the proteome. The activity of NatC is crucial for the correct functioning of its substrates, which include translocation to the Golgi apparatus, the inner nuclear membrane as well as proper mitochondrial function. We show in comparative viability and growth assays that yeast cells lacking MAK3/NAA30 grow poorly in non-fermentable carbon sources and other stress conditions. By using two different experimental approaches and two yeast strains, we show that liquid growth assays are the method of choice when analyzing subtle growth defects, keeping loss of information to a minimum. We further demonstrate that human NAA30 can functionally replace yeast MAK3/NAA30. However, this depends on the genetic background of the yeast strain. These findings indicate that the function of MAK3/NAA30 is evolutionarily conserved from yeast to human. Our yeast system provides a powerful approach to study potential human NAA30 variants using a high-throughput liquid growth assay with various stress conditions.

Classification and phylogeny for the annotation of novel eukaryotic GNAT acetyltransferases.

The enzymes of the GCN5-related N-acetyltransferase (GNAT) superfamily count more than 870 000 members through all kingdoms of life and share the same structural fold. GNAT enzymes transfer an acyl moiety from acyl coenzyme A to a wide range of substrates including aminoglycosides, serotonin, glucosamine-6-phosphate, protein N-termini and lysine residues of histones and other proteins. The GNAT subtype of protein N-terminal acetyltransferases (NATs) alone targets a majority of all eukaryotic proteins stressing the omnipresence of the GNAT enzymes. Despite the highly conserved GNAT fold, sequence similarity is quite low between members of this superfamily even when substrates are similar. Furthermore, this superfamily is phylogenetically not well characterized. Thus functional annotation based on sequence similarity is unreliable and strongly hampered for thousands of GNAT members that remain biochemically uncharacterized. Here we used sequence similarity networks to map the sequence space and propose a new classification for eukaryotic GNAT acetyltransferases. Using the new classification, we built a phylogenetic tree, representing the entire GNAT acetyltransferase superfamily. Our results show that protein NATs have evolved more than once on the GNAT acetylation scaffold. We use our classification to predict the function of uncharacterized sequences and verify by in vitro protein assays that two fungal genes encode NAT enzymes targeting specific protein N-terminal sequences, showing that even slight changes on the GNAT fold can lead to change in substrate specificity. In addition to providing a new map of the relationship between eukaryotic acetyltransferases the classification proposed constitutes a tool to improve functional annotation of GNAT acetyltransferases.

PFN2 and NAA80 cooperate to efficiently acetylate the N-terminus of actin.

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirm this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin, and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics.

Mechanism of actin N-terminal acetylation.

About 80% of human proteins are amino-terminally acetylated (Nt-acetylated) by one of seven Nt-acetyltransferases (NATs). Actin, the most abundant protein in the cytoplasm, has its own dedicated NAT, NAA80, which acts posttranslationally and affects cytoskeleton assembly and cell motility. Here, we show that NAA80 does not associate with filamentous actin in cells, and its natural substrate is the monomeric actin-profilin complex, consistent with Nt-acetylation preceding polymerization. NAA80 Nt-acetylates actin-profilin much more efficiently than actin alone, suggesting that profilin acts as a chaperone for actin Nt-acetylation. We determined crystal structures of the NAA80-actin-profilin ternary complex, representing different actin isoforms and different states of the catalytic reaction and revealing the first structure of NAT-substrate complex at atomic resolution. The structural, biochemical, and cellular analysis of mutants shows how NAA80 has evolved to specifically recognize actin among all cellular proteins while targeting all six actin isoforms, which differ the most at the amino terminus.

Actin Post-translational Modifications: The Cinderella of Cytoskeletal Control.

Actin is one of the most abundant proteins in eukaryotic cells and the main component of the microfilament system. It plays essential roles in numerous cellular activities, including muscle contraction, maintenance of cell integrity, and motility, as well as transcriptional regulation. Besides interacting with various actin-binding proteins (ABPs), proper actin function is regulated by post-translational modifications (PTMs), such as acetylation, arginylation, oxidation, and others. Here, we explain how actin PTMs can contribute to filament formation and stability, and may have additional actin regulatory functions, which potentially contribute to disease development.

Actin polymerization and cell motility are affected by NAA80-mediated posttranslational N-terminal acetylation of actin.

Actin is the most abundant protein in our cells, and also one of the most studied. Nevertheless, an important modifier of actin, the N-terminal acetyltransferase (NAT) for actin, remained unknown until now. The recent identification of the enzyme that catalyzes actin acetylation, has opened up for functional studies of unacetylated actin using knockout cells. This enzyme, called NAA80 (Nα-acetyltransferase 80) or NatH, belongs to the NAT family of enzymes, which together provides N-terminal acetylation for around 80 % of the human proteome. In many cases, N-terminal acetylation is essential. In the case of actin, the acetyl group that NAA80 attaches to actin plays an important role in actin's polymerization properties as well as in actin's function in cell migration.

NAA80 is actin's N-terminal acetyltransferase and regulates cytoskeleton assembly and cell motility.

Actin, one of the most abundant proteins in nature, participates in countless cellular functions ranging from organelle trafficking and pathogen motility to cell migration and regulation of gene transcription. Actin's cellular activities depend on the dynamic transition between its monomeric and filamentous forms, a process exquisitely regulated in cells by a large number of actin-binding and signaling proteins. Additionally, several posttranslational modifications control the cellular functions of actin, including most notably N-terminal (Nt)-acetylation, a prevalent modification throughout the animal kingdom. However, the biological role and mechanism of actin Nt-acetylation are poorly understood, and the identity of actin's N-terminal acetyltransferase (NAT) has remained a mystery. Here, we reveal that NAA80, a suggested NAT enzyme whose substrate specificity had not been characterized, is Nt-acetylating actin. We further show that actin Nt-acetylation plays crucial roles in cytoskeletal assembly in vitro and in cells. The absence of Nt-acetylation leads to significant differences in the rates of actin filament depolymerization and elongation, including elongation driven by formins, whereas filament nucleation by the Arp2/3 complex is mostly unaffected. NAA80-knockout cells display severely altered cytoskeletal organization, including an increase in the ratio of filamentous to globular actin, increased filopodia and lamellipodia formation, and accelerated cell motility. Together, the results demonstrate NAA80's role as actin's NAT and reveal a crucial role for actin Nt-acetylation in the control of cytoskeleton structure and dynamics.

Structural determinants and cellular environment define processed actin as the sole substrate of the N-terminal acetyltransferase NAA80.

N-terminal (Nt) acetylation is a major protein modification catalyzed by N-terminal acetyltransferases (NATs). Methionine acidic N termini, including actin, are cotranslationally Nt acetylated by NatB in all eukaryotes, but animal actins containing acidic N termini, are additionally posttranslationally Nt acetylated by NAA80. Actin Nt acetylation was found to regulate cytoskeletal dynamics and motility, thus making NAA80 a potential target for cell migration regulation. In this work, we developed potent and selective bisubstrate inhibitors for NAA80 and determined the crystal structure of NAA80 in complex with such an inhibitor, revealing that NAA80 adopts a fold similar to other NAT enzymes but with a more open substrate binding region. Furthermore, in contrast to most other NATs, the substrate specificity of NAA80 is mainly derived through interactions between the enzyme and the acidic amino acids at positions 2 and 3 of the actin substrate and not residues 1 and 2. A yeast model revealed that ectopic expression of NAA80 in a strain lacking NatB activity partially restored Nt acetylation of NatB substrates, including yeast actin. Thus, NAA80 holds intrinsic capacity to posttranslationally Nt acetylate NatB-type substrates in vivo. In sum, the presence of a dominant cotranslational NatB in all eukaryotes, the specific posttranslational actin methionine removal in animals, and finally, the unique structural features of NAA80 leave only the processed actins as in vivo substrates of NAA80. Together, this study reveals the molecular and cellular basis of NAA80 Nt acetylation and provides a scaffold for development of inhibitors for the regulation of cytoskeletal properties.

[14C]-Acetyl-Coenzyme A-Based In Vitro N-Terminal Acetylation Assay.

N-terminal acetylation is one of the most abundant co- and posttranslational protein modifications, conserved from prokaryotes to eukaryotes. The functional consequences of this modification are manifold, ranging from protein folding, stability, and interaction to subcellular localization. We describe here an isotope-labeled [14C]-acetyl-Coenzyme A-based acetylation assay, allowing the determination of weak catalytic activities of NATs in vitro. It allows the use of purified recombinant enzymes from Escherichia coli, or co-immunoprecipitated enzymes from various organisms, as well as the determination of the in vitro activity of various cell lysates. Although marked as an old-fashioned biochemical approach, it is the ideal method to hunt for catalytic activities and defining peptide specificities of new potential N-terminal acetyltransferase candidates.

Molecular determinants of the N-terminal acetyltransferase Naa60 anchoring to the Golgi membrane.

Nα-Acetyltransferase 60 (Naa60 or NatF) was recently identified as an unconventional N-terminal acetyltransferase (NAT) because it localizes to organelles, in particular the Golgi apparatus, and has a preference for acetylating N termini of the transmembrane proteins. This knowledge challenged the prevailing view of N-terminal acetylation as a co-translational ribosome-associated process and suggested a new mechanistic functioning for the enzymes responsible for this increasingly recognized protein modification. Crystallography studies on Naa60 were unable to resolve the C-terminal tail of Naa60, which is responsible for the organellar localization. Here, we combined modeling, in vitro assays, and cellular localization studies to investigate the secondary structure and membrane interacting capacity of Naa60. The results show that Naa60 is a peripheral membrane protein. Two amphipathic helices within the Naa60 C terminus bind the membrane directly in a parallel position relative to the lipid bilayer via hydrophobic and electrostatic interactions. A peptide corresponding to the C terminus was unstructured in solution and only folded into an α-helical conformation in the presence of liposomes. Computational modeling and cellular mutational analysis revealed the hydrophobic face of two α-helices to be critical for membranous localization. Furthermore, we found a strong and specific binding preference of Naa60 toward membranes containing the phosphatidylinositol PI(4)P, thus possibly explaining the primary residency of Naa60 at the PI(4)P-rich Golgi. In conclusion, we have defined the mode of cytosolic Naa60 anchoring to the Golgi apparatus, most likely occurring post-translationally and specifically facilitating post-translational N-terminal acetylation of many transmembrane proteins.

First Things First: Vital Protein Marks by N-Terminal Acetyltransferases.

N-terminal (Nt) acetylation is known to be a highly abundant co-translational protein modification, but the recent discovery of Golgi- and chloroplast-resident N-terminal acetyltransferases (NATs) revealed that it can also be added post-translationally. Nt-acetylation may act as a degradation signal in a novel branch of the N-end rule pathway, whose functions include the regulation of human blood pressure. Nt-acetylation also modulates protein interactions, targeting, and folding. In plants, Nt-acetylation plays a role in the control of resistance to drought and in regulation of immune responses. Mutations of specific human NATs that decrease their activity can cause either the lethal Ogden syndrome or severe intellectual disability and cardiovascular defects. In sum, recent advances highlight Nt-acetylation as a key factor in many biological pathways.

The world of protein acetylation.

Acetylation is one of the major post-translational protein modifications in the cell, with manifold effects on the protein level as well as on the metabolome level. The acetyl group, donated by the metabolite acetyl-coenzyme A, can be co- or post-translationally attached to either the α-amino group of the N-terminus of proteins or to the ε-amino group of lysine residues. These reactions are catalyzed by various N-terminal and lysine acetyltransferases. In case of lysine acetylation, the reaction is enzymatically reversible via tightly regulated and metabolism-dependent mechanisms. The interplay between acetylation and deacetylation is crucial for many important cellular processes. In recent years, our understanding of protein acetylation has increased significantly by global proteomics analyses and in depth functional studies. This review gives a general overview of protein acetylation and the respective acetyltransferases, and focuses on the regulation of metabolic processes and physiological consequences that come along with protein acetylation.

The activity of protein phosphatase 5 towards native clients is modulated by the middle- and C-terminal domains of Hsp90.

Protein phosphatase 5 is involved in the regulation of kinases and transcription factors. The dephosphorylation activity is modulated by the molecular chaperone Hsp90, which binds to the TPR-domain of protein phosphatase 5. This interaction is dependent on the C-terminal MEEVD motif of Hsp90. We show that C-terminal Hsp90 fragments differ in their regulation of the phosphatase activity hinting to a more complex interaction. Also hydrodynamic parameters from analytical ultracentrifugation and small-angle X-ray scattering data suggest a compact structure for the Hsp90-protein phosphatase 5 complexes. Using crosslinking experiments coupled with mass spectrometric analysis and structural modelling we identify sites, which link the middle/C-terminal domain interface of C. elegans Hsp90 to the phosphatase domain of the corresponding kinase. Studying the relevance of the domains of Hsp90 for turnover of native substrates we find that ternary complexes with the glucocorticoid receptor (GR) are cooperatively formed by full-length Hsp90 and PPH-5. Our data suggest that the direct stimulation of the phosphatase activity by C-terminal Hsp90 fragments leads to increased dephosphorylation rates. These are further modulated by the binding of clients to the N-terminal and middle domain of Hsp90 and their presentation to the phosphatase within the phosphatase-Hsp90 complex.

Polyglutamine toxicity in yeast induces metabolic alterations and mitochondrial defects.

BACKGROUND: Protein aggregation and its pathological effects are the major cause of several neurodegenerative diseases. In Huntington's disease an elongated stretch of polyglutamines within the protein Huntingtin leads to increased aggregation propensity. This induces cellular defects, culminating in neuronal loss, but the connection between aggregation and toxicity remains to be established. RESULTS: To uncover cellular pathways relevant for intoxication we used genome-wide analyses in a yeast model system and identify fourteen genes that, if deleted, result in higher polyglutamine toxicity. Several of these genes, like UGO1, ATP15 and NFU1 encode mitochondrial proteins, implying that a challenged mitochondrial system may become dysfunctional during polyglutamine intoxication. We further employed microarrays to decipher the transcriptional response upon polyglutamine intoxication, which exposes an upregulation of genes involved in sulfur and iron metabolism and mitochondrial Fe-S cluster formation. Indeed, we find that in vivo iron concentrations are misbalanced and observe a reduction in the activity of the prominent Fe-S cluster containing protein aconitase. Like in other yeast strains with impaired mitochondria, non-fermentative growth is impossible after intoxication with the polyglutamine protein. NMR-based metabolic analyses reveal that mitochondrial metabolism is reduced, leading to accumulation of metabolic intermediates in polyglutamine-intoxicated cells. CONCLUSION: These data show that damages to the mitochondrial system occur in polyglutamine intoxicated yeast cells and suggest an intricate connection between polyglutamine-induced toxicity, mitochondrial functionality and iron homeostasis in this model system.

Selective activators of protein phosphatase 5 target the auto-inhibitory mechanism.

Protein phosphatase 5 (PP5) is an evolutionary conserved serine/threonine phosphatase. Its dephosphorylation activity modulates a diverse set of cellular factors including protein kinases and the microtubule-associated tau protein involved in neurodegenerative disorders. It is auto-regulated by its heat-shock protein (Hsp90)-interacting tetratricopeptide repeat (TPR) domain and its C-terminal α-helix. In the present study, we report the identification of five specific PP5 activators [PP5 small-molecule activators (P5SAs)] that enhance the phosphatase activity up to 8-fold. The compounds are allosteric modulators accelerating efficiently the turnover rate of PP5, but do barely affect substrate binding or the interaction between PP5 and the chaperone Hsp90. Enzymatic studies imply that the compounds bind to the phosphatase domain of PP5. For the most promising compound crystallographic comparisons of the apo PP5 and the PP5-P5SA-2 complex indicate a relaxation of the auto-inhibited state of PP5. Residual electron density and mutation analyses in PP5 suggest activator binding to a pocket in the phosphatase/TPR domain interface, which may exert regulatory functions. These compounds thus may expose regulatory mechanisms in the PP5 enzyme and serve to develop optimized activators based on these scaffolds.

(Hyper)tension release by N-terminal acetylation.

A recent study links N-terminal acetylation and N-end rule degradation to blood pressure regulation. N-terminal mutants of Rgs2, a key G-protein regulator, are differentially processed by N-terminal acetyltransferases and the two branches of the N-end rule pathway. This leads to an imbalance in the signaling governing blood pressure.

Metabolic Response of Escherichia coli upon Treatment with Hypochlorite at Sub-Lethal Concentrations.

Hypochlorite is a reactive oxygen species that is worldwide as an antibacterial disinfectant. Hypochlorite exposure is known to cause oxidative damage to DNA and proteins. As a response to these effects, the metabolite profiles of organisms treated with sub-lethal doses of hypochlorite are assumed to be severely modified; however, the nature of these changes is hardly understood. Therefore, using nuclear magnetic resonance spectroscopy and gas chromatography-coupled mass spectrometry, we analyzed the time-dependent impact of hypochlorite exposure with a sub-lethal concentration (50 µM) on the metabolite profile of the Escherichia coli strain MG1655. Principle component analysis clearly distinguished between the metabolite profiles of bacteria treated for 0, 5, 10, 20, 40, or 60 min. Major changes in the relative amounts of fatty acids, acetic acid, and formic acid occurred within the first 5 min. Comparative gas chromatography-coupled mass spectrometry analyses revealed that the amounts of free methionine and alanine were significantly decreased in the treated cells, demonstrating their susceptibility to hypochlorite exposure. The concentrations of succinate, urea, orotic acid, 2-aminobutyric acid, and 2-hydroxybutyric acid were also severely affected, indicating general changes in the metabolic network by hypochlorite. However, most metabolite levels relaxed to the reference values of untreated cells after 40-60 min, reflecting the capability of E. coli to rapidly adapt to environmental stress factors such as the presence of sub-lethal oxidant levels.

Nematode Sgt1-homologue D1054.3 binds open and closed conformations of Hsp90 via distinct binding sites.

Heat shock protein 90 (Hsp90) is a highly conserved ATP-driven machine involved in client protein maturation, folding, and activation. The chaperone is supported by a set of cochaperones that confer client specificities. One of those proteins is the suppressor of G2 allele of skp1 (Sgt1), which participates together with Hsp90 in the immune responses of plants. Sgt1 consists of three domains: a TPR-, CS-, and SGS-domain, conserved in plants, yeast, and humans. The TPR-domain though is lacking in nematodes and insects. We observe that the Caenorhabditis elegans Sgt1 homologue D1054.3 binds to Hsp90 in the absence of nucleotides but much stronger in the presence of ATP and ATPγS. The latter binding mode is similar to p23, another CS-domain containing Hsp90 cofactor, even though binding is not observable for p23 in the absence of nucleotides. We use point mutations in Hsp90, which accumulate different conformations in the ATPase cycle, to differentiate between binding to open and closed Hsp90 conformations. These data support a strong contribution of the Hsp90 conformation to Sgt1 binding and highlight the ability of this cofactor to interact with all known Hsp90 conformations albeit with different affinities.