N-terminal Acetylation Research Articles

Naa10p Inhibits Beige Adipocyte-Mediated Thermogenesis through N-α-acetylation of Pgc1α.

Diet-induced obesity can be caused by impaired thermogenesis of beige adipocytes, the brown-like adipocytes in white adipose tissue (WAT). Promoting brown-like features in WAT has been an attractive therapeutic approach for obesity. However, the mechanism underlying beige adipocyte formation is largely unknown. N-α-acetyltransferase 10 protein (Naa10p) catalyzes N-α-acetylation of nascent proteins, and overexpression of human Naa10p is linked to cancer development. Here, we report that both conventional and adipose-specific Naa10p deletions in mice result in increased energy expenditure, thermogenesis, and beige adipocyte differentiation. Mechanistically, Naa10p acetylates the N terminus of Pgc1α, which prevents Pgc1α from interacting with Pparγ to activate key genes, such as Ucp1, involved in beige adipocyte function. Consistently, fat tissues of obese human individuals show higher NAA10 expression. Thus, Naa10p-mediated N-terminal acetylation of Pgc1α downregulates thermogenic gene expression, making inhibition of Naa10p enzymatic activity a potential strategy for treating obesity.

Effects of alpha-synuclein post-translational modifications on metal binding.

Parkinson's disease is the second most common neurodegenerative disorder worldwide. Neurodegeneration in this pathology is characterized by the loss of dopaminergic neurons in the substantia nigra, coupled with cytoplasmic inclusions known as Lewy bodies containing α-synuclein. The brain is an organ that concentrates metal ions, and there is emerging evidence that a break-down in metal homeostasis may be a critical factor in a variety of neurodegenerative diseases. α-synuclein has emerged as an important metal-binding protein in the brain, whereas these interactions play an important role in its aggregation and might represent a link between protein aggregation, oxidative damage, and neuronal cell loss. Additionally, α-synuclein undergoes several post-translational modifications that regulate its structure and physiological function, and may be linked to the aggregation and/or oligomer formation. This review is focused on the interaction of this protein with physiologically relevant metal ions, highlighting the cases where metal-AS interactions profile as key modulators for its structural, aggregation, and membrane-binding properties. The impact of α-synuclein phosphorylation and N-terminal acetylation in the metal-binding properties of the protein are also discussed, underscoring a potential interplay between PTMs and metal ion binding in regulating α-synuclein physiological functions and its role in pathology. This article is part of the Special Issue "Synuclein".

Abstract 2614: CCAR2 acetylation establishes a BET/BRD9 acetyl switch in response to combined deacetylase and bromodomain inhibition

Abstract There continues to be interest in targeting epigenetic ‘readers, writers and erasers’ for the treatment of cancer and other pathologies. A mechanistic understanding is frequently lacking, however, for the synergy observed when combining deacetylase and bromodomain inhibitors. We identified cell cycle and apoptosis regulator 2 (CCAR2) as an early target for acetylation in colon cancer cells treated with sulforaphane (SFN). N-terminal acetylation of CCAR2 diminished its interactions with histone deacetylase 3 (HDAC3) and β-catenin, interfering with Wnt coactivator functions of CCAR2, including in cells harboring genetically encoded CCAR2 acetylation. Protein domain arrays and pull-down assays identified acetyl ‘reader’ proteins that recognized CCAR2 acetylation sites, including BRD9 and members of the bromodomain and extraterminal domain (BET) family. The BET inhibitor JQ1 synergized with SFN in colon cancer cells, and suppressed tumor development effectively in a preclinical model of colorectal cancer. Studies with SFN+JQ1 in combination implicated a BET/BRD9 acetyl switch, and a shift in the pool of acetyl ‘reader’ proteins in favor of BRD9 regulated target genes. These results highlight the competition that exists among the ‘readers’ of acetylated histone and non-histone proteins, and provide a mechanistic basis for potential new therapeutic avenues involving epigenetic combination treatments. Citation Format: Gavin S. Johnson, Praveen Rajendran, Li Li, Ying-Shiuan Chen, W. Mohaiza Dashwood, Nhung Nguyen, Ahmet M. Ulusan, Furkan Ertem, Mutian Zhang, Deqiang Sun, Yun Huang, Shan Wang, Hon-Chiu E. Leung, David Lieberman, Laura M. Beaver, Emily Ho, Mark T. Bedford, Kyle Chang, Eduardo Vilar, Roderick H. Dashwood. CCAR2 acetylation establishes a BET/BRD9 acetyl switch in response to combined deacetylase and bromodomain inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2614.

Mass spectral analysis of acetylated peptides: Implications in proteomics.

Sequence determination of peptides using mass spectrometry plays a crucial role in the bottom-up approaches for the identification of proteins. It is crucially important to minimise false detection and validate sequence of the peptides in order to correctly identify a protein. Chemical modification of peptides followed by mass spectrometry is an option for improving the spectral quality. In silico-derived tryptic peptides with different N-terminal amino acids were designed from human proteins and synthesized. The effect of acetylation on the fragmentation of peptides was studied. N-terminal acetylation of the tryptic peptides was shown to form b1-ions, improve the abundance and occurrence of b-ions. In some cases, the intensity and occurrence of some y-ions also varied. Thus, it is demonstrated that acetylation plays an important role in improving the de novo sequencing efficiency of the peptides. The acetylation method was extended to tryptic peptides generated from the proteome of an Antarctic bacterium Pseudomonas syringae Lz4W using the proteomics work flow and mass spectra of the peptides were analysed. Comparison of the MS/MS spectra of the acetylated and unacetylated peptides revealed that acetylation helped in improving the spectral quality and validated the peptide sequences. Using this method, 673 proteins of the 1070 proteins identified were validated.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry with Activated Ion Electron Transfer Dissociation for Large-scale Top-down Proteomics.

Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been recognized as an efficient approach for top-down proteomics recently for its high-capacity separation and highly sensitive detection of proteoforms. However, the commonly used collision-based dissociation methods often cannot provide extensive fragmentation of proteoforms for thorough characterization. Activated ion electron transfer dissociation (AI-ETD), that combines infrared photoactivation concurrent with ETD, has shown better performance for proteoform fragmentation than higher energy-collisional dissociation (HCD) and standard ETD. Here, we present the first application of CZE-AI-ETD on an Orbitrap Fusion Lumos mass spectrometer for large-scale top-down proteomics of Escherichia coli (E. coli) cells. CZE-AI-ETD outperformed CZE-ETD regarding proteoform and protein identifications (IDs). CZE-AI-ETD reached comparable proteoform and protein IDs with CZE-HCD. CZE-AI-ETD tended to generate better expectation values (E values) of proteoforms than CZE-HCD and CZE-ETD, indicating a higher quality of MS/MS spectra from AI-ETD respecting the number of sequence-informative fragment ions generated. CZE-AI-ETD showed great reproducibility regarding the proteoform and protein IDs with relative standard deviations less than 4% and 2% (n = 3). Coupling size exclusion chromatography (SEC) to CZE-AI-ETD identified 3028 proteoforms and 387 proteins from E. coli cells with 1% spectrum level and 5% proteoform-level false discovery rates. The data represents the largest top-down proteomics dataset using the AI-ETD method so far. Single-shot CZE-AI-ETD of one SEC fraction identified 957 proteoforms and 253 proteins. N-terminal truncations, signal peptide cleavage, N-terminal methionine removal, and various post-translational modifications including protein N-terminal acetylation, methylation, S-thiolation, disulfide bonds, and lysine succinylation were detected.

The N-terminus of Sec61p plays key roles in ER protein import and ERAD.

Sec61p is the channel-forming subunit of the heterotrimeric Sec61 complex that mediates co-translational protein import into the endoplasmic reticulum (ER). In yeast, proteins can also be post-translationally translocated by the hetero-heptameric Sec complex, composed of the Sec61 and the Sec63 complexes. The Sec61 channel is also a candidate for the dislocation channel for misfolded proteins from the ER to the cytosol during ER-associated degradation (ERAD). The structure of the Sec61 complex is highly conserved, but the roles of its N-terminal acetylation and its amphipathic N-terminal helix are unknown so far. To gain insight into the function of the Sec61p N-terminus, we mutated its N-acetylation site, deleted its amphipathic helix, or both the helix and the N-acetylation site. Mutation of the N-acetylation site on its own had no effect on protein import into the ER in intact cells, but resulted in an ERAD defect. Yeast expressing sec61 without the N-terminal amphipathic helix displayed severe growth defects and had profound defects in post-translational protein import into the ER. Nevertheless the formation of the hetero-heptameric Sec complex was not affected. Instead, the lack of the N-terminal amphipathic helix compromised the integrity of the heterotrimeric Sec61 complex. We conclude that the N-terminal helix of Sec61p is required for post-translational protein import into the ER and Sec61 complex stability, whereas N-terminal acetylation of Sec61p plays a role in ERAD.

The N-end rule pathway enzyme Naa10 supports epiblast specification in mouse embryonic stem cells by modulating FGF/MAPK.

N-terminal acetylation (Nt-acetylation) refers to the acetylation of the free α-amino group at the N-terminus of a polypeptide. While the effects of Nt-acetylation are multifaceted, its most known function is in the acetylation-dependent N-end rule protein degradation pathway (Ac/N-end rule pathway), where Nt-acetylation is recognized as a degron by designated E3 ligases, eventually leading to target degradation by the ubiquitin-proteasome system. Naa10 is the catalytic subunit of the major Nt-acetylation enzyme NatA, which Nt-acetylates proteins whose second amino acid has a small side chain. In humans, NAA10 is the responsible mutated gene in Ogden syndrome and is thought to play important roles in development. However, it is unclear how the Ac/N-end rule pathway affects the differentiation ability of mouse embryonic stem cells (mESCs). We hypothesized that the balance of pluripotency factors may be maintained by the Ac/N-end rule pathway. Thus, we established Naa10 knockout mESCs to test this hypothesis. We found that Naa10 deficiency attenuated differentiation towards the epiblast lineage, deviating towards primitive endoderm. However, this was not caused by disturbing the balance of pluripotency factors, rather by augmenting FGF/MAPK signaling.

Mass Spectrometric Analysis of Lewy Body-Enriched α-Synuclein in Parkinson's Disease.

Parkinson's disease (PD) is characterized by intraneuronal inclusions of aggregated α-synuclein protein (so-called Lewy bodies) in distinct brain regions. Multiple posttranslational modifications may affect the structure and function of α-synuclein. Mass spectrometry-based analysis may be useful for the characterization and quantitation of α-synuclein forms, but has proven challenging, mainly due to the insolubility of Lewy bodies in aqueous buffer. In the present study, we developed a novel method by combining differential solubilization with immunoprecipitation and targeted proteomics using liquid chromatography and tandem mass spectrometry. Brain tissue homogenization and sample preparation were modified to facilitate analysis of soluble, detergent-soluble, and detergent-insoluble protein fractions (Lewy body-enriched). The method was used to compare α-synuclein forms from cingulate cortex (affected) and occipital cortex (unaffected) in two study sets of PD patients and controls. We identified ∼20 modified α-synuclein variants, including species with N-terminal acetylation and C-terminal truncations at amino acids 103 and 119. The levels of α-synuclein forms Ac-α-syn1-6, α-syn13-21, α-syn35-43, α-syn46-58, α-syn61-80, and α-syn81-96 except α-syn103-119 were significantly increased in PD cingulate region compared to controls in the Lewy body-enriched α-synuclein fraction. In the soluble fraction, only Ac-α-syn1-6 was significantly increased in PD compared to controls. None of the detected α-synuclein variants were Lewy body-specific, but acetylated forms should be examined further as potential biomarkers for abnormal α-synuclein accumulation.

Development and application of a high-throughput liquid chromatography–tandem mass spectrometric method for the simultaneous determination of thymosin α1 and its recombinant human form in plasma and urine

Thymosin α1 (Thymalfasin, Tα1) is a naturally occurring polypeptide widely used as an immune system enhancer for the treatment of HIV/AIDS, hepatitis B and C, and cancer. Recombinant human Tα1 (rh-Tα1) lacking N-terminal acetylation (NTA) displays similar biological activity to Tα1 and has completed phase III clinical trials in China. To compare the pharmacokinetics of rh-Tα1 and Tα1 and establish whether they undergo mutual transmutation in vivo, we developed a novel bioassay based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the simultaneous determination of the two peptides in human plasma and urine. Sample preparation by protein precipitation using a mixture of methanol and perchloric acid was followed by HPLC on a Zorbax 300SB-C18 column (150 × 4.6 mm, 5 μm) maintained at 40 °C. Detection was by multiple reaction monitoring (MRM) of the precursor-to-product ion transitions at m/z 778.0→316.0 for Tα1, m/z 767.3→955.0 for rh-Tα1 and m/z 832.3→159.2 for the internal standard, eptifibatide. The method was linear in the range 2–100 ng/mL for both analytes with good accuracy and precision. High sample throughput was facilitated by inclusion of a parallel two-column chromatographic system. The method was successfully applied to a comparative pharmacokinetic study involving single subcutaneous injections of either Tα1 or rh-Tα1 to two groups of healthy male volunteers. The results indicate that rh-Tα1 undergoes NTA in vivo to form Tα1 but that Tα1 is not deacetylated to rh-Tα1.

Characterization of Evolutionarily Conserved Trypanosoma cruzi NatC and NatA-N-Terminal Acetyltransferase Complexes.

Protein N-terminal acetylation is a co- and posttranslational modification, conserved among eukaryotes. It determines the functional fate of many proteins including their stability, complex formation, and subcellular localization. N-terminal acetyltransferases (NATs) transfer an acetyl group to the N-termini of proteins, and the major NATs in yeast and humans are NatA, NatB, and NatC. In this study, we characterized the Trypanosoma cruzi (T. cruzi) NatC and NatA protein complexes, each consisting of one catalytic subunit and predicted auxiliary subunits. The proteins were found to be expressed in the three main life cycle stages of the parasite, formed stable complexes in vivo, and partially cosedimented with the ribosome in agreement with a cotranslational function. An in vitro acetylation assay clearly demonstrated that the acetylated substrates of the NatC catalytic subunit from T. cruzi were similar to those of yeast and human NatC, suggesting evolutionary conservation of function. An RNAi knockdown of the Trypanosoma brucei (T. brucei) NatC catalytic subunit indicated that reduced NatC-mediated N-terminal acetylation of target proteins reduces parasite growth.

Identification of Amino Acid Residues in Influenza A Virus PA-X That Contribute to Enhanced Shutoff Activity.

The influenza virus protein PA-X modulates the host immune responses and viral pathogenicity through suppression of host protein expression. The endonuclease active site in the N-terminal region, the basic amino acid cluster in the C-terminal PA-X-specific region, and N-terminal acetylation of PA-X by NatB are important for the shutoff activity of PA-X. Here, we focused on the shutoff activity of PA-X derived from the A/California/04/2009 and A/WSN/33 viruses because these two PA-X proteins differ in their shutoff activity. Mutagenesis analysis revealed that proline and serine at positions 28 and 65, respectively, play a central role in this difference. Furthermore, we found that P28 and S65 also affect the shutoff activity of PA-X derived from other influenza virus subtypes. These data demonstrate that P28 and S65 contribute to enhanced shutoff activity of PA-X.

Top-down Mass Spectrometry of Sarcomeric Protein Post-translational Modifications from Non-human Primate Skeletal Muscle.

Sarcomeric proteins, including myofilament and Z-disk proteins, play critical roles in regulating muscle contractile properties. A variety of isoforms and post-translational modifications (PTMs) of sarcomeric proteins have been shown to be associated with modulation of muscle functions and the occurrence of muscle diseases. Non-human primates (NHPs) are excellent research models for sarcopenia, a disease associated with alterations in sarcomeric proteins, due to their marked similarities to humans. However, the sarcomeric proteins in NHP skeletal muscle have not been well characterized. To gain a deeper understanding of sarcomeric proteins in NHP skeletal muscle, we employed top-down mass spectrometry (MS) to conduct a comprehensive analysis on isoforms and PTMs of sarcomeric proteins in rhesus macaque skeletal muscle. We identified 23 protein isoforms with 46 proteoforms of sarcomeric proteins, including 6 isoforms with 18 proteoforms from fast skeletal troponin T. Particularly, for the first time, a novel PDZ/LIM domain protein isoform, PDLIM7, was characterized with a newly identified protein sequence. Moreover, we also identified multiple PTMs on these proteins, including deamidation, methylation, acetylation, tri-methylation, phosphorylation, and S-glutathionylation. Most PTM sites were localized, including Asn13 deamidation on MLC-2S; His73 methylation on αactin; N-terminal acetylation on most identified proteins; N-terminal tri-methylation on MLC-1S, MLC-1F, MLC-2S, and MLC-2F; Ser14 phosphorylation on MLC-2S; and Ser15 and Ser16 phosphorylation on MLC-2F. In summary, a comprehensive characterization of sarcomeric proteins including multiple isoforms and PTMs in NHP skeletal muscle was achieved by analyzing intact proteins in the top-down MS approach.

Acetylation of CCAR2 Establishes a BET/BRD9 Acetyl Switch in Response to Combined Deacetylase and Bromodomain Inhibition.

There continues to be interest in targeting epigenetic "readers, writers, and erasers" for the treatment of cancer and other pathologies. However, a mechanistic understanding is frequently lacking for the synergy observed when combining deacetylase and bromodomain inhibitors. Here we identify cell cycle and apoptosis regulator 2 (CCAR2) as an early target for acetylation in colon cancer cells treated with sulforaphane. N-terminal acetylation of CCAR2 diminished its interactions with histone deacetylase 3 and β-catenin, interfering with Wnt coactivator functions of CCAR2, including in cells harboring genetically encoded CCAR2 acetylation. Protein domain arrays and pull-down assays identified acetyl "reader" proteins that recognized CCAR2 acetylation sites, including BRD9 and members of the bromodomain and extraterminal domain (BET) family. Treatment with the BET inhibitor JQ1 synergized with sulforaphane in colon cancer cells and suppressed tumor development effectively in a preclinical model of colorectal cancer. Studies with sulforaphane+JQ1 in combination implicated a BET/BRD9 acetyl switch and a shift in the pool of acetyl "reader" proteins in favor of BRD9-regulated target genes. SIGNIFICANCE: These results highlight the competition that exists among the "readers" of acetylated histone and nonhistone proteins and provide a mechanistic basis for potential new therapeutic avenues involving epigenetic combination treatments.

NAA40 contributes to colorectal cancer growth by controlling PRMT5 expression

N-alpha-acetyltransferase 40 (NAA40) catalyzes the transfer of an acetyl moiety to the alpha-amino group of serine 1 (S1) on histones H4 and H2A. Our previous studies linked NAA40 and its corresponding N-terminal acetylation of histone H4 (N-acH4) to colorectal cancer (CRC). However, the role of NAA40 in CRC development was not investigated. Here, we show that NAA40 protein and mRNA levels are commonly increased in CRC primary tissues compared to non-malignant specimens. Importantly, depletion of NAA40 inhibits cell proliferation and survival of CRC cell lines and increases their sensitivity to 5-Fluorouracil (5-FU) treatment. Moreover, the absence of NAA40 significantly delays the growth of human CRC xenograft tumors. Intriguingly, we found that NAA40 knockdown and loss of N-acH4 reduce the levels of symmetric dimethylation of histone H4 (H4R3me2s) through transcriptional downregulation of protein arginine methyltransferase 5 (PRMT5). NAA40 depletion and subsequent repression of PRMT5 results in altered expression of key oncogenes and tumor suppressor genes leading to inhibition of CRC cell growth. Consistent with this, NAA40 mRNA levels correlate with those of PRMT5 in CRC patient tissues. Taken together, our results establish the oncogenic function of the epigenetic enzyme NAA40 in colon cancer and support its potential as a therapeutic target.

Impact of N-Terminal Acetylation on α-Synuclein Amyloid Formation

The accumulation of α-synuclein (αSyn) as β-sheet-rich amyloid in Lewy bodies is one of the hallmarks of Parkinson's disease. Recent data have demonstrated that αSyn is constitutively N-terminally acetylated in mammalian cells, a co-translational modification that has been shown to increase α-helical propensity in the N-terminal region of the protein and modulate membrane-binding behavior. The results of in vitro experiments comparing the amyloid formation kinetics of acetylated (Ac-αSyn) and the more widely studied non-acetylated a-synuclein (NH3-αSyn) have been contradictory. However, these assays largely rely on the extrinsic dye thioflavin T (ThT), which displays a dramatic increase in quantum yield upon binding to the cross-β sheet structure in amyloids. We find that the ThT response is approximately tenfold lower in Ac-αSyn, and that Ac-αSyn aggregates more slowly than NH3-αSyn at physiological pH. Intrinsic fluorescence of Trp mutants confirms that ThT emission accurately reports on the kinetics of amyloid formation for both forms of the protein. Seeding experiments indicate that the ThT fluorescence intensity is determined by the final fibrillar structure templated from the seeds rather than the presence or absence of the N-terminal acetyl group. Structural differences of the de novo and seeded fibrils are corroborated by Raman spectroscopy, limited proteolysis experiments, and transmission electron microscopy. These results indicate that N-terminal acetylation has important consequences for the aggregation propensity and amyloid structure of αSyn, and that nonacetylated αSyn is not a substitute for the biological form of the protein.

Using Site-Specific Vibrational Spectroscopy to Determine Structure of Membrane-Bound N-Terminally Acetylated Alpha-Synuclein

Despite a demonstrated connection to the etiology of Parkinson's Disease (PD), the structure and function of the neuronal brain protein alpha-synuclein (aS) remain elusive and confuse attempts to elucidate the link between the protein and PD. Structure determination is difficult because aS is an intrinsically-disordered protein in solution and because the recently ubiquity of N-terminal acetylation in vivo questions the relevance of many previous experiments that used unacetylated aS. Most previous research on aS membrane-bound structure, primarily using NMR, also lacks the ability to directly assess conformational dynamics on very short timescales. Site-specific vibrational spectroscopy presents a new technique that could reveal new details of the membrane-bound conformational distribution of aS. Here, we first attempted to obtain N-terminally acetylated aS (Nt-Ac aS) via the coexpression of human aS and yeast N-acetyltransferase B (NatB) in E. coli cells but failed to complete the acetylation. An adapted expression protocol using a sequence of increasing-volume cultures (according to the lab of David Eliezer) and E. coli cells pretransformed to express NatB before transformation of aS (according to the lab of Elizabeth Rhoades) successfully yielded several single-cysteine mutants of N-terminally acetylated aS, and we converted the thiol to the SCN vibrational probe group. We used infrared spectroscopy to measure solvent exposure of the SCN group at several sites along membrane-bound unacetylated and acetylated aS and compared the results. SCN-labeled variants of aS should be useful to explore aS's membrane-bound conformations in contact with a wide variety of lipid systems.

N-terminal acetylation and the N-end rule pathway control degradation of the lipid droplet protein PLIN2

Perilipin 2 (PLIN2) is a major lipid droplet (LD)-associated protein that regulates intracellular lipid homeostasis and LD formation. Under lipid-deprived conditions, the LD-unbound (free) form of PLIN2 is eliminated in the cytosol by an as yet unknown ubiquitin (Ub)-proteasome pathway that is associated with the N-terminal or near N-terminal residues of the protein. Here, using HeLa, HEK293T, and HepG2 human cell lines, cycloheximide chase, in vivo ubiquitylation, split-Ub yeast two-hybrid, and chemical cross-linking-based reciprocal co-immunoprecipitation assays, we found that TEB4 (MARCH6), an E3 Ub ligase and recognition component of the Ac/N-end rule pathway, directly targets the N-terminal acetyl moiety of Nα-terminally acetylated PLIN2 for its polyubiquitylation and degradation by the 26S proteasome. We also show that the TEB4-mediated Ac/N-end rule pathway reduces intracellular LD accumulation by degrading PLIN2. Collectively, these findings identify PLIN2 as a substrate of the Ac/N-end rule pathway and indicate a previously unappreciated role of the Ac/N-end rule pathway in LD metabolism.

Biochemical and structural analysis of N-terminal acetyltransferases.

N-terminal acetylation is a co- and post-translational modification catalyzed by the conserved N-terminal acetyltransferase (NAT) family of enzymes. A majority of the human proteome is modified by the human NATs (NatA-F and H), which are minimally composed of a catalytic subunit and as many as two auxiliary subunits. Together, NATs specifically regulate many cellular functions by influencing protein activities such as their degradation, membrane targeting, and protein-protein interactions. This chapter will describe methods developed for their preparation, and their biochemical and structural characterization. This will include methodologies for expression and purification of recombinant NAT protein, kinetic assays, biochemical and biophysical assays, and strategies for structural studies.

Expression and Characterization of Human Fragile X Mental Retardation Protein Isoforms and Interacting Proteins in Human Cells.

Fragile X mental retardation protein is an mRNA-binding protein associated with phenotypic manifestations of fragile X syndrome, an X-linked disorder caused by mutation in the FMR1 gene that is the most common inherited cause of intellectual disability. Despite the well-studied genetic mechanism of the disease, the proteoforms of fragile X mental retardation protein have not been thoroughly characterized. Here, we report the expression and mass spectrometric characterization of human fragile X mental retardation protein. FMR1 cDNA clone was transfected into human HEK293 cells to express the full-length human fragile X mental retardation protein. Purified fragile X mental retardation protein was subjected to trypsin digestion and characterized by mass spectrometry. Results show 80.5% protein sequence coverage of fragile X mental retardation protein (Q06787, FMR1_HUMAN) including both the N- and C-terminal peptides, indicating successful expression of the full-length protein. Identified post-translational modifications include N-terminal acetylation, phosphorylation (Ser600), and methylation (Arg290, 471, and 474). In addition to the full-length fragile X mental retardation protein isoform (isoform 6), two endogenous fragile X mental retardation protein alternative splicing isoforms (isoforms 4 and 7), as well as fragile X mental retardation protein interacting proteins, were also identified in the co-purified samples, suggesting the interaction network of the human fragile X mental retardation protein. Quantification was performed at the peptide level, and this information provides important reference for the future development of a targeted assay for quantifying fragile X mental retardation protein in clinical samples. Collectively, this study provides the first comprehensive report of human fragile X mental retardation protein proteoforms and may help advance the mechanistic understanding of fragile X syndrome and related phenotypes associated with the FMR1 mutation.

Ribosome-NatA architecture reveals that rRNA expansion segments coordinate N-terminal acetylation.

The majority of eukaryotic proteins are N-terminally α-acetylated by N-terminal acetyltransferases (NATs). Acetylation usually occurs co-translationally and defects have severe consequences. Nevertheless, it is unclear how these enzymes act in concert with the translating ribosome. Here, we report the structure of a native ribosome-NatA complex from Saccharomyces cerevisiae. NatA (comprising Naa10, Naa15 and Naa50) displays a unique mode of ribosome interaction by contacting eukaryotic-specific ribosomal RNA expansion segments in three out of four binding patches. Thereby, NatA is dynamically positioned directly underneath the ribosomal exit tunnel to facilitate modification of the emerging nascent peptide chain. Methionine amino peptidases, but not chaperones or signal recognition particle, would be able to bind concomitantly. This work assigns a function to the hitherto enigmatic ribosomal RNA expansion segments and provides mechanistic insights into co-translational protein maturation by N-terminal acetylation.