Comparative Proteomic Analysis of Lysine Acetylation in

Article pubs.acs.org/jpr

Cite This: J. Proteome Res. 2018, 17, 374−385

Comparative Proteomic Analysis of Lysine Acetylation in Trypanosomes Nilmar Silvio Moretti,†,‡ Igor Cestari,‡ Atashi Anupama,‡ Ken Stuart,‡ and Sergio Schenkman*,† †

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, R. Pedro de Toledo 669 L6A, 04039-032 São Paulo, SP, Brazil ‡ Center for Infectious Disease Research, 307 Westlake Avenue North, Suite 500, Seattle, Washington 98109, United States S Supporting Information *

ABSTRACT: Protein acetylation is a post-translational modification regulating diverse cellular processes. By using proteomic approaches, we identified N-terminal and ε-lysine acetylated proteins in Trypanosoma cruzi and Trypanosoma brucei, which are protozoan parasites that cause significant human and animal diseases. We detected 288 lysine acetylation sites in 210 proteins of procyclic form, an insect stage of T. brucei, and 380 acetylation sites in 285 proteins in the form of the parasite that replicates in mammalian bloodstream. In T. cruzi insect proliferative form we found 389 ε-lysine-acetylated sites in 235 proteins. Notably, we found distinct acetylation profiles according to the developmental stage and species, with only 44 common proteins between T. brucei stages and 18 in common between the two species. While Kac proteins from T. cruzi are enriched in enzymes involved in oxidation/reduction balance, required for the parasite survival in the host, in T. brucei, most K-ac proteins are enriched in metabolic processes, essential for its adaptation in its hosts. We also identified in both parasites a quite variable N-terminal acetylation sites. Our results suggest that protein acetylation is involved in differential regulation of multiple cellular processes in Trypanosomes, contributing to our understanding of the essential mechanisms for parasite infection and survival. KEYWORDS: Trypanosoma brucei, Trypanosoma cruzi, acetylation, mass spectrometry, glycolysis, histone, oxidation/reduction

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INTRODUCTION Environment adaptation is crucial for survival. It can be achieved by alterations in physiological processes, and it is regulated at different levels, from modifications in gene expression to alterations in enzymatic activities.1 A fast mechanism to control the activity of proteins is through reversible post-translational modifications, affecting distinct pathways, including those related to metabolism.2 Broad spectra of post-translational modifications have been described including acetylation and methylation, which have a prominent regulatory role for enzymatic activities.3,4 Acetylation can occur at the Nα-group of the first residue of a protein, called Ntacetylation, or at Nε-amine group of lysine residues. While Ntacetylation can affect protein degradation, complex formation, protein folding and protein subcellular localization, Nεacetylation acts by neutralizing lysine charge and can alter protein−protein interactions, protein complex formation and enzymatic activities.1,5 Lysine protein acetylation was first described in N-terminal domains of histones and it was shown to function in gene expression regulation.6 However, in the last 10 years, lysine acetylation appeared as a post-translational modification affecting hundreds of proteins in addition to histones.1 The advance of new proteomics approaches revealed the presence of this modification not only in the nuclear compartment but also in non-nuclear compartments in human cell lines, Saccha© 2017 American Chemical Society

romyces cerevisiae, Escherichia coli, and protozoan parasites, such as Toxoplasma gondii and Plasmodium falciparum.7−11 These compiled proteomic studies, called acetylomes, describe a set of lysine-acetylated proteins involved in different cellular processes, although most of them were found in enzymes of several metabolic pathways.1,8,12,13 Trypanosoma cruzi and Trypanosoma brucei, the etiologic agents of Chagas Disease and Sleeping Sickness in the human, respectively, have a complex life cycle shuttling between invertebrate and mammalian hosts. T. cruzi has two major evolutive forms that develop in the insect gut; the epimastigotes (EPI) and infective metacyclic- trypomastigote. In contact with the mammalian host, the parasite invades cells and multiplies in the host cells as amastigotes. It is released as a nondividing trypomastigote form, which circulates in the host bloodstream.14 T. brucei also develops in the insect vector gut as the procyclic trypomastigote form (PCF), which reaches the insect salivary gland when it generates dividing epimastigotes and infective metacyclic-trypomastigotes. In the mammalian host, T. brucei trypomastigotes proliferate in bloodstream, and are called bloodstream form (BSF).15 These parasite forms have evolved elaborated and effective mechanisms to cope with and adapt to the diverse environReceived: August 24, 2017 Published: November 23, 2017 374

DOI: 10.1021/acs.jproteome.7b00603 J. Proteome Res. 2018, 17, 374−385

Article

Journal of Proteome Research

Preparation of Protein Lysates and Immunoblotting

mental conditions in their hosts. Changes in temperature, pH, osmotic pressure and nutrient availability during the transition from one to another host coincide with morphological, gene expression, translation and metabolic changes in the parasite that allows its survival.16,17 For example, T. brucei BSF that replicates in the blood faces high glucose levels and relies on glycolysis to generate ATP.18 In contrast, PCF that replicates in the insect gut relies on amino acids as the main nutrient source and generate ATP by oxidative phosphorylation.19 When T. cruzi invade mammalian cells by forming a phagolysosome before escaping to the cytosol, it faces oxidative species generated by the immunological system. A highly oxidant environment may be present when the parasites is exposed to feces of the insect vector.20,21 It is still largely unknown how Trypanosomes deal with these different states during their life cycles. We hypothesized that protein acetylation could have a role in controlling enzymatic and protein activities related to these changes. To investigate this possibility, we performed a comparative proteome-wide analysis between PCF and BSF of T. brucei and epimastigotes forms of T. cruzi to identify their acetylated proteins. Our data revealed a very distinct set of acetylated proteins when comparing T. cruzi and T. brucei. Acetylation was observed in many antioxidant enzymes from T. cruzi, an essential biological process for this parasite during its life cycle. Moreover, large differences were observed in enzymes involved in the primary metabolism when comparing PCF and BSF known to use different sources of nutrients. The identification of differently acetylated residues in these organisms will help to understand how Trypanosomatids adapt to variable conditions encountered during their life cycles.

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Total and enriched fractions of cytoplasm and nucleus were obtained from 1 × 108 parasites from T. cruzi epimastigotes and T. brucei PCF and BSF forms, as described.25 Enriched fractions of mitochondrion were obtained using 1 × 109 exponentially growing T. cruzi epimastigotes and T. brucei PCF forms.26 BSF mitochondrion enriched fraction was not used due the difficult to obtain consistent amounts of samples to be used in the MS analysis. Total protein fractions were obtained by washing 1 × 108 parasites (T. cruzi and T. brucei forms) in phosphatebuffered saline (PBS) followed by incubation in lyses buffer (1% Triton X-100 in 50 mM Tris-HCl, 150 mM NaCl, pH 7.4, with EDTA-free protease inhibitor cocktail, Roche). To avoid protein deacetylation, 20 mM of nicotinamide, 20 mM butyric acid, two lysine deacetylases inhibitors, were added to the lyses buffer. Cell lysates were centrifuged at 10 000g for 10 min at 4 °C, and the cleared lysate were prepared for mass spectrometry. Isolated mitochondrial fractions were lysed similarly. Protein extracts described above were submitted to SDSPAGE, followed by immunoblotting using nitrocellulose membranes further probed with rabbit anti-acetyl-lysine antibodies (Millipore Corporation), polyclonal antibody anticytoplasmic heat shock protein 70 (Hsp70). Blots were revealed using anti-immunoglobulin peroxidase conjugates and ECL (Millipore) as described.27 LC−MS/MS Analysis

Protein samples were precipitated with 10 volumes of acetone and dissolved in 8 M of urea, followed by reduction and alkylation. Trypsin digestion was performed at 37 °C during 18 h and the reaction was stopped by addition of 5% of formic acid. Peptides were vacuum-dried and cleaned using Zip Tip micro-C18 (Millipore Corporation) before subsequent MS analysis. LC−MS/MS analyses were performed with an EasynLC 1000 (Thermo Scientific) coupled to an Orbitrap Elite mass spectrometer (Thermo Scientific). The LC system was configured in a vented format28 and consisted of a fused-silica nanospray needle (PicoTip emitter, 50 μm ID, New Objective) packed in-house with Magic C18 AQ 100 Å reverse-phase media (25 cm, Michrom Bioresources Inc.) and a trap (IntegraFrit Capillary, 100 μm ID, New Objective), containing Magic C18 AQ 200 Å (2 cm). The peptide sample was diluted in 10 μL of 2% acetonitrile and 0.1% formic acid in water and 8 μL was loaded onto the column and separated using a twomobile-phase system consisting of 0.1% formic acid in water and 0.1% acetic acid in acetonitrile. A 90 or 180 min gradient from 7% to 35% acetonitrile in 0.1% formic acid at a flow rate of 400 nL/min was used for chromatographic separations. The mass spectrometer was operated in a data-dependent MS/MS mode over the m/z range of 400−1800. The mass resolution was set at 120 000. For each cycle, the 15 most abundant ions from the scan were selected for MS/MS analysis using 35% normalized collision energy. Selected ions were dynamically excluded for 30 s.

EXPERIMENTAL PROCEDURES

Parasite Cultivation

T. brucei single-marker Lister 427 bloodstream forms were grown in vitro at 37 °C in Hirumi’s modified Iscove’s medium (HMI-9)22 supplemented with 10% fetal bovine serum in the presence of 2 μg/mL of G418 at 37 °C and 5% CO2. T. brucei 29.13 procyclic forms were grown in vitro at 27 °C in SDM-79 medium23 containing hemin (7.5 μg/mL) and 10% fetal bovine serum in the presence of G418 (15 μg/mL) and hygromycin (25 μg/mL). T. cruzi CL Brener strain epimastigotes were cultivated in liver infusion tryptose (LIT) medium, containing 10% fetal bovine serum at 28 °C. KATs and KDACs Inhibition Assays

T. brucei BSF (100 μL at 2.0 × 104 parasites per ml) were plated in 96-well plates and mixed with 100 μL of compounds at different concentrations, as described earlier.24 Salermide, nicotinamide and trichostatin-A (TSA-A) were obtained from (Sigma-Aldrich), anacardic acid, 1,2-Bis(isothiazol-5-y)disulfane and CPTH2 were obtained from (Cayman Chemicals). The compounds were diluted in HMI-9 medium with 10% fetal bovine serum. Parasites not treated with compounds were also plated as controls. After 48 h of incubation, 20 μL of Alamar Blue (Thermo Fisher Scientific) were added, and the assays were developed for 4 h. Fluorescence measurements were recorded using a SpectraMax M3 microplate reader (Molecular Devices) with excitation at 544 nm and emission at 590 nm (590 nm cutoff). Data were analyzed using GraphPad 6.0 Prism.

Data Processing

MS spectra were evaluated using MASCOT 2.5 (MATRIX SCIENCE). Peptides were matched to T. brucei TREU927 proteins and T. cruzi CL Brener.29 Enzyme specificity was set to trypsin/P, with up to two missed cleavages permitted. A peptide mass tolerance of ±5 ppm and MS/MS tolerance of ±0.5 Da was used. Carbamidomethyl cysteine was specified as a fixed modification, with oxidized methionine and acetylation of lysine residues allowed. The results were further analyzed with 375


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Figure 1. T. cruzi and T. brucei acetylome. (A) Protein fractionations were performed to obtain nuclear, cytoplasmic and mitochondrial samples of T. cruzi epimastigote forms and PCF and nuclear and cytoplasmic samples of BSF. The samples were submitted to LC−MS/MS analysis to generate the acetylome of T. cruzi and T. brucei. (B) Venn-diagram representing the total number of K-ac proteins identified in T. cruzi and T. brucei insect and mammalian forms. (C) Charts of proteins containing different numbers of lysine-acetylated sites in each parasite as indicated by the colored legend. (D) Schematic representation of proteins detected with the highest acetylation levels in T. cruzi and T. brucei. Predicted functional domains of each protein are indicated in color. Numbers indicate the K-ac position detected for each protein. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH); fructose-bisphosphate aldolase (ALD); hexokinase 1 (HK1); paraflagellar rod protein 1 (PFR1).

the Scaffold software (Proteome Software), which utilizes a Bayesian approach. High-scoring peptide identifications with protein probability ≥90 and peptide probability ≥90 were considered for the analysis. Acetylated proteins were classified according to gene ontology (GO) annotation in TritrypDB (http://tritrypdb. org) to include molecular function and cellular localization data. For KEGG pathways enrichment total lysine-acetylated proteins from EPI, PCF and BSF were analyzed using the EuPathDB Galaxy Data Analysis Service (http://eupathdb.org). Sequence logos were generated in PSSM format without the use of pseudo counts via the Seq2Logo Web server.30 PSSM format logos give a visual representation of amino acid enrichments at the positions around the K-ac. To obtain the peptide sequence for generation of sequence logos, −7/+7 amino acid from the K-ac position were extracted from the protein sequence and used for an alignment using ClustalX31

with default parameters set before logo generation. In the logos generated, Y-axis describes the amount of information in bits and the X-axis the position of the amino acids in the alignment. At each position, there is a stack of symbols representing the amino acids, where large symbols represent frequently observed amino acids, big stacks represent conserved positions and small stacks variable positions. The heat maps were generated using the software Java TreeView32 and RStudio. Protein structure analyses were made using ChimeraX software33 and protein structures were retrieve from Protein Data Base bank.

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RESULTS

Mapping Lysine-Acetylated Proteins of T. brucei and T. cruzi

We performed a proteome-wide survey of T. brucei and T. cruzi lysine-acetylated proteins using whole cell lysates, cytoplasmic, nuclear and mitochondrial enriched fractions of PCF and EPI, 376


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in T. cruzi, PCF and BSF forms (Figure 1B). These proteins are listed in the Table S2. The low number of common K-ac proteins already occurred for PCF and BSF with only 44 K-ac proteins in common. This distinct acetylation profile is clearly evidenced by immunoblots of total protein extracts of PCF and BSF using a polyclonal anti-acetyl lysine antibody (Figure S2). These results suggested to us that acetylation is variable among species and cellular stages in Trypanosomes. We found that over 80% of the acetylated proteins contained only one acetylation site in both T. brucei life cycle stages (180 in PCF and 237 in BSF). This number was 67% (158 proteins) in T. cruzi (Figure 1C). Glyceraldehyde-3-phosphate dehydrogenase (9 K-ac sites), fructose-bisphosphate aldolase (7 K-ac sites), histone H2A (6 K-ac) and hexokinase 1 (6 K-ac sites) were among the proteins with highest levels of acetylation identified in PCF. In contrast, in BSF, highly acetylated proteins were the paraflagellar rod protein 1 (10 K-ac sites), alphatubulin (7 K-ac sites), beta-tubulin (5 K-ac sites) and histone H2A (7 K-ac sites). In T. cruzi, heat-shock protein 70 (12 K-ac sites), pyruvate phosphate dikinase (9 K-ac sites), metallopeptidase, clan MH (8 K-ac sites) were the proteins with highest lysine-acetylated sites detected. The acetylation positions in some of these proteins are shown in Figure 1D.

and whole cell lysates, cytoplasmic and nuclear enriched fractions of BSF (Figure 1A). Fractionations of parasites were done to decrease sample complexity, thus increasing identification of less abundant proteins. Proteins were trypsindigested and analyzed by mass spectrometry for peptide and acetylation site identification. Proteomics followed by bioinformatics analyses identified a total of 235,210 and 285 lysineacetylated proteins in EPI, PCF and BSF, respectively. We considered multigenic proteins, such histones, as single proteins. Analysis of acetylation sites identified 389 lysineacetylated sites (K-ac) in T. cruzi EPI, 288 K-ac sites in T. brucei PCF proteins and 380 K-ac sites in T. brucei BSF proteins. A detailed list of lysine-acetylated peptides and the position of Kac sites are provided in Table S1. Some representative spectra detected are shown in the Figure S1. Table 1 summarizes the Table 1. Comparative Acetylomes of Different Organisms organism

biological sample analyzed

sites

proteins

Trypanosoma cruzi

epimastigote forms

369

235

Trypanosoma brucei

procyclic forms

288

210

Trypanosoma brucei

bloodstream forms

380

285

Plasmodium falciparum Plasmodium falciparum Toxoplasma gondii

erythrocytic stage

421

230

this study this study this study 9

erythrocytic stage

2876

1146

11

intracellular tachyzoite forms extracellular tachyzoite forms − yeast forms −

411

274

10

571

386

75

1070 1073 226

349 477 137

56 76 77

adult worm forms

2393

1109

78

SL2 cells

1096

1013

79

yeast forms

2878

1059

8

Toxoplasma gondii Escherichia coli Candida albicans Mycobacterium tuberculosis Schistosoma japonicum Drosophila melanogaster Saccharomyces cerevisiae

reference

Nt-Acetylation in Trypanosomes

One advantage to use the employed proteomic approach was to detect acetylations that happen in residues present at the Nterminal region of the proteins, called N-terminal (Nt)acetylation, which would not have been detected using approaches based on antibody enrichment of lysine-acetylated peptides. The N-terminal (Nt)-acetylation is the irreversible addition of an acetyl group to the N-α -amino group of a polypeptide. It usually happens during translation when the first peptide emerges from the ribosome tunnel34 with acetylation of methionine. Alternatively, the second amino acid of the protein can be Nt-acetylated after methionine removal, by methionine aminopeptidases. This is called the N-methionine excision mechanism and the most common acetylated residues are Ser, Ala, Thr, Val and Pro.35 These residues are likely acetylated by a set of enzymes called NATs (N-terminal acetyltransferases) that perform Nt-acetylation at specific amino acid residues. Our proteome analyses identified 114 proteins containing acetylation in N-terminal residues in BSF, 141 in PCF and 172 in EPI. In BSF proteins, 47 were acetylated in the first Met, 38 were acetylated in the Ser2, 24 in Ala2 and 5 in Thr2 residues (Figure 2A) and Table S3. A similar proportion of Nacetylations was observed in PCF, with 56 proteins acetylated in Met, 51 in Ser 2; 24 in Ala, 8 in Thr 2 and 2 in Val 2 residues and in T. cruzi with 64 proteins acetylated in Met; 55 in Ser 2; 42 in Ala; 7 in Thr 2 and 4 in Val 2 residues. These results are consistent with the presence in the T. brucei and T. cruzi genome of homologues to almost all NATs subunits with more than 25% amino acid identity with their human and S. cerevisiae homologues. The Nt-acetylated proteins identified in T. cruzi and T. brucei were found in several cellular compartments based in the Gene ontology (GO) terms. In T. cruzi they appeared enriched in the cytosol and in the mitochondrion whereas in T. brucei they are found in cytosolic and flagellar fractions (Figure 2B). Moreover, these proteins are distributed in diverse cellular functional groups according GO in both parasites, that include metabolism, DNA/RNA metabolism, motility in PCF, motility

number of acetylated proteins and sites of acetylation found in T. cruzi and T. brucei generated in this study compared with those reported for other organisms. The number of acetylated proteins and acetylated sites that we identified in both parasites was similar to that identified in some bacteria and other protozoan parasites but less than seen in yeast and in metazoan. It is important to mention that we detected in our proteomic analysis about 30% of total expressed proteins of T. cruzi (2714 of 10 338) and T. brucei (3,293 for PCF and 2883 for BSF of 11 074). From that 8.65, 6.4 and 9.9% corresponds to acetylated proteins in EPI, PCF and BSF, respectively. Similar observations were obtained in other protozoa. Since we only used trypsin to digest our samples prior MS/MS analysis, one possibility is that some of the identified modifications could be trimethylations, as we did not enrich our samples with antiacetyl-lysine antibodies. However, to avoid that, we did our MS/MS analysis using a mass resolution of 120 000 with mass accuracy for the instrument within 5 ppm. This resolution was sufficient to separate the trimethylations from acetylations. Notably, we found distinct acetylation profiles of T. cruzi and T. brucei, with 65 K-ac proteins in common, but only 18 mutual 377


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Figure 3. Cellular compartment and cellular functional distribution of lysine-acetylated proteins in T. cruzi and T. brucei. (A) Gene Ontology distribution showing the number of lysine-acetylated proteins of T. cruzi and T. brucei (PCF and BSF) identified in our analysis according to cellular compartments. Gene ontology enrichment of T. cruzi and T. brucei (PCF and BSF) lysine-acetylated proteins based on KEGG pathways (B).

example, while carbohydrate/glycolysis metabolic processes, fatty acid elongation and citrate cycle processes were enriched in PCF; pentose phosphate pathway, inositol phosphate metabolism and amino acid metabolic processes were enriched in BSF and oxidation/reduction and amino acid metabolism were prevalent in T. cruzi. (Figure 3B). These results further support the idea that protein acetylation is involved in the differential regulation of multiple cellular processes in Trypanosomes, as observed for other organisms.1,9,10

Figure 2. Nt-acetylation in T. cruzi and T. brucei. (A) Pie charts showing the number of proteins with Nt-acetylation in the serine (S), alanine (A), methionine (M), and threonine (T) detected in T. cruzi and T. brucei parasite forms. Distribution of Nt-acetylated proteins of T. cruzi (purple), PCF (yellow) and BSF (green) according to cellular compartment (B) and cellular functional group (C) based on GO.

and translation in BSF and metabolism, transport, translation in T. cruzi (Figure 2C). Several proteins were found exclusively in EPI, PCF and BSF, suggesting that these modifications are preferentially enriched depending on the species and on the developmental stage.

Acetylation Motifs

We analyzed the sequences adjacent to the K-ac sites in the identified peptides (−7 to +7 residues) using the SeqLogo2 program and subcategorized these K-ac sites according to their protein predicted subcellular location based on GO enrichment analysis. The generated patterns indicated distinct trends for each cellular compartment (Figure 4). While PCF cytosolic proteins have G, K or R at −1 and +1, BSF has S, D and R at −1 and D or E predominantly at +1. C, G and D predominate at −1 and C, P and A at +1 in T. cruzi EPI. PCF and BSF mitochondrial proteins are enriched in G at −1 positions, while in T. cruzi, the −1 positions contains mainly W, N or G. T. brucei nuclear proteins were enriched in G at −1 and −2 position and R and P, at positions +1 and +2, respectively, while in T. cruzi, nuclear proteins are enriched respectively in W/H and G at −1 and −2, and K and G at +1 and +2 positions. T. cruzi glycosomal proteins are enriched in W at −2 and +2, which is different from PCF and BSF that are enriched in A and P at −2 and G and T at +2 positions, respectively.

Cellular Compartment and Functional Distribution of K-ac Proteins

The lysine-acetylated proteins were assigned to a cellular compartment group based on GO analysis. The Figure 3A shows that the K-ac proteins have a distribution in essentially all cellular compartments of both parasites, although most of identified acetylated proteins were predicted cytosolic localization. In BSF, we found enrichment in nuclear followed by flagellar proteins. In contrast, in both insect stages (EPI and PCF), there was large number of acetylated proteins in the glycosome and mitochondrion when compared to BSF. Analysis of the K-ac proteins was also done according to KEGG pathways enrichment. In general, acetylation was found enriched in several molecular pathways in both parasites with distinct profiles comparing EPI, PCF and BSF (Figure 3B). For 378


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Figure 4. Sequence motifs of T. cruzi and T. brucei acetylation sites ±7 amino acids from the targeted lysine residue. Relative abundance of amino acid residues at adjacent sites of acetylated lysine separated according to cytoplasmic, glycosome, mitochondrial and nuclear protein localization generated by Seq2Logo web based software.

These data suggest that distinct specificity of acetyltransferases and deacetylases enzymes act on the different stages and species of the parasite or that they are located at distinct cellular compartments in these parasites.36 Histone Acetylations

Histones are well-known targets of lysine acetylation and play an essential role in regulating their function. These posttranslational modifications sites are well conserved in Trypanosomes, but differ from other eukaryotes, such as mammals.37 We identified new sites of histone acetylation in T. brucei, including new acetylations in histone variants (Figure 5A). We confirmed all previously described acetylations of histones H2A in both parasite stages and identified a new acetylation at K20 of histone H2A. We also identified two new acetylated sites, K48 and K104, of histone H2B in BSF; but none of the other previously reported N-terminus acetylation of this protein. The identified acetylations of H2Av were at K35, K43, K45, K47, K49, K55 and K59. We also found that H2Bv was acetylated at K4, K7, K15 and K19 positions. Modifications of H2 variants may be significant for T. brucei given the enrichment of these histones at transcription start sites.38 It is also noteworthy the enrichment of histone acetylation in BSF compared to PCF, except for the modifications in the Cterminus of H2A (Figure 5B). In the histone H3 we identified two new acetylation sites, K61 and K76 in BSF and confirmed acetylation of K23 in PCF and BSF; but the previously identified acetylations in K2 K4, K5, K10, K14, K17 and K18 of histone H4 were not detected.39 However, we identified three new acetylations in histone H4, K57 and K76 in BSF and K77 in both stages. In T. cruzi, besides to detect previously identified K10 and K14 in H4; K7, K15 and K17 in H2B.v, we also observed a new H3 acetylation site at position K79 (Figure 5C). Given that histones are widespread in the genome and their acetylation may vary according to their specific function at each genomic location,38,40 the diversity of histone acetylation sites identified here reveals the dynamic and complex role that acetylation may have in regulating histone function in trypanosomes.

Figure 5. Histone acetylation detected in T. brucei parasite forms. (A) Schematic representation of lysine acetylated sites identified in the amino acid sequence of each histone of T. brucei. Red numbers indicate the acetylation sites identified in PCF and green numbers those detected in BSF. Closed and open blue circles indicate the new and previously found acetylations, respectively. (B) Heat-map of lysine acetylation spectrum counts of histones H3, H4, H2A and H2B comparing PCF and BSF. K-ac intensity is the ratio from the total acetylated spectra counts detected for each protein versus the total spectra counts detected for each protein. (C) Histone acetylation sites identified in T. cruzi compared to previously described analysis.

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Journal of Proteome Research Glycolysis in T. brucei

distinct pattern of acetylation in these enzymes when comparing PCF and BSF. Several acetylated sites were detected in most PCF enzymes, few of which were conserved in both stages (Figure 6B). A semiquantitative analysis of the acetylation levels based on the identification frequency ratio of the total numbers of acetylated spectra counts relative to the total number of spectra counts of each protein suggested that PCF proteins have higher levels of acetylation than BSF for these enzymes (Figure 6C). To gain insight in the function of these acetylations, we analyzed the position of all K-ac sites in the three glycolytic enzymes of T. brucei with solved 3D structures; fructose-1,6-bisphosphate aldolase, glycerol-3-phosphate dehydrogenase, and triosephosphate isomerase, and found that mainly in the case of aldolase, at least one acetylation site is close to the active site (Figure 7). The other sites are located in the protein external surface (Movies S1− S3), which could also have a role in the formation of complexes and compartmentalization of the glycolytic enzymes in T. brucei. Thus, these findings suggest an additional mechanism for the

We analyzed the acetylation of proteins that compose the Embden−Meyerhof (E−M) segment of glycolysis, which in T. brucei takes place in glycosomes. 19 This segment encompasses the conversion of glucose to glucose-6-phosphate by hexokinase (HK) to the formation of 1,3 phosphoglycerate by glyceraldehydehyde-3-phosphate dehydrogenase (GAPDH) (Figure 6A). The pathway utilizes one ATP for the HK reaction

Figure 6. Protein acetylation in glycolytic enzymes of T. brucei. (A) Schematic representation of the Embden−Meyerhof segment of glycolysis in T. brucei, comparing the acetylation marks identified in each parasite form. Each acetyl group (Ac) shown in yellow represents a lysine-acetylated position detected in each enzyme. (B) Position of the lysine-acetylated residues identified in each protein of PCF and BSF. Hexokinase 1 and 2 (HK 1/2); glucose phosphate isomerase (PGI); phosphofructokinase (PFK); fructose-1,6-bisphosphate aldolase (ALD); glycerol-3-phosphate dehydrogenase (GPDH); triosephosphate isomerase (TIM); glycerol kinase (GK); glyceraldehyde-3phosphate dehydrogenase (GAPDH). Enzymes of PCF are represented in blue and of BSF in green. *Indicates the absence of acetylated peptides, although nonacetylated peptides were detected, including those containing the identified acetylated sites. (C) Heatmap comparing the protein acetylation levels (ratio) of the glycolytic enzymes between the two parasites forms. The ratio of the total number of lysine-acetylated spectra counts versus the total spectra counts observed for each protein is shown.

and one for the ATP-dependent phosphofructokinase (PFK) catalyzed reaction, and generates a net of two ATPs and two NADH+s. In eukaryotes, HK and PFK are allosterically regulated by their products and other downstream metabolites, which conserves ATP, but this metabolite regulation does not occur in T. brucei.19 We found that all E−M enzymes, except glucose phosphate isomerase (PGI) contained acetylated lysines (Figure 6A and B). More important, there was a

Figure 7. Lysine-acetylated sites detected in glycolytic enzymes of T. brucei. Distribution of K-ac sites (purple) identified in our analysis according to protein structure of fructose-1,6-bisphosphate-aldolase (A); glycerol-3-phosphate dehydrogenase (B); and triosephosphate isomerase (C). Protein structures were obtained from Protein Data Bank (PDB): 1F2J (fructose-1,6-bisphosphate-aldolase); 2J27 (triosephosphate isomerase) and 1EVY (glycerol-3-phosphate dehydrogenase). Orange represents substrate binding sites at catalytic domains. 380


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To verify the relevance of protein acetylation for T. brucei, we also tested some of these compounds on the parasite viability by using Alamar Blue. We observed a reduction in parasite viability for nicotinamide and salermide, inhibitors of NAD+dependent lysine deacetylases, and trichostatin an inhibitor of Zn+-dependent lysine deacetylases (Table 2). We also studied

regulation of the glycolytic enzymes in PCF and BSF of T. brucei. T. cruzi eIF5A Acetylation

The eukaryotic translation initiation factor 5A (eIF5A) is an essential and well-conserved protein, which has been implicated in translation elongation of proteins containing polyprolyl, proline-alanine and glycine tracts in mammals and yeast.41−43 eIF5A is the only protein modified by hypusination, which occurs at K50 and K51 in human and S. cerevisiae, respectively, and is essential for eIF5A activity.44 Hypusination is generated via two sequential reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydrolase (DOHH).45 In our proteomics analysis, we detected that the lysine 50 (K50) is acetylated in the T. cruzi eIF5A (TceIF5A), which corresponds to the acetylation detected in human eIF5A at K47 (Figure 8A), as both proteins share 78% of similarity. Based on

Table 2. Comparative Effect of KDACs and KATs in T. brucei BSF and Human Cellsa EC50 compound KDACs inhibitors Nicotinamide Salermide Trichostatin A KATs inhibitors CPTH2 Anacardic acid 1,2-Bis(isothiazol-5-y) disulfane

T. brucei

human (reference)

6.0 mM 29.2 μM 1.5 μM

5−15 mM80 100 μM81 124 nM82

13.2 μM 26.3 μM 26.2 μM

500−800 μM83,b 64 μM ≥ 200 μM84,b 5−10 μM85

a

BSF were incubated 24 h in the presence of variable amounts of the indicated inhibitors, and the EC50 and survival calculated using AlamarBlue as described in Experimental Procedures. bEffect observed using recombinant enzymes

the effects of inhibitors of acetyltransferases. All tested inhibitors affected BSF viability (Table 2). These results support the notion that acetylation events are required also for Trypanosome proliferation.

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DISCUSSION Here we investigated the acetylation profile of T. cruzi epimastigotes and T. brucei insect and mammalian parasite forms using proteomic analysis and observed large differences in lysine-acetylated and Nt-acetylated protein profiles comparing the parasites. We found large differences in the acetylations of the two developmental forms of T. brucei, which likely reflects stage-specific adaptions during this parasite life cycle. One of the major differences was the lower acetylation of glycolytic enzymes in the parasite stage that grows in blood and uses glycolysis as a major metabolic pathway to generate energy in comparison with parasites that develop in the insect vector. We also detected a different pattern of lysine-acetylated Ntacetylated proteins and the respective sites of acetylation when comparing the two species of Trypanosoma, with an enrichment of modified proteins related to metabolic processes and transport for EPI, metabolic processes and translation for PCF and motility and DNA/RNA metabolism for BSF, demonstrating a possible distinct role of this modification in each parasite forms. Although our findings could not cover a complete set of acetylated modifications in face of the present acetylomes, which detected similar numbers of acetylated proteins, we might have detected the most abundant modifications, indicating that each particular species and development contains a specific set of modifications, which might reflect a different physiological situation in each case. The distinct acetylation profile was obtained with nonenriched samples of lysine-acetylated peptides of different organelles from T. cruzi and T. brucei. Our strategy allowed us to detect similar numbers of lysine-acetylated peptides compared to other organisms using acetyl-lysine enrichment techniques. Importantly, we obtained a similar number of acetylated proteins in both parasites; however, only 18 proteins

Figure 8. T. cruzi eIF5A lysine-acetylation. (A) Amino acid alignment of T. cruzi and human eIF5A presenting the hypusination (green) and lysine-acetylated sites (red) identified in parasite protein. The hypusination loop is highlighted by orange square. (B) Human eIF5A protein structure (left) compared to predicted T. cruzi eIF5A protein structure (right) showing hypusination (red) and acetylation sites (green).

the crystal structure of the human protein46 and the models of Leishmania braziliensis, another Trypanosomatid (Protein Data Bank 1X60), these acetylation sites are found in the “hypusine loop” (STSK47TGK50HGHAK) located at N-terminal region of the protein (Figure 8B). In the human protein, the K47 acetylation is implicated in negative regulation of human eIF5A activity by affecting DHS biding, which results in inhibition of eIF5A hypusination.47,48 It is therefore possible that in T. cruzi eIF5A, in which K53 is hypusinated at lysine 53,49 the same modulation might occur. Nevertheless, unmatched acetylations sites were found in both proteins (T. cruzi, K42 and human, K68). Effect of KATs and KDACs Inhibitors on T. brucei Growth

Acetylation-mediated cellular signaling involves lysine acetyltransferases, which add acetyl groups to proteins and lysine deacetylases, which remove acetyl groups from proteins.50−52 Several compounds have been described to inhibit these enzymes in numerous organisms, including parasites.53−55 Recently, we demonstrated that salermide, specific NAD+dependent lysine deacetylase inhibitor, affects T. cruzi epimastigote multiplication and progression of parasite infection in vitro and in vivo.27 381


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Another important example is that T. cruzi eIF5A was also acetylated near the hypusination site, further suggesting that acetylation could be involved in the control of translation in this parasite. As in Trypanosomatids post-transcriptional regulation is the main way to regulate gene expression regulation,69 it would be interesting to investigate the role of acetylation in TceIF5A activity. The balance of acetylation/ deacetylation of human eIF5A is controlled by the acetyltransferase PCAF, and by the class III deacetylases, SIRT2 and the class I/II, HDAC6,48,70 Preliminary results from our lab demonstrated that cytoplasmic sirtuin, called TcSir2rp1,27 interacts with Tc-eIF5A, reinforcing the idea that acetylation could affects eIF5A activity as demonstrated for Hs-eIF5A. An overall preference to some amino acids around the acetylated site was detected for each cellular compartment, indicating that different acetylases and deacetylases could act, depending on the parasite organelle. In fact, these enzymes have been found in different T. brucei and T. cruzi compartments participating in vital processes of the parasite.36 Our results confirmed previous findings that deacetylases inhibitors affect growth of T. brucei53 and validate the importance of εlysine acetylation playing a role in this parasite. We and others have also found large effects in parasite growth and infective by overexpressing sirtuin deacetylases, or when using inhibitors of these deacetylases.27,71 Here, we further extend these observations to acetylase inhibitors, providing additional tools for development of drugs against trypanosomes. Besides the importance of ε-lysine acetylation shown here, we also demonstrated that T. cruzi and T. brucei have a distinct Nt-acetylation profiles. Further studies are necessary to investigate the importance of Nt-acetylation in trypanosomes, a modification involved in different functions, such as, regulation of protein degradation by recruitment of ubiquitin ligases,72 regulation of protein translocation from the cytosol to the ER73 and in the assembly of protein complexes,74 processes that are important during parasite differentiation.

are common for EPI T. cruzi, PCF and BSF. This scenario is also observed in PCF and BSF, which shared only 45 K-ac proteins, supporting the notion that acetylation may participate in the control of stage-specific processes from gene expression to metabolism in PCF and BSF.17 Indeed, the acetylated proteins identified are from different cellular compartments and they are predicted to function in various cellular processes. Similar results have been obtained for other organisms,7,56 highlighting the importance of this post-translational modification as a regulatory process. The most abundant nonhistone K-ac proteins from T. cruzi are cytosolic, mitochondrial and glycosomal. Interesting, a large number of these proteins are related to oxidative stress responses, which is expected to be critical for T. cruzi survival during its life cycle.57 Among these proteins we found tryparedoxin peroxidase, trypanothione synthase, glutathione peroxidase and mitochondrial iron superoxide dismutase A (TcSOD-A). Previous works have demonstrated that manganese superoxide dismutase (MnSOD) activity from mammals is regulated by acetylation,58 and we observed that K-ac residues identified in T. cruzi are present in similar regions of those that regulate MnSOD activity,59 We are currently working to validate the effect of these acetylations in Tc-SOD-A activity. Our analysis detected previously described histone modifications in T. cruzi as K10 and K14 at histone H4 and K7, K15 and K17 at histone H2B.v.60,61 We identified new histone acetylation sites, which were not previously described in Trypanosomatids.39,61 For example, we detected acetylations at K32 and acetylation at K79 of the histone H3, acetylation at K57 of the histone H4 and acetylations at K48 of H2B. For the first time, we also demonstrated new acetylations at H2A and H2B variants in T. brucei. In addition, and more relevant, we observed differences in the number of histone acetylation hits in BSF as compared to PCF, as observed before for T. brucei.62 Whether this could be related to differences in gene expression, it should be further evaluated. A striking result was that most of glycolytic enzymes present in the glycosome of T. brucei are acetylated. Interestingly, in BSF, which use glycolysis as main energy source, less and different acetylations were found when compared to PCF, which relies on oxidative phosphorylation for ATP production. This observation might suggest that acetylation could play a role in the regulation of glycolytic enzymes activity as already demonstrated in other organisms.13,63−66 In fact, when we analyzed K-ac sites position in the protein structure of fructose1,6-bisphosphate aldolase, glycerol-3-phosphate dehydrogenase and triosephosphate isomerase, we found some acetylated residues close to catalytic site of these enzymes. Indeed, acetylations near the active site have been demonstrated to modulate the activity of some glycolytic enzymes.13,64,65,67 Furthermore, acetylation of these proteins might reduce their abnormal positive charge in T. brucei proposed to be related to their localization inside glycosomes, which are related to peroxisomes.68 New experiments are necessary to know how and if acetylation can regulate glycolytic enzymes in T. brucei. We also detected acetylation of glycolytic enzymes from T. cruzi, which includes glucose-phosphate isomerase (K2); fructose-1,6-bisphosphate-aldolase (K38; K163; K240 and K327); triosephosphate isomerase (K4) and glyceraldehyde 3-phosphate dehydrogenase (K101 and K242) most of them, not found in T. brucei, indicating specific functions or regulatory mechanism between the two species.

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CONCLUSIONS In conclusion, this work adds a piece in the puzzle of the knowledge by studying ε-lysine acetylation and Nt-acetylation in trypanosomes, protozoan parasites that branched early in the evolution. We demonstrated distinct sets of acetylations that could correlate with the environmental fitness of each developmental stage of these organisms. More important, our study can contribute to define, using more specific experiments the role of how each of the detected modifications, act on each enzyme. In the case of T. brucei, our study opens the possibility to study fine-tuning of metabolic enzymes in response to environmental changes. The same would be relevant in the case of T. cruzi known to depend on antioxidant enzymes activity for survival and are main targets of the available drugs, which reinforce the notion that the development of specific acetyltransferases and deacetylases inhibitors could help in the treatment of neglected diseases.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.7b00603. 382



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Figure S1: Representative acetylated spectra detected in our acetylome analysis; Figure S2: Immunoblotting of total protein extracts from T. brucei PCF and BSF with antiacetyl-lysine antibody (PDF) Table S1: Lysine acetylated peptides identified in EPI T. cruzi and T. brucei PCF and BSF; Table S2: T. cruzi and T. brucei common K-ac proteins; Table S3: Nterminal acetylated proteins found in T. cruzi EPI and T. brucei PCF and BSF (XLS) Movie S1: Lysine-acetylated sites found in fructose-1,6bisphosphate aldolase from T. brucei; the purple regions indicate the detected acetylation sites and the red the enzyme active site (AVI) Movie S2: Lysine-acetylated sites found in glycerol-3phosphate dehydrogenase from T. brucei; the purple regions indicate the detected acetylation sites and the red the enzyme active site (AVI) Movie S3: Lysine-acetylated sites found in triosephosphate isomerase from T. brucei; the purple regions indicate the detected acetylation sites and the red the enzyme active site (AVI)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +55-11-55764870. ORCID

Sergio Schenkman: 0000-0001-9353-8480 Author Contributions

N.S.M. contributed to the conception and design of the study, to analysis and interpretation of the data and to write the manuscript. I.C. contributed to conception and design of the study. A.A. contributed to analysis of the data. K.S. contributed to conception and design of the study. S.S. contributed to conception and design of the study and to write the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Yuko Ogata for helping in the MS analysis, Dr. Elton J. R. Vasconcelos for helping with R analysis, and members of Stuart and Schenkman’s lab for helpful discussions. This work was supported by Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo − FAPESP (Grants 2011/ 51973-3 and 2015/22031-0 to S.S., 2012/09403-8 and 2013/ 20074-9 to N.S.M.), and by Conselho Nacional de ́ Desenvolvimento Cientifico e Tecnológico − CNPq (Grants 477143/2011-3 and 445655/2014-3 to S.S), and Instituto Nacional de Ciência e Tecnologia de Vacinas from Brazil. K.S. was funded by a National Institutes of Health Grant R01 AI078962. I.C. was supported by an American Heart Association fellowship (14POST18970046).

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ABBREVIATIONS EPI, epimastigotes form of T. cruz; PCF, procyclic form of T. brucei; BSF, bloodstream form of T. brucei; LIT, liver infusion tryptose; TSA-A, trichostatin-A; Nt, N-terminal; NATs, N-terminal acetyltransferases; GO, gene ontology; DHS, deoxyhypusine synthase; DOHH, deoxyhypusine hydrolase. 383


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Comparative Proteomic Analysis of Lysine Acetylation in

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