Identification of sulfenylation patterns in trophozoite stage Plasmodium falciparum using a non-dimedone based probe

Abstract

Plasmodium falciparum causes the deadliest form of malaria. Adequate redox control is crucial for this protozoan parasite to overcome oxidative and nitrosative challenges, thus enabling its survival. Sulfenylation is an oxidative post-translational modification, which acts as a molecular on/off switch, regulating protein activity. To obtain a better understanding of which proteins are redox regulated in malaria parasites, we established an optimized affinity capture protocol coupled with mass spectrometry analysis for identification of in vivo sulfenylated proteins. The non-dimedone based probe BCN-Bio1 shows reaction rates over 100-times that of commonly used dimedone-based probes, allowing for a rapid trapping of sulfenylated proteins. Mass spectrometry analysis of BCN-Bio1 labeled proteins revealed the first insight into the Plasmodium falciparum trophozoite sulfenylome, identifying 102 proteins containing 152 sulfenylation sites. Comparison with Plasmodium proteins modified by S-glutathionylation and S-nitrosation showed a high overlap, suggesting a common core of proteins undergoing redox regulation by multiple mechanisms. Furthermore, parasite proteins which were identified as targets for sulfenylation were also identified as being sulfenylated in other organisms, especially proteins of the glycolytic cycle. This study suggests that a number of Plasmodium proteins are subject to redox regulation and it provides a basis for further investigations into the exact structural and biochemical basis of regulation, and a deeper understanding of cross-talk between post-translational modifications.

Graphical Abstract

1. Introduction

Malaria is caused by apicomplexan parasites of the genus Plasmodium. 5 different human pathogenic species are known, of which Plasmodium falciparum (P. falciparum) causes the deadliest form [1]. The parasite has a complex life cycle and protein expression patterns change dramatically with each stage; 20-33% of all proteins are stage specific [2]. At the same time, the parasite is subjected to different sources of nitrosative and oxidative challenges imposed by its mosquito vector Anopheles, its human host, and its own high metabolic rate [3]. To overcome these challenges, Plasmodium has developed a multifarious antioxidant system mainly based on the NADPH-dependent thioredoxin and glutathione systems [4,5]. Furthermore, the parasite possesses 5 peroxiredoxins [6,7]. Thus, despite lacking the classical mammalian antioxidant and redox regulating enzymes such as catalase, glutathione peroxidase, and sulfiredoxin, the parasite manages to exert control over its intracellular redox state [8].

Post-translational modifications (PTMs) of proteins are an important aspect of cellular regulation, allowing adaptation to changing environmental conditions through increasing proteome’s complexity and diversity. A number of Plasmodium PTMs have previously been investigated, revealing involvement in many crucial pathways (reviewed in [9]). These studies identified a high susceptibility of Plasmodium proteins to phosphorylation (30% of all proteins) [10], lysine acetylation (14%) [11], and ubiquitination (5%) [12]. Cysteine residues are particularly susceptible amino acids for PTM, and contribute to redox signaling [13]. Previous studies focusing on cysteine PTMs in P. falciparum investigated interaction partners of thioredoxin, glutaredoxin, and plasmoredoxin and identified several proteins which seem to be particularly prone to redox PTMs [14]. Glutathionylation and nitrosation pull-down experiments identified 491 and 317 targets, respectively [15,16]. There was a high overlap in the proteins identified in these studies, demonstrating the significance of redox regulation and the particular susceptibility to oxidation of proteins such as glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH), which is involved in the central carbon metabolism of cells. Other redox PTMs have not been studied in detail in the Plasmodium system, and in this current study we remedy this by identifying the Plasmodium sulfenylome.

Sulfenic acid (-SOH), as an intermediate for several other PTMs, plays a crucial role in signal transduction and transcriptional regulation. Both reversible and what currently are thought to be irreversible oxidative modifications at cysteine sites (e.g., oxidation to sulfinic or sulfonic acids with some exceptions) [17,18] regulate protein activity through multiple mechanisms, whereas irreversible oxidation targeting a broader range of protein amino acids and nucleotides is associated with failure of protective mechanisms and thus oxidative imbalance [19,20]. Sulfenic acids represent an intermediate en route to other oxidation states. They serve as a regulator of several transcription factors, a sensor of oxidative stress, and a chemical species relevant in redox signaling [21,22]. Their transient nature is due to their metastable and highly reactive functional groups. Due to the formal oxidation state 0 of its sulfur atom, electrophilic as well as nucleophilic chemical activity can be exhibited [23]. Certain proteins, such as peroxidases and peroxiredoxins, belonging to the cell’s enzymatic reducing system, form -SOH intermediates during their catalytic cycle [24]. Prokaryotes such as Escherichia coli show similar sulfenylation mechanisms, emphasizing the importance of this transient modification [25].

1,3-dicarbonyl reagents, such as 5,5-dimethyl-1,3-cyclohexadione (dimedone), have been conventionally used to trap protein sulfenic acids [26]. Over the last decades, various derivatives linked to fluorescent or affinity-based probes have been developed, enabling enrichment and visualization of sulfenylated proteins (reviewed in [19]). Nevertheless, trapping this often-unstable modification requires reagents with faster kinetics such as that achieved with several more recently developed compounds [27,28]. Amongst these, the biotin-linked strained bicyclononyne BCN-Bio1 shows reaction rates with protein sulfenic acids over 100-fold higher than dimedone-based derivatives and was therefore selected for the studies included here [28].

We performed labeling of parasite lysates with BCN-Bio1 followed by pulldown of labeled sulfenylated proteins and MS/MS analysis to provide insight into the Plasmodium falciparum trophozoite sulfenylome. We identified 102 sulfenylated proteins and 152 sulfenylation sites, partially overlapping with previously identified redox-regulated proteins. As sulfenic acids are common intermediates in a broad range of protein redox modifications, their identification is a crucial step in further understanding the parasite’s redox metabolism and its role in regulation of cellular processes.

2. Methods

Chemicals

All chemicals used were of the highest available purity and were obtained from Roth (Karlsruhe, Germany), Sigma-Aldrich (Steinheim, Germany), BioRad (Munich, Germany), Fluka (Steinheim, Germany), or Merck (Darmstadt, Germany). RPMI 1640 medium and Albumax were from Gibco (Paisley, United Kingdom), gentamycin from Invitrogen (Karlsruhe, Germany). BCN-Bio1 was synthesized as previously described and is available from Xoder Technologies, LLC (Winston-Salem, North Carolina, USA) [28]. N-ethylmaleimide (NEM) was purchased from Fluka (Steinheim, Germany), bovine catalase from Sigma-Aldrich (Steinheim, Germany), high capacity streptavidin agarose from Invitrogen (California, USA), anti-biotin (33):sc-101339 from Santa Cruz (Texas, USA), and anti-mouse antibody from Dianova (Hamburg, Germany).

Cultivation of Plasmodium falciparum

Intraerythrocytic stages of the chloroquine sensitive strain 3D7 were maintained in continuous culture according to Trager and Jensen with slight modifications [29]). RPMI 1640 medium was completed with 0.5% lipid-rich bovine serum albumin (Albumax), 9 mM (0.16%) glucose, 0.2 mM hypoxanthine, and 22 μg/mL gentamycin. Parasites were grown in red blood cells (A+) at 3.3% hematocrit with a parasitemia between 1 to 10%. Incubations were performed at 37 °C in 3% O2, 3% CO2, and 94% N2. Synchronization to ring stages occurred through treatment with 5% (w/v) sorbitol [30]. Trophozoite stage infected erythrocytes with a parasitemia of 8-10% were harvested by lysing red blood cells for 10 min at 37 °C in a 20-fold volume of saponin lysis buffer (7 mM K2HPO4, 1 mM NaH₂PO₄, 11 mM NaHCO₃, 58 mM KCl, 56 mM NaCl, 1 mM MgCl₂, 14 mM glucose, and 0.02% saponin, pH 7.4). Saponin lysis was repeated twice before three washings with PBS. 360 mL cultured cells were harvested to get one pellet. Pellets were stored until use at −80 °C.

Pull-down of sulfenylated proteins using BCN-Bio1

The previously described sulfenylation pull-down protocol [31,32] was optimized for Plasmodium. A parasite pellet was lysed in degassed mRIPA lysis buffer (150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 25 mM NaF, 10 μM ZnCl₂, 50 mM HEPES, 10% glycerol, 1% Triton-X-100, pH 7.5) freshly supplemented with 200 U/mL bovine liver catalase, 20 mM NEM, complete protease inhibitor, and 250 μM BCN-Bio1. Cells were disrupted with 4 freezing-thawing cycles. The lysate was centrifuged at 100,000 x g for 30 min at 4 °C to obtain a clear supernatant. Ice cold acetone was added in a 5-fold volume and protein precipitation was carried out for 1 h at −20 °C. Upon 10 min centrifugation at 11,000 x g at 4 °C proteins were pelleted, and supernatant was discarded. After complete acetone evaporation, the pellet was resuspended in 700 μL PBS/2% SDS. Protein concentration was determined using a NanoDrop™ 2000. Sample concentration was calculated according to Lambert-Beer’s law using the software’s extinction coefficient for protein solutions.

To enrich biotinylated proteins, high capacity streptavidin agarose beads with a binding capacity of 10 mg/mL were used. Prior to incubation with the proteins, beads were washed 3x with 4 bed volumes of PBS/0.1% SDS. In between the washing steps, beads were separated from supernatant by 1 min centrifugation at 11,000 x g at room temperature (RT). Overnight incubation was carried out on a rotator at 4 °C, after diluting the sample 1:2 with PBS. The following day beads were spun down for 1 min at 11,000 x g at RT, and the supernatant containing unbound proteins removed and collected. In order to remove nonspecifically bound proteins, beads were washed with 4-fold bed volume of PBS (30 min) followed by 2 M urea, 1 M NaCl, 0.1% SDS with 10 mM DTT, all in PBS and at RT. One final washing step was then carried out in PBS. In between, beads were spun down for 1 min at 11,000 x g, and supernatant was removed and collected. Elution was conducted by boiling the beads at 95 °C for 10 min in 100 mM Tris, 500 mM NaCl, pH 8.0. To ensure complete removal of bound proteins, elution was performed three times. Protein concentrations of the eluates and wash steps were determined as described above. Western blot analysis confirmed successful performance prior to mass spectrometry analysis. Each of the 3 biological replicates was split into 2 fractions to give technical replicates in mass spectrometry analysis. Eluates were then stored at −20 °C until further processed for mass spectrometry analysis.

Immunodetection

To confirm labeling and washing efficiency of the performed pull-down, immunodetection using α-biotin antibody was performed. Briefly, 25 μL of protein sample with sample buffer containing DTT was denaturated for 5 min at 95 °C. Following 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. Blocking was carried out for 1 h at room temperature or overnight at 4 °C with 5% nonfat milk in Tris-buffered saline 0,05% (v/v) Tween-20 (TBST) with constant agitation. To visualize BCN-Bio1 tagged proteins, the membrane was incubated for 1 h at RT with anti-biotin antibody, diluted 1:500 in 5% non-fat milk in TBST, and for 1 h at RT with anti-mouse antibody-HRP, 1:10,000 in 5% non-fat milk in TBST. In between incubations, the membrane was washed with TBST 3x for 8 minutes. The membrane was developed with luminol and 0.0011% coumaric acid.

Sample preparation for mass spectrometry analysis

Eluates were sent on dry ice to the Scripps Research Institute, La Jolla, California. Samples were thawed and 125 μL were removed. Urea (60.06 mg) was added, and after urea was dissolved, 6.25 μL of 100 mM TCEP was added. Samples were shaken at RT for 30 min. Iodoacetamide (IAA, 6 μL of 250 mM) was added and the samples were covered in aluminum foil and shaken at RT for 30 min. EndoLysC (Wako, 1 μg) was added and the reactions were shaken at 37 °C for 4 hours. Samples were diluted 4x with 100 mM Tris and 1 μg trypsin was added. Samples were shaken overnight at 37 °C and stored in the freezer.

Mass spectrometry analysis

Samples were thawed, acidified with 7 μL of formic acid, shaken and spun at highest speed for 15 min. Samples were transferred to a new tube. Each sample (25 μg) was pressure loaded onto a 250-μm inside diameter (i.d.) fused silica microcapillary column (Polymicro Technologies) that was packed with 2.0 cm of Partishpere strong cation exchange resin (Whatman) followed by 2.0 cm of 10 cm Aqua C18 resin (Phenomenex). The column was washed for 30 min with 5% acetonitrile/0.1% formic acid (Buffer A).

A 100 μm i.d. capillary with a 5 μm pulled tip packed with 15 cm of 3 μm Aqua C18 material (Phenomenex) was attached via a union, and the entire split-column was placed in line with an Agilent 1100 quaternary high-performance liquid chromatography (HPLC) and analyzed using a 9-step separation. The buffer solutions used for separation were: 5% acetonitrile/0.1% formic acid (Buffer A); 80% acetonitrile/0.1% formic acid (Buffer B); and 500 mM ammonium acetate/5% acetonitrile/0.1% formic acid (Buffer C). Step 1 consisted of a 90 min gradient from 0% to 100% buffer B. Steps 2–9 had the following profile: 10 min of X% buffer C, a 15 min gradient from 0% to 5% buffer B, and a 95 min gradient from 15% to 100% buffer B. The 10 min buffer C percentages (X) were: 10%, 20%, 30%, 40%, 50%, 60%, 70%, and 100%, respectively, for the 9-step analysis. As peptides eluted from the microcapillary column, they were electrosprayed directly into an Orbitrap Velos Pro mass spectrometer (ThermoFisher) with the application of a distal 2.4 kV spray voltage. A cycle of one full-scan mass spectrum (400–1800 m/z) followed by 15 data-dependent tandem mass spectrometry spectra at a 35% normalized collision energy was repeated continuously throughout each step of the multidimensional separation. The application of mass spectrometer scan functions and HPLC solvent gradients were controlled by the XCalibur 2.2 SP1 data system.

Mass spectrometry data analysis

Collected tandem mass spectra were analyzed with Integrated Proteomics Pipeline (IP2; Integrated Proteomics Applications, Inc., San Diego, CA. http://www.integratedproteomics.com) using ProLuCID [33] and DTASelect 2.0 [34,35]. Using RawExtract 1.9.9 (http://fields.scripps.edu/downloads.php) [36] spectrum raw files were extracted into MS1 and MS2 files prior to searching them against the PlasmoDB database (release date 03-30-2015). In order to estimate peptide probabilities and false discovery rates, a decoy database consisting of the reversed sequence of all proteins appended to the target database was used. As human proteins may be present in the sample, a human database was included as well. A peptide confidence of 99.9% was set as the minimum threshold, and only peptides with a precursor delta mass less than 20 ppm were accepted, including only peptides with a minimum peptide length of 6 amino acids. The search space included all fully and half-tryptic peptide candidates that fell within the mass tolerance window with maximal 3 missed internal cleavage sites. Following modifications on cysteine were taken into account: +392.1759 (BCN-Bio1 bound to sulfenic acid, see figure 2B), and +141.04261 (NEM bound to sulfenic acid [31]). The false discovery rate was calculated as the percentage of reverse decoy peptide/spectrum matches (PSM) among all the PSM that passed the 99.9% confidence threshold. After this last filtering step, the peptide false discovery rate was estimated to be below 1%.

Figure 2 –

A: Gel electrophoresis of pull-down steps. Samples collected during pull-down were subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie brilliant blue. B: Immunodetection of pull-down eluates from 3 biological replicates using α-biotin.

Protein clustering

Identified target proteins for sulfenylation were clustered according to their molecular function and their involvement in biological processes using gene ontology (GO) slim annotations. At the same time enrichment analysis was performed using the enrichment tool by PlasmoDB (www.plasmodb.org). Pie charts were produced using Microsoft Excel 2019.

3. Results

Identification of sulfenylated proteins in P. falciparum cell extracts

To gain an insight into P. falciparum’s trophozoite sulfenylome, the protein sulfenylation labeling probe BCN-Bio1 was used. In this approach, modified proteins were specifically labeled prior to enrichment and mass spectrometry analysis (Figure 1). Only proteins for which the modified sulfenylated peptide was identified by a specific mass shift of +392.1759 for BCN-Bio1 bound to a sulfenylated cysteine, and +141.0426 for NEM bound to a sulfenylated cysteine were accepted as true hits. +141.0426 was included because Reisz and coworkers have shown that alkylating agents, such as NEM show similar rate constants toward sulfenic acid as do sulfenic acids detection probes [31]. Protein persulfides, which have also been shown to react with strained cycloalkynes as BCN-Bio1 were not included in the search [37]. Stringent search parameters (detailed in materials and methods) identified 167 peptides containing a modification, representing 152 modification sites on 102 proteins (Table 1). 38 of these modification sites were detected in 2/3 replicates and 39 in all 3 biological replicates. Supplementary S2 lists all proteins with identified modification, the respective peptides including sulfenylation sites and statistics.

Figure 1 –

Schematic workflow of pull-down protocol Sulfenylated proteins of P. falciparum trophozoites were covalently labeled by BCN-Bio1, Free thiols were blocked by N-ethylmaleimide (NEM). Enrichment using avidin beads was performed. Throughout stringend washing steps unspecifically bound proteins were removed. Eluted proteins were analysed using multidimensional protein identification technique (MudPIT). Sulfenylated peptides labeld by BCN-Bio1 can be detected in MudPIT analysis by a mass shift of +392.1759 Da.

Table 1.

Proteins identified by mass spectrometry as being sulfenylated sorted according to their biological function (UniProt.org).

N°	PlasmoDB Accession N°	Protein name	Molar mass (kDa)	Sequence coverage in each replicate a	SOH site b	Protein nitrosylated [16] c	Protein glutathionylated [15] d	Predicted localisation e
Biosynthetic pathway
1	PF3D7_0511800	Inositol-3-phosphate synthase	69.1	69.9% / 76.0% / 71.9%	C560	+	+
2	PF3D7_0513300	Purine nucleoside phosphorylase	26.9	71.4% / 69.8% / 82.0%	C71 C141 C208	+	+
3	PF3D7_0608800	Ornithine aminotransferase	46.0	65.9% / 54.1% / 40.6%	C63	+	+
4	PF3D7_0621200	Pyridoxine biosynthesis protein PDX1	33.0	63.1% / 62.5% / 63.1%	C16 C177	+	+
5	PF3D7_0810800	Dihydropteroate synthetase	83.4	43.2% / 51.4% / 42.1%	C473		+
6	PF3D7_0918900	Gamma-glutamylcysteine synthetase	124.5	22.8% / 29.6% / 32.8%	C103 6 + C103 8	+
7	PF3D7_0922200	S-adenosylmethionine synthetase (PfSAMS)	44.8	71.9% / 73.1% / 68.4%	C264	+ C113	+
8	PF3D7_0922600	Glutamine synthetase, putative	63.2	57.3% / 61.7% / 50.3%	C66 C346 C479
9	PF3D7_0926700	Glutamine-dependent NAD(+) synthetase, putative	97.8	24.7% / 22.8% / 11.9%	C91		+
10	PF3D7_1343000	Phosphoethanolamine N-methyltransferase	31.0	82.0% / 82.0% / 86.1%	C70 C87 C161	+	+
11	PF3D7_1354500	Adenylosuccinate synthetase	50.1	46.4% / 52.9% / 35.5%	C283 C396		+
Cell cycle/Proliferation
12	PF3D7_0511000	Translationally-controlled tumor protein homolog	20.0	60.2% / 60.2% / 67.3%	C14	+ C14	+
13	PF3D7_0619400	Cell division cycle protein 48 homologue, putative	92.4	56.2% / 61.4% / 67.4%	C418 + C425 C425 C575	+ C695	+
Cellular redox homeostasis
14	PF3D7_0802200	1-cys peroxiredoxin	25.2	58.6% / 61.4% / 45.0%	C101	+ C184	+
15	PF3D7_0814900	Superoxide dismutase [Fe]	22.7	72.2% / 90.9% / 72.7%	C85 C116		+
16	PF3D7_0923800.1 PF3D7_0923800.2	Thioredoxin reductase	68.7	44.7% / 34.2% / 39.7% 51.0% / 39.0% / 45.3%	C422		+
17	PF3D7_1438900	Thioredoxin peroxidase 1	21.8	79.0% / 86.2% / 63.6%	C170	+	+
Cytoskeleton
18	PF3D7_0422300	Alpha tubulin 2	49.7	33.6% / 35.8% / 35.8%	C376
19	PF3D7_0903700	Alpha tubulin 1	50.3	45.3% / 49.0% / 43.9%	C376	+ C65
20	PF3D7_1008700	Tubulin beta chain	49.8	42.9% / 50.8% / 43.8%	C127 C129	+
21	PF3D7_1473300	CKK domain-containing protein, putative	114.3	X / 1.4% / 1.4%	C456
DNA and RNA metabolism and processing
22	PF3D7_0624600	SNF2 helicase, putative	315.6	22.6% / 25.7% / 23.3%	C147 + C150*	+
23	PF3D7_0711500	Regulator of chromosome condensation, putative	78.9	30.1% / 24.9% / 30.5%	C66
24	PF3D7_0717700	Serine-tRNA ligase, putative	62.5	45.1% / 60.1% / 51.0%	C247	+	+
25	PF3D7_0812500	RNA-binding protein, putative	117.1	7.1% / 8.5% / 6.4%	C182'	+
26	PF3D7_1224300	polyadenylate-binding protein, putative	97.2	40.6% / 50.3% / 48.9%	C410	+ C234	+
27	PF3D7_1346300	DNA/RNA-binding protein Alba 2	25.0	51.2% / 46.0% / 46.4%	C133	+	+
28	PF3D7_1347500	DNA/RNA-binding protein Alba 4	42.2	70.7% / 77.4% / 74.2%	C63 C286	+	+
29	PF3D7_1461900	Valine--tRNA ligase, putative	128.1	45.0% / 53.1% / 45.2%	C428		+
Glycolysis
30	PF3D7_0626800	Pyruvate kinase (PfPK)	55.7	60.3% / 56.6% / 58.7%	C222 C302 C343 C433	+	+
31	PF3D7_0915400	6-phosphofructokinase	159.5	55.7% / 54.5% / 53.2%	C645 C1321 C1038	+ C777	+
32	PF3D7_1015900	Enolase	48.7	81.8% / 83.9% / 87.2%	C157 C370	+	+
33	PF3D7_1324900	L-lactate dehydrogenase (PfLDH)	34.1	74.4% / 72.5% / 71.8%	C59 C260	+	+	SP
34	PF3D7_1439900	Triosephosphate isomerase	27.9	72.2% / 73.8% / 76.2%	C217	+	+
35	PF3D7_1444800	Fructose-bisphosphate aldolase	40.1	84.0% / 83.2% / 82.1%	C247	+
36	PF3D7_1462800	Glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH)	36.6	78.9% / 81.9% / 81.6%	C58 C157 C250	+	+
Kinase/ Signal transduction
37	PF3D7_0818200	14-3-3 protein	30.2	65.6% / 63.0% / 61.1%	C41	+	+
38	PF3D7_0826700	Receptor for activated c kinase	35.7	57.6% / 58.5% / 60.4%	C162 C177 C216 C384	+	+
39	PF3D7_1103700	Casein kinase II beta chain	28.4	29.4% / 34.3% / 27.3%	C122		+
40	PF3D7_1124600	Ethanolamine kinase	49.9	39.5% / 53.2% / 57.4%	C283	+	+
41	PF3D7_1144500	Ubiquitin activating enzyme (E1) subunit Aos1, putative	39.7	28.1% / 25.7% / 29.9%	C297
42	PF3D7_1242800	Rab specific GDP dissociation inhibitor	52.3	54.5% / 64.7% / 56.0%	C250		+
43	PF3D7_1414400	Serine/threonine protein phosphatase PP1	34.9	50.3% / 58.2% / 53.3%	C169
Miscellaneous
44	PF3D7_0406400	Cytosolic glyoxalase II	30.5	45.6% / 42.6% / 42.2%	C4	+	+
45	PF3D7_1012400	Hypoxanthine-guanine phosphoribosyltransferase	26.4	72.7% / 81.8% / 73.2%	C134	+	+
46	PF3D7_1033400	Haloacid dehalogenase-like hydrolase	32.8	56.9% / 55.9% / 63.5%	C92	+
47	PF3D7_1311900	Vacuolar ATP synthase subunit a	68.6	72.5% / 79.5% / 67.3%	C526	+	+
48	PF3D7_1406300	Glycerophosphodiester phosphodiesterase	56.3	67.4% / 68.2% / 53.9%	C91		+	SP
Pathogenesis / Evasion / Protein export / Antigen
49	PF3D7_0929400	High molecular weight rhoptry protein 2	162.7	61.3% / 60.4% / 62.6%	C1344/ C1344'	+	+	SP/Rhop
50	PF3D7_0930300	Merozoite surface protein 1	195.7	41.5% / 60.6% / 63.7%	C173	+	+	SP/PM
51	PF3D7_1028700	Merozoite TRAP-like protein	58.1	X / 8.4% / 26.3%	C47			SP
52	PF3D7_1029600	Adenosine deaminase	42.5	68.7% / 80.7% / 70.3%	C173 C281	+	+
53	PF3D7_1126000	Threonine-tRNA ligase	119.5	43.8% / 47.0% / 40.4%	C428 C589 C767		+	SP/AP
54	PF3D7_1229400	Macrophage migration inhibitory factor	12.8	94.8% / 95.7% / 95.7%	C3/C3' C4/C4' C3+C4	+	+
Protein / hemoglobin degradation
55	PF3D7_0207600	Serine repeat antigen 5	111.8	48.4% / 58.8% / 57.4%	C672	+	+	SP/PV
56	PF3D7_1115400	Cysteine proteinase falcipain 3	56.7	42.9% / 48.4% / 41.9%	C331	+	+	FV
57	PF3D7_1118300	Insulinase, putative	173.6	36.6% / 31.3% / 31.9%	C322 C1364	+	+
58	PF3D7_1225800	Ubiquitin-activating enzyme E1	131.8	48.6% / 51.2% / 46.5%	C641		+
59	PF3D7_1311800	M1-family alanyl aminopeptidase	126.1	76.9% / 80.0% / 78.9%	C140 C617	+	+
60	PF3D7_1360800	Falcilysin	138.9	51.4% / 57.3% / 61.3%	C438	+	+	FV
61	PF3D7_1407800	Plasmepsin IV	51.0	47.0% / 48.8% / 43.0%	C370	+	+	FV
62	PF3D7_1446200	M17 leucyl aminopeptidase	67.8	59.0% / 53.9% / 55.4%	C121	+	+
Protein degradation
63	PF3D7_0317000	Proteasome subunit alpha type-3, putative	29.3	46.0% / 42.9% / 39.3%	C222		+
64	PF3D7_0727400	Proteasome subunit alpha type-5, putative	28.4	82.8% / 75.0% / 82.8%	C76 C112	+	+
65	PF3D7_0807500	Proteasome subunit alpha type-6, putative	29.5	81.9% / 77.3% / 68.8%	C172		+
66	PF3D7_0932300	M18 aspartyl aminopeptidase	65.6	52.6% / 52.3% / 49.5%	C226		+
67	PF3D7_1248900	26S protease regulatory subunit 8, putative	49.5	33.1% / 32.2% / 35.6%	C391
68	PF3D7_1306400	26S protease regulatory subunit 10B, putative	44.7	35.6% / 42.2% / 38.9%	C111'
69	PF3D7_1402300	26S proteasome regulatory subunit RPN6	78.4	20.6% / 23.0% / 16.2%	C377
Protein folding
70	PF3D7_0214000	T-complex protein 1, putative	61.0	48.2% / 51.3% / 40.8%	C184 C499
71	PF3D7_0320300	T-complex protein 1 epsilon subunit, putative	59.2	40.4% / 25.6% / 32.7%	C458	+
72	PF3D7_0322000	Peptidyl-prolyl cis-trans isomerase	19.0	64.3% / 49.1% / 49.1%	C69	+ C168	+	ER
73	PF3D7_0527500	Hsc70-interacting protein	51.1	29.7% / 30.8% / 23.4%	C17 C187	+ C187	+
74	PF3D7_0627500	Protein DJ-1	20.3	86.2% / 86.2% / 81.5%	C85 C106/ C106'	+	+
75	PF3D7_0708400	Heat shock protein 90	86.2	58.3% / 63.0% / 59.9%	C393 C616	+	+
76	PF3D7_0708800	Heat shock protein 110	100.0	50.2% / 57.4% / 59.9%	C265 C606	+	+
77	PF3D7_0818900	Heat shock protein 70 (PfHsp70-1)	73.9	77.3% / 65.6% / 68.5%	C28 C583	+	+
78	PF3D7_0827900	Protein disulfide isomerase	55.5	68.1% / 69.8% / 75.2%	C384 C387	+	+	SP/ER
79	PF3D7_1118200	Heat shock protein 90, putative	108.5	43.2% / 48.6% / 39.2%	C556	+
80	PF3D7_1344200	Heat shock protein 110, putative	108.2	62.4% / 72.4% / 73.6%	C23	+	+	SP/ER
Protein transport
81	PF3D7_0210000	Secretory complex protein 61 gamma subunit	9.3	34.6% / 34.6% / 34.6%	C19			ER
82	PF3D7_0214100	Protein transport protein SEC31	166.7	25.8% / 33.6% / 29.4%	C309
83	PF3D7_0524000	Karyopherin beta	127.4	59.1% / 60.1% / 52.9%	C212 C452	+
84	PF3D7_1361100	Protein transport protein Sec24A	106.7	22.6% / 28.5% / 29.0%	C251
Transcription
85	PF3D7_1011800	PRE-binding protein	131.6	49.0% / 46.8% / 51.8%	C505	+	+
Translation
86	PF3D7_0309600	60S acidic ribosomal protein P2	11.9	92.0% / 92.0% / 82.1%	C12	+	+
87	PF3D7_1103100	60S acidic ribosomal protein P1, putative	13.0	85.6% / 83.1% / 83.1%	C19		+
88	PF3D7_1204300	Eukaryotic translation initiation factor 5A	17.6	51.6% / 51.6% / 54.0%	C73	+ C73 + C148	+
89	PF3D7_1302800	40S ribosomal protein S7, putative	22.5	48.5% / 22.2% / 33.0%	C23
90	PF3D7_1323400	60S ribosomal protein L23	22.1	31.6% / 25.3% / 19.5%	C134
91	PF3D7_1338300	Elongation factor 1-gamma, putative	47.8	46.0% / 55.2% / 54.3%	C70	+	+
92	PF3D7_1357000 / PF3D7_1357100	Elongation factor 1-alpha	49.0	68.8% / 63.9% / 67.5%	C356	+	+
93	PF3D7_1434600	Methionine aminopeptidase 2	71.7	33.9% / 30.4% / 25.3%	C447
94	PF3D7_1438000	Eukaryotic translation initiation factor eIF2A, putative	76.8	36.6% / 36.9% / 31.6%	C176		+
95	PF3D7_1451100	Elongation factor 2	93.5	59.3% / 63.3% / 67.1%	C130 C362 C653 C725 C786	+ C565	+
96	PF3D7_1468700	Eukaryotic initiation factor 4A	45.3	67.3% / 67.3% / 63.8%	C291	+	+
Unknown
97	PF3D7_0321800	WD repeat-containing protein, putative	322.2	X / X / 0.6%	C2321
98	PF3D7_0811400	Conserved protein, unknown function	71.4	45.9% / 54.1% / 43.9%	C517		+
99	PF3D7_0813300	Conserved Plasmodium protein, unknown function	36.3	32.4% / 32.4% / 32.4%	C61	+	+
100	PF3D7_1023900	Chromodomain-helicase-DNA-binding protein 1 homolog, putative	381.3	16.0% / 18.4% / 19.4%	C1375	+	+
101	PF3D7_1205600	Conserved Plasmodium protein, unknown function	39.0	28.5% / 37.2% / 27.6%	C213	+	+
102	PF3D7_1361800	Conserved Plasmodium protein, unknown function	291.0	42.7% / 39.8% / 41.5%	C821 C1337 C1549

^aIndicates the percentage of a protein’s sequence in one of three independently conducted experiments which has been covered in mass spectrometry analysis.

^bCysteines which were identified as being sulfenylated due to a mass shift of C = SO-NEM (+141.0426); C* = SO-BCN-Bio1 (+392.177), and their position in a protein’s amino sequence are listed. “C + C” indicates two cysteines in the same peptide sequence which were found to be simultaneously sulfenylated.

^c“+” indicates proteins which were also identified by Wang et al. as being S-nitrosated. If available, the modified cysteine is given. Proteins found to be non-specifically enriched were subtracted [16].

^d“+” indicates proteins which were also identified by Kehr et al. as being S-glutathionylated. Proteins found to be non-specifically enriched were subtracted [15].

^eSP, signal peptide; AP, apicoplast; PV, parasitophorous vacuole; PM, plasma membrane; ER, endoplasmic reticulum; FV, food vacuole; Rhop, rhoptry. Annotation based on PlasmoDB/SIgnalP analyses.

Throughout preparation collected samples were subjected to protein concentration measurements, SDS-PAGE and immunodetection targeting the biotin moiety of BCN-Bio1 with an anti-biotin antibody. Thus, the efficiency of the protocol throughout the 3 independently performed experiments was confirmed (Figure 2B, Supplementary S1). The eluates of the 3 biological replicates showed similar lane patterns of proteins of varying sizes ranging from low to high mass, indicating a broad distribution of sulfenylated proteins (Figure 2B). Notably, 2 bands between 66 and 166 kDa appeared more prominent in all 3 samples. One would expect those proteins to be highly abundant in mass spectrometry analysis, potentially overlapping other protein signals. Therefore, MS data were searched for the highest abundance proteins. L-lactate dehydrogenase (PF3D7_1324900, PfLDH) showed the highest spectrum count but has only a molecular mass of only 34.1 kDa, not represented by a remarkably prominent lane in immunodetection. P. falciparum proteins in a mass range of 66 to 110 kDa such as heat shock protein 70 (PfHsp70-1, PF3D7_0818900), elongation factor 2 (PF3D7_1451100), and inositol-3-phosphate synthase (PF3D7_0511800) showed spectrum counts less than half that of PfLDH. Regarding human proteins, only human glyceraldehyde-3-phosphate-dehydrogenase (HsGAPDH, 36.1 kDa) showed a spectrum count in the same range as PfHsp70-1 (see S2). Bovine serum albumin (BSA), which is a constituent of the cell culture medium, has a molecular mass of 69.3 kDa. It was identified in all 3 replicates but at a spectrum count 100 times lower than that of PfHsp70-1. It is thus not totally clear what the dominant bands in WB analysis represent, but they do not seem to influence MS analysis.

Classification of sulfenylated proteins in P. falciparum

In order to obtain a better overview of the proteins’ characteristics, identified sulfenylation targets were clustered according to their gene ontology (GO) slim annotations depicting their role in biological processes and molecular functions (Figure 3, Supplementary S3). Sulfenylated proteins of P. falciparum seem to be involved in a large variety of biological processes such as biosynthesis, locomotion, translation, response to stress, and protein folding (Figure 3A). A GO (gene ontology) enrichment analysis using slim GO terms (biological processes) reveals that our dataset is highly enriched in proteins involved in carbohydrate metabolism and nucleobase-containing compound catabolic processing. The first group, which is enriched more than 10-fold, comprises mainly glycolytic enzymes, and will be discussed in more detail later. The second group, enriched over 8-fold, contains the enzymes purine nucleoside phosphorylase, adenosine deaminase, and adenylosuccinate synthase

Figure 3 -.

Functional classification and enrichment of sulfenylated proteins in the trophozoite stage of P. falciparum. Sulfenylated proteins identified were clustered according to their gene ontology (GO) annotations in biological process (A) and molecular function (B). The number in brackets indicates the enrichment of the respective GO slim term compared to all P. falciparum proteins. GO slim enrichment analysis was performed using the online enrichment tool of PlasmoDB (www.plasmodb.org)

Clustering according to the involvement in molecular function gives a different pattern (Figure 3B). More than a quarter of identified sulfenylated proteins participate in ion binding, such as SNF2 helicase and S-adenosylmethionine synthase. The second largest group is formed by proteins involved in RNA binding, followed by proteins with peptidase activity. Enrichment analysis reveals more than 6-fold enrichment of proteins with transferase activity such as dihydropteroate synthase, ethanolamine kinase, and elongation factor 1-gamma. Proteins with hydrolase activity such as glycerophosphodiester phosphodiesterase and insulinase are also enriched more than 5-fold compared to background.

4. Discussion

Redox PTMs play a major role in cellular regulation as they affect the structure and function of numerous proteins [38]. Sulfenylation, as a reversible oxidative modification, has been shown to serve as a regulator of transcription factors, a sensor for oxidative stress, and mediate in general redox signaling [21]. In this study BCN-Bio1, a highly strained bicyclo[6.1.0]nonyne, showing high reactivity with sulfenylated proteins [28], was used to identify the P. falciparum trophozoite sulfenylome.

Previous redox studies focusing on the malaria parasite P. falciparum revealed 32 putative interaction partners for the redox enzymes thioredoxin (PfTrx), glutaredoxin (PfGrx), and plasmoredoxin (PfPlrx) [14]. Furthermore, in pull-down analysis of whole cell lysates of the trophozoite stage, 491 and 317 putative targets for S-glutathionylation and S-nitrosation were identified, respectively [15,16]. To deepen our understanding of the interplay between redox PTMs in P. falciparum, this current study focused on the identification of sulfenylated proteins to enable comparison with other previously identified redox PTM targets.

A common issue in bottom-up proteomics is the over-representation of abundant proteins in datasets. Therefore, the identified sulfenylation targets in this study were first checked for their abundance in the trophozoite stage. Global proteomic studies revealed abundance levels of Plasmodium proteins over different stages of development [39]. S2 shows the broad distribution of abundance levels of proteins identified in this study, thus verifying the enrichment method applied, which enables identification of even low abundance proteins.

Of 167 detected modified peptides around 50% were detected in one out of three biological replicates, but with high confidence. This underlines the difficulties in detecting modification sites. As sulfenylation is a highly dynamic modification, trapping it remains highly challenging. Some of these peptides may well be of low abundance, further complicating the identification. Alternative approaches such as direct detection of biotin-containing tags (DiDBiT) can improve the detection of modification sites. In this approach proteins labeled by a biotin-tag containing probe are digested prior to enrichment. This is reported to increase detection efficiency by up to 50% [40].

While comparing overall P. falciparum sulfenylation patterns of trophozoites with sulfenylation patterns of other organisms, comparable levels of modified proteins can be observed. Out of P. falciparum isolate 3D7’s predicted 5,449 proteins, 102 are found to be sulfenylated, representing approximately 1.9% of the proteome. In Homo sapiens (H. sapiens) 1,285 out of 74,788 proteins could be detected as sulfenylation targets, representing around 1.7% [41]. In Arabidopsis thaliana (A. thaliana, 39,361 proteins), 1,394 proteins were identified as targets for sulfenylation, giving a coverage of around 3.5% [42].

Clustering of sulfenylated proteins revealed large distribution amongst different biological processes and molecular functions (Figure 3). At the same time certain GO slim terms are more enriched than others. Clustering sulfenylated proteins according to their molecular functions shows around 6-fold enrichment of transferase and hydrolase activity while carbohydrate metabolic process is enriched more than 10-fold. Proteins belonging to those GO terms may be especially susceptible towards oxidative modification. Whether this modification is required for regulation these proteins has to be elucidated in detail

Proteins of the glycolytic pathway appear to be particularly targeted for sulfenylation; 7% of P. falciparum’s sulfenylated proteins belong to this pathway, despite representing only 0.001% of all total predicted proteins. Glycolysis is crucial for the survival of the intracellular parasite, as Plasmodium is reliant on the metabolization of glucose for ATP production [43]. A comparative study of glycolytic enzymes amongst different organisms reveals conserved sulfenylation patterns and sites. Table 2 compares the patterns of glycolytic enzymes phosphofructokinase, aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), phosphoglycerate kinase, enolase, and pyruvate kinase (PK) in H. sapiens, A. thaliana, and P. falciparum. Six proteins were found to be sulfenylated in H. sapiens as well as in A. thaliana [44]. When these findings are compared with our dataset for P. falciparum, a high overlap can be observed; 7 out of 11 glycolytic enzymes were identified as targets for sulfenylation, including 6-phosphofructokinase (PfPFK1, PF3D7_0915400), fructose-bisphosphate aldolase (Pf3D7_1444800), triosephosphate isomerase (Pf3D7_1439900), PfGAPDH (PF3D7_1462800), enolase (PF3D7_1015900), PfPK (PF3D7_0626800), and PfLDH (PF3D7_1324900). With the exception of PfPFK1 and PfLDH, homologues of the other 5 proteins were also identified as being sulfenylated in H. sapiens and A. thaliana, indicating a strong conservation of sulfenylation patterns in proteins of the glycolysis pathway across kingdoms. The amino acid sequences surrounding the sulfenylated residue ot homologue proteins showed a high level of conservation (data not shown), and this together with limitations in a) the number of sequences available for comparison and, b) the lack of knowledge of specific sulfenylation patterns in all organisms studied, hindered us in being able to specify a “sulfenylation motif”, i.e a conserved pattern potentially required for sulfenylation.

Table 2.

Sulfenylated glycolysis enzymes previously found in different organisms. (+): Protein has been found to be sulfenylated. Modified according to [44].

	H. sapiens	A. thaliana	P. falciparum
Phosphofructokinase
Aldolase	+	+	+
Triosephosphate isomerase	+	+	+
Glyceraldehyde-3-P-dehydrogenase	+	+	+
Phosphoglycerate kinase	+	+
Enolase	+	+	+
Pyruvate kinase	+	+	+

Glycolytic enzymes identified as being sulfenylated in this current study have previously been identified as targets of S-glutathionylation (8/11) and S-nitrosation (10/11). These findings are in accordance with previous publications and imply a strong redox regulation of proteins in this key energy metabolism pathway [15,16]. Glycolytic proteins show high sequence conservation at their catalytic sites, but contain unique structural differences between parasitic and host homologues, rendering these proteins attractive targets for the development of antimalarials. Alam et al. describe the moonlighting functions of glycolytic proteins which may arise due to structural changes induced by PTMs [45]. Besides catalyzing the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, eukaryotic GAPDH is involved in, amongst others, gene regulation, vesicular transport, and cell signaling, as reviewed in [46]. The respective activity seems to be dependent on its cellular localization, influenced through PTMs such as phosphorylation, acetylation, and S-nitrosation [45]. PfGAPDH is expressed in all life stages [47] and out of 8 cysteine residues, 3 were identified as being sulfenylated (C56, C157, C250). PfGAPDH has previously also been confirmed as a target for S-glutathionylation. Depending on the concentration and the incubation time, the protein is reversibly inhibited by oxidized glutathione (GSSG) [15]. PfGAPDH’s active site cysteines were shown to be susceptible to S-nitrosation, leading to an inhibition of its enzymatic activity. Low molecular weight thiols such as reduced glutathione can reverse this effect [48]. In general, sulfenylation as a consequence of oxidative disbalance may lead to a rapid adaption of different metabolic pathways and thus help organisms deal with this challenge [49].

When our protein hit list is compared with previously reported targets for S-nitrosation and S-glutathionylation, we find that out of 102 sulfenylated proteins, 55 were also found to be S-glutathionylated and S-nitrosated; 17 were found to be S-glutathionylated [15], and 10 to be S-nitrosated [16], meaning that around 80% of Plasmodium sulfenylated proteins are susceptible to more than one redox modification (Table1, Figure 4). For example, one of the sulfenylated proteins identified by this study was S-adenosylmethionine synthetase (PfSAMS). The enzyme catalyzes the formation of S-adenosylmethionine from methionine and ATP and thus participates in multiple processes (epigenetic, metabolism, signaling) supporting parasite growth. Its structure is highly homologous to other SAMS, especially those of plants and other protozoans [50]. Pretzel et al. demonstrated the contribution of Cys52, Cys113, and Cys187 to its redox regulation by influencing its structure and activity. Upon S-glutathionylation and S-nitrosation PfSAMS is inhibited, emphasizing the importance of redox regulation [51].

Figure 4 -.

Venn diagram depicting the overlap between P. falciparum proteins susceptible to different redox modifications Pull-down experiments identified 101 targets for sulfenylation, 491 target proteins for S-glutathionylation, and 317 target proteins for S-nitrosation [15,16]. 55 proteins were identified containing all 3 PTM. The Venn diagram was generated using the online tool http://bioinformatics.psb.ugent.be/webtools/Venn/.

Our study also identified the macrophage migration inhibitory factor (PfMIF, PF3D7_ 1229400) as a target for sulfenylation. This protein is a homologue of the human cytokine MIF and is released into the host’s blood stream upon schizont rupture. It is predicted to modulate the human immune system, making it an interesting target for further study [52,53]. Of its four cysteines, Cys3 and Cys4 were identified as being susceptible to sulfenylation. These two cysteines have previously been shown to be crucial for antioxidant activities as well as for the Trx-like oxidoreductase activity of this protein [54]. PfMIF has also been identified as being glutathionylated and nitrosated in the trophozoite stage [15,16]. As this protein appears highly susceptible to redox modifications while itself participating in antioxidant activities, further research is needed to understand its putative regulatory mechanisms.

Thioredoxin peroxidase 1 (PfTrxPx [aka PfPrx1a], Pf3D7_1438900) belongs to the family of cysteine-dependent Prx enzymes. They reduce peroxides and can be recycled by PfTrx or PfGrx [55]. PfTrxPx is a 2-Cys Prx, meaning that two cysteines are involved in its catalytic cycle [56]. Its resolving cysteine Cys170 has been identified as being sulfenylated in this study. This cysteine, as well as its peroxidatic cysteine Cys50, are important for the Prx-based redox regulation of 127 cytosolic target proteins as shown by Brandstaedter and coworkers [57].

These findings indicate possible general crosstalk between redox modifications. As sulfenic acids are often intermediates en route to more stable modifications, it is likely that proteins are initially sulfenylated under unstressed conditions and upon stress are further modified, becoming for example S-glutathionylated or S-nitrosated upon exposure to glutathione or NO-donors. Both modifications are thought to protect thiols against irreversible oxidation [58,59]. In general, the exposure of a cysteine, its pK_a and its electrophilicity determine its reactivity [60]. Surface exposure of cysteines, and thus accessibility for modification can change depending on structural modifications of the protein [61,62]. In some cases, redox sensitive proteins are able to shuttle between different compartments of a cell and are thus subjected to changing redox environments, such as proximity to metal centers, rapid changes in local pH, and changes in solvent access. These factors may all affect the pK_a and reactivity of critical cysteine(s) [63]. Further studies have investigated non-redox PTMs in Plasmodium, including phosphorylation, acetylation, ubiquitination, and sumoylation, as reviewed in [9,64]. Today, more than 300 PTMs are known across a variety of systems, massively expanding a proteome’s diversity and complexity [65]. Nevertheless, their interplay still remains ambiguous, rendering additional research necessary. As PTMs are so consequential, significant efforts in antimalarial drug discovery focus on inhibition of enzymes mediating these modifications [66].

Some proteins detected in at least 2 replicates as being sulfenylated were previously found as targets for other PTMs, including PfSNF2 helicase (PF3D7_0624600). This nuclear protein is suggested to be part of large functional complexes involved in transcriptional regulation, and has been confirmed as a target for phosphorylation in Plasmodium [10,67]. Gene expression must be highly regulated to meet the needs of the parasite at each stage of the life-cycle, therefore it is not surprising that proteins involved in transcription are especially targets for sulfenylation, allowing for fine tuning of gene regulation.

The cyclophilin peptidyl-prolyl-cis-trans-isomerase (PF3D7_0322000) accelerates the folding of Plasmodium proteins through cis-trans isomerase activity and shows a chaperone function in a comparable manner to heat shock proteins [68]. This activity is inhibited by the cyclic peptide cyclosporine A, which also has an antimalarial effect [69]. Besides sulfenylation, S-glutathionylation, and S-nitrosation, this protein has been detected as a target for mono-ubiquitination [12]. This rapidly reversible modification can label a protein for degradation [12]. Furthermore, this protein has recently been identified as being an interaction partner of peroxiredoxin 1 a (PfPrx1a) [57]. It is thought that PfPrx, which undergoes sulfenylation during its catalytic cycle, can transfer this oxidation to an interacting protein, thus regulating its activity [70].

We investigated the predicted sub-cellular localisation of targets of sulfenylation (Table 1). This analysis revealed that sulfenylation can take place on proteins with a wide range of localisations, including plasma membrane, food vacuole, apicoplast, rhoptries and endoplasmic reticulum (ER), and highlights that sulfenylation takes place in at least several cellular compartments, and that sulfenylation activity is not restricted merely to the parasite cytosol.

In a previous study, Sturm et al. identified 32 proteins interacting with the redox enzymes thioredoxin (PfTrx), glutaredoxin (PfGrx), and plasmoredoxin (PfPlrx) [14]. Those which interact with more than one of the redox enzymes are also more likely to be sulfenylated, giving an overlap of 14 proteins (Table 3). One of them is the enzyme pyruvate kinase (PfPK) (PF3D7_0626800). It has been shown that S-glutathionylation leads to inhibition of PfPK in a concentration dependent reversible mode [15]. Additionally, PfPK is susceptible to S-nitrosation and 3 of its 15 cysteines have been found to be sulfenylated [16]. These data indicate a strong susceptibility towards redox regulation of this rate-limiting enzyme, which is needed to adapt the glycolytic flux to changing environments, such as those experienced as the parasite passes from vertebrate host to mosquito vector and vice versa [71].

Table 3.

P. falciparum sulfenylated proteins identified as interaction partners of thioredoxin (Trx), glutaredoxin (Grx), or plasmoredoxin (Plrx) [14].

	PlasmoDB Accession n°	Protein name	Trx	Grx	Plrx	SOH
1	PF3D7_0608800	Ornithine aminotransferase	+	+	+	+
2	PF3D7_0626800	Pyruvate kinase	+	+	+	+
3	PF3D7_0708400	Heat shock protein 90	+	+	+	+
4	PF3D7_0802200	1-cys peroxiredoxin	+			+
5	PF3D7_0818200	14-3-3 protein	+	+	+	+
6	PF3D7_0818900	Heat shock protein 70	+	+	+	+
7	PF3D7_0826700	Receptor for activated c kinase		+		+
8	PF3D7_0827900	Protein disulfide isomerase			+	+
9	PF3D7_0922200	S-adenosylmethionine synthetase	+	+	+	+
10	PF3D7_1324900	L-lactate dehydrogenase		+		+
11	PF3D7_1343000	Phosphoethanolamine N-methyltransferase		+		+
12	PF3D7_1438900	Thioredoxin peroxidase 1	+			+
13	PF3D7_1451100	Elongation factor 2		+	+	+
14	PF3D7_1462800	Glyceraldehyde-3-phosphate dehydrogenase		+	+	+

As mentioned above, a certain conservation of sulfenylation can be observed across different kingdoms. However, when comparing the hit list for P. falciparum with identified sulfenylated proteins of H. sapiens, differences become obvious. Of the 102 P. falciparum target proteins, 20 are parasite-specific and do not have a human homologue, for 52 proteins there is evidence that the human homologues are also subject to sulfenylation [27,72]. The remaining 26 proteins were found to be sulfenylated in the parasite but not in its host. It may be that those proteins have distinct regulatory mechanisms. One of these proteins is M17 leucyl aminopeptidase (PF3D7_1446200). This protein is a known target for the development of antimalarial drugs, as inhibition leads to death of P. falciparum in culture [73]. The protein catalyzes the release of neutral amino acids from the N-terminus of peptides, thus participating in hemoglobin digestion, pivotal for parasite survival [74], but a role of redox PTM in modulation of its activity has not yet been described.

Proteomic analyses of P. falciparum have revealed susceptibility of its proteins towards multiple PTMs, whereas possible cross talk between different modifications remains to be elucidated in detail. Our identification of sulfenylation targets give an interesting new insight into the redox regulation of the parasite’s proteins and allow for further structural and functional analysis of identified target proteins. Nevertheless, proteomic approaches need to be supplemented with in vivo data, verifying the biological function of the respective modification. A better understanding of these complex mechanisms is pivotal for combatting the pathogen and thus fighting malaria.

Highlights.

• Pull-down to identify sulfenylome of P. falciparum trophozoites.

• 102 sulfenylated proteins identified including 152 modification sites.

• High overlap between target proteins for S-glutathionylation, S-nitrosation and sulfenylation.

• Especially glycolytic cycle seems to be modified.