Scanning Quadrupole Data Independent Acquisition â Part B

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Scanning Quadrupole Data Independent Acquisition – Part B Application to the Analysis of the Calcineurin Interacting Proteins during Treatment of Aspergillus fumigatus with Azole and Echinocandin Antifungal Drugs Praveen Juvvadi, M. Arthur Moseley, Christopher J. Hughes, Erik J. Soderblom, Sarah Lennon, Simon R. Perkins, J. Will Thompson, Scott J Geromanos, Jason Wildgoose, Keith Richardson, James I. Langridge, Johannes P.C. Vissers, and William J. Steinbach J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00499 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

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Title Scanning Quadrupole Data Independent Acquisition – Part B Application to the Analysis of the Calcineurin Interacting Proteins during Treatment of Aspergillus fumigatus with Azole and Echinocandin Antifungal Drugs

Authors Praveen R. Juvvadi1,ǂ,*, M. Arthur Moseley2,ǂ, Christopher J. Hughes3,ǂ, Erik J. Soderblom2,ǂ, Sarah Lennon3, Simon R. Perkins4, J. Will Thompson2, Scott J. Geromanos5, Jason Wildgoose3, Keith Richardson3, James I. Langridge3, Johannes P.C. Vissers3,ǂ,*, William J. Steinbach1,6,* 1.

Division of Pediatric Infectious Diseases, Department of Pediatrics, Duke University Medical Center, Durham, NC

2.

Proteomics and Metabolomics Shared Resource Center for Genomic and Computational Biology, Duke University Medical Center, Durham, NC

3.

Waters Corporation, Wilmslow, United Kingdom

4.

Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom

5.

Waters Corporation, Milford, MA

6.

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC

ǂ

these authors have contributed equally to this work

* to

whom

correspondence

should

be

addressed

[email protected] and [email protected])

ACS Paragon Plus Environment

([email protected],

Journal of Proteome Research

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Abstract Calcineurin is a critical cell signalling protein that orchestrates growth, stress response, virulence, and antifungal drug resistance in several fungal pathogens. Blocking calcineurin signalling increases the efficacy of several currently available antifungals and suppresses drug resistance. In this study, we demonstrate the application of a novel scanning quadrupole DIA method for the analysis of changes in the proteins co-immunoprecipitated with calcineurin during therapeutic antifungal drug treatments of the deadly human fungal pathogen Aspergillus fumigatus. Our experimental design afforded assessment of precision of the method as demonstrated by peptide and protein centric analysis from eight replicates of the study pool QC samples. Two distinct classes of clinically-relevant antifungal drugs that are guide-line recommended for the treatment of invasive “aspergillosis” caused by Aspergillus fumigatus, the azoles (voriconazole) and the echinocandins (caspofungin and micafungin) that specifically target the fungal plasma membrane and the fungal cell wall, respectively,

were

chosen

to

distinguish

variations

occurring

in

the

proteins

coimmunoprecipitated with calcineurin. Novel potential interactors were identified in response to the different drug treatments that are indicative of the possible role for calcineurin in regulating these effectors. Notably, treatment with voriconazole showed increased immunoprecipitation of key proteins involved in membrane ergosterol biosynthesis with calcineurin. In contrast, echinocandin (caspofungin or micafungin) treatments caused increased immunoprecipitation of proteins involved in cell wall biosynthesis and septation. Furthermore, abundant co-immunoprecipitation of ribosomal proteins with calcineurin occurred exclusively in echinocandins treatment indicating reprogramming of cellular growth mechanisms during different antifungal drug treatments. While variations in the observed calcineurin immunoprecipitated proteins may also be due to changes in their expression levels under different drug treatments, this study suggests an important role for calcineurindependent cellular mechanisms in response to antifungal treatment of A. fumigatus that warrants future studies. Keywords: label-free quantitation, anti-fungal drug treatment, Aspergillus fumigatus Abbreviations: DIA, oa-TOF, VOR, CSP, MFG, SPQC


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Introduction In this study, the utility of the scanning quadrupole DIA method, as described in part A, is demonstrated by the quantitative analysis of the immunoprecipitated calcineurin protein complexes resulting from three different antifungal drug treatments for Aspergillus fumigatus. Invasive aspergillosis (IA), caused by the fungus A. fumigatus, is a leading infectious cause of death in immunocompromised patients [1-3]. Voriconazole (VOR) is the consensus guideline-recommended first-line antifungal therapy against IA [4]. However, the emergence of azole antifungal resistance over the last decade has prompted the use of echinocandin antifungals (e.g., caspofungin (CSP), micafungin (MFG)) as much-needed second-line therapeutic options [1,5-7]. While the azoles inhibit ergosterol biosynthesis and cause membrane stress by compromising the integrity of the fungal cell membrane, the echinocandins target fungal cell wall β-glucan synthesis [8-10]. The calcineurin pathway has an established role in cell wall integrity in different fungi [11], and calcineurin inhibitors, cyclosporine (CsA) and tacrolimus (FK506), have shown an in vitro synergistic effect against A. fumigatus in combination with CSP [12]. Resistance to antifungals has also been linked to the calcineurin signaling pathway [13], and the combination of calcineurin inhibitors with antifungals has been shown to inhibit the growth of various drug resistant fungi [14-18]. More recently, calcineurin inhibitors were active against multidrug-resistant strains of A. fumigatus [19] and also dimorphic pathogenic fungi [20], implicating the importance of calcineurin control over drug response machinery. Hyphal growth and extension are necessary to cause invasive fungal disease, and the importance of calcineurin for hyphal growth and cell wall biosynthesis is well-established in A. fumigatus [11]. Although the calcineurin pathway has been shown to impact both the cell membrane and cell wall integrity through the regulation of effectors that influence the biosynthesis of ergosterol, chitin and β-glucan, the exact mechanism of how calcineurin controls these processes is not clearly understood [11,21]. Combinatorial strategies involving the use of the major antifungal classes (azoles and echinocandins) in combination with calcineurin-specific inhibitors (FK506 and cyclosporine A) to counteract the emergence of resistance in fungal pathogens warrants further investigations to aid our understanding and potential exploitation of the calcineurin signaling network in these pathogens [12,13,22]. Calcineurin has, in summary, clearly been established as an important regulator of hyphal growth under stress conditions and in conferring drug resistance in A. fumigatus. Protein level changes in the fungus in direct response to antifungal treatments can provide valuable insights into drug actions and also potential biomarkers of drug efficacy. While earlier studies were focused on the genomic approaches to decipher alterations in



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protein profiles as a response to antifungal drug treatment [23-25], few recent studies have examined the response of A. fumigatus to antifungal agents such as amphotericin B [26], CSP [27] and VOR [23]. However, no study to date has examined comparative changes in the proteome in response to different drug exposures in A. fumigatus. The alterations in the interactors of calcineurin following treatment of A. fumigatus with the leading antifungals targeting the cell wall (CSP and MFG) and the cell membrane (VOR) were therefore determined, and the relationship between changes in the immunoprecipitated calcineurin protein complements following different drug exposures examined. To accomplish this, the A. fumigatus strain expressing the calcineurin catalytic subunit (CnaA) tagged to EGFP at its native locus was utilised. The three guideline-recommended antifungals used in clinical management, CSP, MFG and VOR, were used at slightly sub-minimal inhibitory concentrations in order to observe the full effect of their mechanism of action.

Experimental Conditions Organism and construction of CnaA-GFP expression strain A. fumigatus wild-type strain akuBKU80 was used for the construction of CnaA-GFP expression strain from its native locus and Escherichia coli DH5α competent cells used for subcloning. Cellular extracts preparation and GFP-Trap purification For all the proteomic analyses experiments and protein level comparisons between the different drug treatments, the A. fumigatus strain expressing CnaA-GFP fusion protein was utilized. The CnaA-GFP expression strain cultured in the absence of the drugs served as the control. To identify proteins binding to calcineurin the strain was cultured separately in 200 ml GMM liquid medium (in a 500 ml culture flask) for 24 h, shaking at 250 rpm, 37°C, by inoculating a defined amount of spore suspension (107/ml or 108/ml) in the absence or presence of the different antifungals. Voriconazole (Pfizer, New York, NY) was used at 0.125 µg/ml; Caspofungin (Merck, Kenilworth, NJ) and Micafungin (Astellas, Tokyo, Japan) were each used at 1 µg/ml concentrations. The 24 h mycelial cultures were collected by vacuum filtration using a miracloth (CalBiochem, Billerica, Massachusetts) over a sintered glass funnel and washing extensively with ice-cold distilled water (with 250-500 ml for each).The mycelia were semi-dried on filter paper and approximately 1-2 g (wet weight) mycelia was used for protein extraction. The mycelia were homogenized to a fine powder using liquid nitrogen in a mortar and pestle. Later 5 ml of lysis buffer, comprising 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.01% Triton X-100, 1 mM DTT, 1 mM PMSF, 1:100 Protease


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Inhibitory Cocktail (Sigma Aldrich, St. Louis, MO), was added and mixed well. The homogenized mycelial suspension was centrifuged (5000 rpm for 10 min at 4°C) to remove mycelial debris and the crude supernatant was again clarified by centrifugation at 7000 rpm for 15 min at 4°C. The supernatant (crude extract) was carefully collected into a fresh tube and the total protein in the crude extract was quantified by Bradford method and normalized to contain ~10 mg protein/5 ml in the sample before GFP-Trap affinity purification (Chromotek, Planegg-Martinsried, Germany). Fifty µl GFP-Trap resin was equilibrated by washing three times in 500 µl ice-cold dilution buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1 mM PMSF, 1:100 Protease Inhibitory Cocktail) according to the manufacturer instructions and finally re-suspended in 100 µl ice cold dilution buffer. The GFP-Trap resin suspension was then mixed with total crude cell lysate containing ~10 mg total protein and incubated at 4°C by gentle agitation for 2 h. After incubation for 2 h, the suspension was centrifuged at 2000 rpm for 10 min at 4°C and the pelleted GFP-Trap resin was washed once in 500 µl of ice-cold dilution buffer and then twice with 500 µl of wash buffer (10 mM Tris-HCl pH 7.5, 350 mM NaCl, 0.5 mM EDTA, 1 mM PMSF, 1:100 Protease Inhibitory Cocktail). Protein bound GFP-Trap resin was finally washed three times with 100 µl 50 mM ammonium bicarbonate, pH 8.0, and then re-suspended in 50 µl 50 mM ammonium bicarbonate, pH 8.0. Protein digestion A. fumigatus immunoprecipitated samples were supplemented with RapiGest SF (Waters Corporation, Milford, MA) surfactant to a final concentration of 0.1%, reduced with 10 mM DTT for 30 min at 80°C and alkylated with 20 mM IAA for 45 min at room temperature. On resin proteolytic digestions were accomplished by addition of 500 ng sequencing grade trypsin (Promega) for 18 h at 37°C as previously described [27]. Following removal of supernatants, peptides were acidified to pH 2.5 with TFA, incubated at 60˚C for 1 h to hydrolyse the RapiGest SF, which was removed by centrifugation. Next, all samples were lyophilized to dryness and resuspended in 40 µl 1%TFA/2% acetonitrile LC-MS configuration LC separations were performed using a nanoACQUITY system (Waters Corporation) equipped with a Symmetry C18 5 µm, 2 cm x 180 µm precolumn and an HSS T3 C18 1.8 µm, 20 cm x 75 µm analytical column. The samples were transferred with aqueous 0.1% (v/v) formic acid to the precolumn at a flow rate of 5 µl/min. Mobile phase A was water containing 0.1% (v/v) formic acid, whilst mobile phase B was acetonitrile containing 0.1% (v/v) formic acid. The peptides were eluted from the precolumn to the analytical column and



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separated with a gradient of 5 to 40% mobile phase B over 90 min at a flow rate of 300 nl/min. The analytical column temperature was maintained at 35ºC. The lock mass compound, [Glu1]-Fibrinopeptide B (200 fmol/µl), was delivered at 600 nl/min to the reference sprayer source of the mass spectrometer. Mass spectrometric analysis of tryptic peptides was performed using a Xevo G2-XS QTOF mass spectrometer (Waters Corporation, Wilmslow, United Kingdom). The mass spectrometer was operated with a resolution of 35,000 FWHM and all analyses were performed in positive ion mode ESI. The ion source block temperature and capillary voltage were set to 100ºC and 3.2 kV, respectively. The time-of-flight (TOF) mass analyzer of the mass spectrometer was externally calibrated with a NaCsI mixture from m/z 50 to 1990. LCMS data were collected using a novel data independent mode of acquisition (SONAR). In this acquisition mode the quadrupole was continuously scanned between m/z 400 to 900, with a quadrupole transmission width of approximately 24 Da. The oa-TOF records mass spectra as the quadrupole scans and stores these MS data into two hundred discrete bins. Two data functions (modes) are acquired in an alternating fashion, differing only in the collision energy applied to the gas cell. In the low energy MS1 mode, data are collected with a constant gas cell collision energy of 6 eV. In the elevated energy MS2 mode, the gas cell collision energy is ramped from 14 eV to 40 eV (per unit charge). As such, the resulting data contains both peptide precursor ions and all associated fragment ions. The spectral acquisition time in each mode was 0.5 s with a 0.02 s interscan delay. The reference sprayer was sampled every 60 s and the data post-acquisition lock mass corrected. Data processing and database searching SONAR quadrupole scanning DIA data were processed using Progenesis QI for proteomics v2 (PQIp) (Nonlinear Dynamics, Newcastle upon Tyne, United Kingdom) using optimized threshold and search parameters. The data acquisition methodology and associated analytical validation are presented in extensive detail in the accompanying part A manuscript. Briefly, in PQIp, all data features are aligned between samples based on their accurate mass and retention time values. All quantitative values are calculated from the high-resolution and quadrupole isolated selected ion chromatograms (SIC), expressed as the area-under the curve of these SICs from the MS1 analyses. Protein and peptides identifications were obtained by searching an A. fumigatus NCBI (9150 RefSeq entries, April 2016) database. The results have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [28] with dataset identifier PXD005848. All abundance values were normalized to calcineurin across all samples. All fold changes refer to protein-based values derived from the fold changes of the peptides to which they map. Relative protein


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abundances, log2 and square root transformed, assuming Poisson distribution, were considered significant when the 95% confidence interval was exceeded. Significance (p) fold change values were calculated using a z-test with the median CV value of all replicate protein abundance measurements (n = 2144) used as an estimation of the biological variance. Results and Discussion Study

Pool

QC

sampling

and

data

analyses

to

define

proteins

specifically

immunoprecipitated with calcineurin To define how the echinocandin (caspofungin, micafungin) and azole (voriconazole) antifungals influence the interaction of calcineurin with different proteins within the fungal cell, candidate interactors were qualitatively and quantitatively characterized by quadrupole scanning DIA LC-MS following GFP-Trap affinity purification. In order to identify proteins coimmunoprecipitated with calcineurin we utilized an A. fumigatus strain expressing the catalytic subunit of calcineurin (CnaA) tagged to GFP at its native locus. The experimental strategy, as outlined in Figure 1, included culturing of the CnaA-GFP expression strainseparately in the absence (control) or presence of caspofungin (CSP), micafungin (MFG) and voriconazole (VOR) for a period of 24 h. Proteins extracted from the respective cultures were normalized across all the samples prior to GFP-Trap affinity purification in order to characterize the enrichment of proteins immunoprecipitating with calcineurin under different antifungal treatments. To provide clear metrics on the qualitative and quantitative reproducibility of the overall analyses, a study pool QC (SPQC) sample, comprised of equal aliquots of all samples in this study was run eight times while collecting control and test data. The corresponding eight results files, along with all control and drug treatment sample data analysis results are included in Supplemental Table 1, sheet 1. An identification summary of proteins qualitatively and quantitatively characterized, both as a total number (538 proteins) and those with 2 or more peptides (430 proteins), as well as a histogram of the SPQC coefficient of variation for all proteins with a protein and peptide FDR < 1% is shown in Figures 2A and 2B. The complete data set, including the qualitative and quantitative results in mzIdentML and mzQuantML format, respectively, are available in the PRIDE proteomics data repository (PXD005848). For the proteins quantitated by 2 or more peptides, the median %CV was 8.3%, and for those identified with 1 or more peptides, the %CV was 8.9%. These values are similar in magnitude to the quantitative results described in part A of this manuscript, where the quantitative analysis of a four protein mixture was examined in detail and reinforcing the precision that can be achieved using a SONAR label-free DIA LC-



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MS approach for the quantitative analysis of complex biological samples. In this study, the majority (79.9%) of the proteins quantified had at least two sequence unique peptides. Moreover, approximately 92% of all detected features, i.e. charge state groups, with a total close to about 27,000 features, were co-detected in all eight SPQC samples, demonstrating very consistent sampling of the multi-dimensional retention time, m/z and quadrupole position space. In all quantitative analyses of the control and antifungal drug-treated samples, all data were normalized to the level of calcineurin, shown in the Supplementary Table 1, sheet 1, and those with a fold-change greater than or less than 2-sigma (2σ) and p < 0.05 were considered to be of interest for hypothesis generation. A graphical summary of the qualitative analysis is shown by the unsupervised PCA and hierarchical clustering/heat map analysis of the data in Figure 3, illustrating that both the echinocandins, caspofungin (CSP) and micafungin (MFG), induced the largest changes in the immunoprecipitated calcineurin protein complements of the samples. The PCA results also indicate that voriconazole (VOR) treatment had little effect at the protein level on the calcineurin interactors of A. fumigatus. Following the SPQC analysis, and normalization of the values with respect to calcineurin, the proteins identified as co-immunoprecipitating with calcineurin were compared between untreated (control) versus the different drug treatment conditions as shown in Supplemental Table 1, sheets 2-4. The complete list of proteins identified and quantified using AUC are also shown in the Supplemental Table 1, sheet 1. The biological variance, determined as outlined in the Experimental Conditions section, was used to assign significance (p) values to the measured protein abundance fold change values for the various drug treatment vs. control comparisons. The results are graphically summarised in the volcano-style distributions presented in Figure 4, showing significance as a function of fold change. Proteins exclusively detected and quantified that coimmunoprecipited with calcineurin are highlighted in black. The insets of Figures 4 (A), (B) and (C) illustrate the application of a t-test to determine fold change significance. Considering a ≥ 2σ change, equalling a ± 1.7 fold change on an absolute scale, and p < 0.05 to be significant, treatment with echinocandins showed an overlap of 164 proteins that were common to both CSP and MFG; Supplementary Table 1, sheets 5-6), with 63 proteins exclusively co-immunoprecipitating with calcineurin in CSP treatment (Table 1) vs. 87 proteins exclusively co-immunoprecipitating with calcineurin in MFG treatment (Table 2). Treatment with VOR resulted in a total of 52 proteins (with ≥ 2σ change; Supplementary Table 1, sheet 7) co-immunoprecipitating with calcineurin, of which 9 proteins were exclusive to VOR treatment (Table 3). While 26 proteins with ≥ 2σ change that co-immunoprecipitated with calcineurin were common to all three drug treatments (Supplementary Table 2), 14 proteins were bound to calcineurin exclusively under CSP and VOR treatments and 3


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proteins exclusively bound to calcineurin under MFG and VOR treatments. The results of these binary comparisons are summarised in Figure 5, supporting the results shown in Figure 3 in that echinocandins induced changes shared the greatest similarity in their altered quantitative protein complement. Echinocandin treatments differentially influenced the interaction of calcineurin with proteins regulating fungal cell wall biosynthesis and hyphal septum organization In accordance with the known role for echinocandins in targeting cell wall biosynthesis machinery [29], we noted that treatment with echinocandins enhanced binding of calcineurin to Rho1 (the regulatory subunit of β-1,3-glucan synthase complex), known to regulate β-1,3-glucan synthase activity [30]. Though not very significant in the case of CSP treatment, MFG treatment resulted in a 2.3-fold increase of Rho1 binding to calcineurin (Supplementary Table 3). In the presence of CSP, Gel1 (a β(1-3) glucanosyltransferase) and Scw11 (a β-1,3-glucan-modifying enzyme) showed a 6.7-fold and 2.9-fold respective increase in co-immunoprecipitation with calcineurin. Gel1 β(1-3) glucanosyltransferase plays a major role in the elongation of cell wall β-1,3-glucan chains [31]. Intriguingly, although MFG treatment did not show such an increase in binding to calcineurin with respect to Gel1 and Scw11 proteins, it did increase the interaction of calcineurin with glucosamine fructose6-phosphate aminotransferase (GFAT) by 2.7-fold. GFAT catalyses the formation of glucosamine 6-phosphate, the first rate-limiting step in the biosynthesis of cell wall chitin and its expression was increased under cell wall stress [32]. Another enzyme that catalyses the synthesis of

GDP-mannose in cell wall biosynthesis [33], mannose-1-phosphate

guanylyltransferase (Srb1), also showed 2.7-fold enhanced interaction under CSP and MFG treatments. In contrast, VOR treatment did not result in any significant increase in the binding of the respective proteins to calcineurin (Supplementary Table 3), indicating the probability of calcineurin-mediated regulation of these effector proteins only under cell wall stress induced upon echinocandin treatments. The increased interaction observed between calcineurin and these key cell wall biosynthesis proteins supports previous reports on the potential role of calcineurin in the regulation of cell wall integrity pathway [12, 34-35]. Increased interaction of calcineurin with septins AspB, AspC and AspD, the key cytoskeletal GTPase proteins that localize at the hyphal septa [36], and are involved in the organization of septation was also evident in the presence of both the echinocandins but not in the case of VOR treatment. A recent study from our laboratory has also revealed the interaction of AspB septin with calcineurin during caspofungin treatment [37]. Opposed to the observed increased interactions, a 2-fold decrease in binding of a α1,2-mannosyltransferase, Ktr4, with calcineurin was noted in the presence of CSP and MFG


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in comparison to the VOR treatment (Supplementary Table 3). Mannosyltransferases play a crucial role in the modification of cell wall proteins through the addition of N-linked and/or Olinked oligosaccharides and contribute to fungal virulence [38]. Ktr4 is responsible for glycosylation of cell wall mannoproteins and cell wall-integrity [39], and its deletion resulted in thinner cell walls and hypersensitivity cell wall stress. Previous studies in our laboratory have revealed the localization of calcineurin at the hyphal tips and septa [40], where active cell wall synthesis occurs lending support to the observed cell wall and septation related protein interactions in this study. The mechanism of how calcineurin regulates these important cell wall and septation associated proteins remains the subject of future investigations. We hypothesize that calcineurin being a protein phosphatase is involved in the dephosphorylation of these substrates. Treatment with the echinocandins altered the immunoprecipitation of key proteins related to cytoskeleton organization and membrane trafficking with calcineurin We have previously demonstrated that calcineurin localized at the active points of fungal hyphal growth, the hyphal tip and septum. Dynamic movement of calcineurin within the hyphal compartments and its co-localization with the major cytoskeletal component, actin, during septation revealed the possibility of calcineurin’s interaction with yet unknown proteins involved in cytoskeleton structure and function [41]. It was noted that calcineurin interacted with a number of cytoskeletal proteins and others involved in membrane trafficking, such as ArpA (an actin-related protein), tubulin (α-1, β and TubB subunits), Rac Rho GTPase, Rab GTPase (Vps21/Ypt51) and the monomeric GTPase SarA under normal growth conditions (Supplementary Table 3). From among these proteins, tubulin α-1 and tubulin β showed a 3 to 4-fold and 4 to 6-fold decreased calcineurin binding following treatment with CSP and MFG, respectively. These results suggest that the mode of action of the echinocandins may also involve alterations in the interaction of calcineurin with the tubulin cytoskeletal components and proteins responsible for membrane trafficking. A previous study showed the interaction of calcineurin with α-1 tubulin [42]. The precise mechanism of how calcineurin interaction with these cytoskeletal proteins regulates their function remains to be investigated. Calcineurin immunoprecipitated protein complexes revealed differential enrichment of proteins involved in membrane biosynthesis during echinocandin and voriconazole treatments Important trends that also emerged from these analyses are the interaction of calcineurin with the key regulatory enzymes such as the 14α-sterol demethylases (Cyp51A


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and Cyp51B), squalene monoxygenase (Erg1) and hydroxymethylglutaryl-CoA synthase (Erg13) involved in the biosynthesis of membrane ergosterol. While the interaction of Cyp51A and Cyp51B was particularly enhanced in the presence of VOR when compared to the control (Supplementary Table 3), indicative of the important role for calcineurin in membrane stress response, we cannot rule out the possibility that the expression levels of these proteins in the presence of VOR may also be a contributing factor to the observed increase in calcineurin binding to these proteins. It is known that prolonged VOR exposure induces the expression of both cyp51A and cyp51B mRNAs [43]. The cyp51A and cyp51B genes encode for 14α-sterol demethylases involved in the synthesis of ergosterol required for fungal membrane structure, and mutations in the cyp51A gene are responsible for emergence of azole resistance [5-7]. Following VOR treatment, there was a 3.2-fold increase observed in the amount of Cyp51A immunoprecipitating with calcineurin in comparison to the control (Supplementary Table 3). The interaction of calcineurin with a putative transmembrane protein, UsgS, increased by 10-fold in the VOR treatment sample. Although we did notice a 2-fold increase in the interaction of UsgS under CSP treatment, a 4-fold decrease in interaction was noted in MFG treatment again indicating differential interactions under different drug treatments. These results suggest that although CSP and MFG antifungals belong to the same echinocandin class, they may cause differential effects on calcineurin interactions with different proteins. In support of this, it has been previously demonstrated that CSP at higher concentrations triggers a differential calcium response in comparison to MFG, and results in differential phosphorylation of calcineurin in vivo [44]. Furthermore, while these comparative analyses indicate drug-dependent variations in the interactors of calcineurin, it is also unknown if the echinocandin treatments or other factors contribute to the observed decrease in the interaction of Cyp51A and Cyp51B proteins. Previous reports from Candida species [45], C. neoformans [46] and Mucorales [47] suggested fungicidal activity of calcineurin inhibitors (cyclosporine A and FK506) in combination with azoles that are usually fungistatic. However, in A. fumigatus clinical isolates from transplant and non-transplant patients exposed to a combination of VOR with calcineurin inhibitors was not synergistic [48-49], but azole-resistant strains were susceptible to calcineurin inhibitors [12], indicating calcineurin inhibition as a potential effective strategy to overcome azole resistance. In addition, a recent study showed that combination of FK506 with VOR had synergistic inhibitory activity against Aspergillus biofilm formation [50].



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Significant alterations in other calcineurin interactors were evident during different antifungal treatments In the CSP and MFG treatments a significant number of ribosomal proteins (41 proteins) with ≥ 2σ change were identified to interact with calcineurin (Supplementary Table 4). Such an increase with respect to any of these ribosomal proteins was not observed in VOR treatment. The observed increase in ribosomal protein interactions with calcineurin in CSP and MFG treatments but not in VOR treatment in particular indicates the possibility of reprogramming of cellular growth mechanisms during different drug treatments. A previous study on profiling the proteome of A. fumigatus in response to CSP treatment also showed an increase in the abundance of ribosomal proteins following exposure for 24 h [27]. The Pma1 plasma membrane H+-ATPase, which was previously shown to be down-regulated in the presence of CSP [27] but up-regulated in the presence of amphotericin B [26] showed increased interaction with calcineurin in the presence of CSP (3.7-fold) and MFG (2.1-fold) but decreased binding to calcineurin in the presence of VOR (3.5-fold). Pma1has been previously shown to be a target of calcineurin in S. cerevisiae and A. fumigatus [51-52]. The endoplasmic reticulum calcium ATPase was increased by 2.5-fold and 2.3-fold following CSP and VOR treatment, respectively, but not in treatment with MFG. Another well know interactor of calcineurin, the calcineurin binding protein (CbpA), which belongs to the RCAN (regulator of Calcineurin) family of proteins and has been shown to both positively and negatively impact calcineurin function in fungi [27,53], was also found to interact with calcineurin in our analyses. However, we did not observe any significant difference in its binding to calcineurin under the different antifungal drug treatments. Only in the presence of CSP and MFG, a 9.6-fold and 3.9-fold respective increase was noted in the G-protein complex beta subunit CpcB protein binding to calcineurin (Supplemental Table 1). The Gβ-CpcB protein was recently shown to be involved in cell wall integrity and virulence of A. fumigatus [54]. Interestingly, the increased fold change in the interaction of calcium/calmodulin-dependent protein kinase with calcineurin only in the presence of CSP (5.6-fold; Supplemental Table 1, sheet 5) and MFG (5.2-fold; Supplemental Table 1, sheet 6), but not following VOR treatment, also indicated differential calcineurin interactions between the azole and the echinocandin treatments. The DUF89 protein, whose function remains unknown, was also increased 2-fold in VOR treatment in comparison to the control (Table 3), but not in CSP and MFG treatment. There was also a 2.6-fold increase in binding of calcineurin to the COP II vesicle protein Yip3 in the VOR treatment only (Supplemental Table 1, sheet 7). Several hypothetical proteins were also identified as calcineurin interactors in the presence of different antifungals. However, due to insufficient annotation available for these proteins in the Aspergillus genome database, no significance


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could be assigned to these proteins. A schematic representation of the major quantitative proteomic findings of calcineurin inhibition and antifungal drug-dependent alterations in the calcineurin interactome is shown in Figure 6.

Conclusions Recent studies on the identification of new substrates for calcineurin through advanced proteomic technologies both in the model yeast S. cerevisiae [55], and in human pathogenic yeast, C. neoformans [56] provided novel insights into additional functions of calcineurin in vivo. Here, we performed a comparative study to probe the proteome of A. fumigatus for potential calcineurin interactors under treatment with two important classes of clinical guideline-recommended antifungal drugs, the echinocandins and the azoles that are currently being used to treat patients suffering from invasive aspergillosis. This is the first label-free quantitative proteomics study to utilize a new scanning quadrupole DIA method to distinguish important changes in the calcineurin-dependent proteome of A. fumigatus in response to different drug treatments. Novel and specific interactors of calcineurin in response to the drug treatments indicative of calcineurin’s role in regulating these effectors have been detected. The only well characterized substrate of calcineurin to date is Crz1p (ortholog of mammalian NFAT), which mediates the transcriptional response triggered by calcineurin activation in response to stress conditions [57-58]. While Rcn1 belonging to the calcipressin family of proteins has been shown to bind to calcineurin and contribute to fine tuning calcineurin signaling in the yeast [59], other known stress regulated substrates of calcineurin in the yeast are Hph1 and Hph2 [60], Slm1 and Slm2 [61]. Although the genes encoding Crz1 (CrzA) and Rcn1 (CbpA) proteins have been characterized in A. fumigatus [27,62-64], several calcineurin substrates remain unknown in A. fumigatus. This study contributes to our understanding of the important, but yet unknown, link between calcineurin and antifungal drug mechanisms. How calcineurin regulates these proteins will be an important aspect for future investigation. Overall, the presented results for the three antifungals at clinically relevant doses provide relevant biological context on how these drugs can differentially modulate protein-protein interactions in vivo and lend further insight into both drug efficacy and resistance. While this study identified previously unknown substrates of calcineurin, strengthening the link between calcineurin, cell membrane and cell wall biosynthesis, stress and repair mechanisms, further studies are nevertheless required to confirm the exact mechanism of calcineurin-mediated regulation of these substrates, as the identified interactors may be regulated through a phosphorylation-dephosphorylation mechanism.



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Acknowledgement This project was funded, in part, through an NIH/NIAID R21 award AI127551 to PRJ and WJS and an Astellas Investigator-Initiated Trial to WJS. Richard Denny of Waters Corporation is kindly acknowledged for discussions on significance tests. Conflict of Interest CJH, SL, SJG, JW, KR, JIL, and JPCV are employed by Waters Corporation, which operates in the field covered by the article. The remaining authors declare no competing financial interests. Supporting Information The following files are available free of charge at ACS website http://pubs.acs.org: Table S1:

Summary of all data and all binary and multi-condition comparisons.

Table S2:

Proteins

Found

Coimmunoprecipitated

with

Calcineurin

In

all

Drug

Treatments Table S3:

Drug Induced Changes in Cell Wall, Septation, Cytoskeletal and Membrane Associated Proteins Immunoprecipitated with Calcineurin

Table S4:

Ribosomal Proteins Coimmunoprecipitated with Calcineurin in the Presence of CSP and MFG

Captions Figure 1. Scheme of the experimental strategy to identify proteins binding to calcineurin under different antifungal drug treatments in Aspergillus fumigatus. Figure 2. Numbers of quantifiable proteins (A) and %CV histogram for the eight SPQC analyses (B). Figure 3. Unsupervised PCA scores distribution (control (CTRL, purple), voriconazole (VOR, green), caspofungin (CSP, red), micafungin (MFG, blue) and study pool QC (SPQC, grey)) (A) and hierarchical clustering (B) of the identified and quantified calcineurin proteins affected by drug treated A. fumigatus.


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Figure 4. Fold change significance (p; z-score) vs. log2 protein fold change distributions for caspofungin (A), micafungin (B), voriconazole (C) antifungal drug treated vs. non-treated (control; CTRL) A. fumigatus. Black = proteins exclusively co-immunoprecipitated with Calcineurin overviewed in Tables 1, 2 and 3, respectively, Grey = not significantly regulated or not exclusively co-immunoprecipitated with Calcineurin. Shown inset, as references, are volcano, single degree t-test based distribution for the same drug treatment vs. control comparisons, respectively. Size = fold change. Figure 5. Venn intersection of proteins co-immunoprecipitating with calcineurin in the three drug treatments comparing to the control with a fold change of greater than or less than 2σ and p < 0.05. Figure 6. Schematic representation of calcineurin inhibition and antifungal drug-dependent alterations in the proteins co-immunoprecipitating with calcineurin. The major echinocandin antifungals (caspofungin and micafungin) target β-glucan synthesis in the fungal cell wall (green arrow) and the azoles (voriconazole) target the ergosterol biosynthesis in the fungal cell membrane (orange arrow). Calcineurin inhibition abolishes azole and echinocandin resistance making it a suitable drug target for overcoming antifungal drug resistance. Calcineurin is a heterodimer composed on CnaA (catalytic) and CnaB (regulatory) subunits and its activity is inhibited by the immunosuppressants, FK506 and cyclosporine A (CsA). Calcineurin plays a major role through the binding and activation of several yet unknown downstream effector proteins involved in the regulation of cell wall integrity, hyphal growth, drug resistance, stress response and virulence. How the echinocandins and azoles differentially influence the calcineurin interactome is unknown (shown in dotted arrows). Proteomic profiling of calcineurin interactors in the presence of the antifungals (echinocandins and azoles) revealed alterations in its interactions (shown in bold solid arrows). Bold dotted arrow indicates the reduced interaction of calcineurin with different cytoskeletal proteins in the presence of echinocandins. Only some relevant proteins are shown. References 1.

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Bultynck, G.; Heath, V.L.; Majeed, A.P.; Galan, J.M.; Haguenauer-Tsapis, R.; Cyert, M.S. Slm1 and slm2 are novel substrates of the calcineurin phosphatase required for heat stress-induced endocytosis of the yeast uracil permease. Mol. Cell. Biol. 2006, 26, 4729-4745.

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Soriani, F.M.; Malavazi, I.; da Silva, Ferreira, M.E.; Savoldi, M.; Von Zeska Kress, M.R.; de Souza Goldman, M.H.; Loss, O.; Bignell, E.; Goldman, G.H. Functional characterization of the Aspergillus fumigatus CRZ1 homologue, CrzA. Mol. Microbiol. 2008, 67, 1274-1291.

63.

Cramer, R.A.; Perfect, B.Z.; Pinchai, N.; Park, S.; Perlin, D.S.; Asfaw, Y.G.; Heitman, J.; Perfect, J.R.; Steinbach, W.J. Calcineurin target CrzA regulates conidial germination, hyphal growth, and pathogenesis of Aspergillus fumigatus. Eukaryot Cell. 2008, 7, 1085-1097.

64.

Pinchai, N.; Perfect, B.Z.; Juvvadi, P.R.; Fortwendel, J.R.; Cramer, R.A.; Asfaw, Y.G.; Heitman, J.; Perfect, J.R.; Steinbach, W.J. Aspergillus fumigatus calcipressin CbpA is involved in hyphal growth and calcium homeostasis. Eukaryot Cell. 2009, 8, 511-519.


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Table 1. Proteins exclusively co-immunoprecipitated with Calcineurin in CSP treatment exceeding the 2σ (95% probability) regulation confidence interval. Accession Number 70994944 70990422 71000343 70992617 70984376 70990780 70999105 146323681 70989077 71000100 70990924 70996594 70990790 70999706 70986482 70984092 70991753 70990216 70998348 70998192 70989089 70992211 70990662 70983670 70989229 70996256 70993698 71002366 70985282 70984832 70995926 71000068 70991661

Protein locus and description XP_752248.1 glycerol-3-phosphate dehydrogenase, mitochondrial XP_750060.1 ATP synthase delta chain, mitochondrial precursor XP_754866.1 peptidyl-prolyl cis-trans isomerase/cyclophilin XP_751157.1 NADH-ubiquinone oxidoreductase 39 kDa subunit XP_747702.1 amidophosphoribosyltransferase XP_750239.1 proliferating cell nuclear antigen (PCNA) XP_754274.1 translation initiation factor 2 alpha subunit XP_001481556.1 NADH-ubiquinone oxidoreductase 21 kDa subunit XP_749388.1 casein kinase I XP_754767.1 glucose-6-phosphate 1-dehydrogenase XP_750311.1 phenylalanyl-tRNA synthetase, beta subunit XP_753052.1 TCTP family protein XP_750244.1 serine/threonine protein phosphatase PP1 XP_754570.1 DNA damage response protein (Dap1) XP_748734.1 pyruvate dehydrogenase E1 beta subunit PdbA XP_747566.1 mitochondrial import receptor subunit (tom40) XP_750725.1 thiazole biosynthesis enzyme XP_749957.1 cytochrome C1/Cyt1 XP_753896.1 NADH-ubiquinone oxidoreductase B12 subunit XP_753823.1 protein transport protein Sec61 alpha subunit XP_749394.1 aspartyl-tRNA synthetase Dps1 XP_750954.1 ATP citrate lyase subunit (Acl), putative XP_750180.1 40S ribosomal protein S8e XP_747362.1 cell wall glucanase (Scw11) XP_749464.1 14-3-3 family protein ArtA XP_752883.1 mitochondrial phosphate carrier protein (Mir1) XP_751696.1 Aha1 domain family XP_755864.1 DUF636 domain protein XP_748147.1 histone H2A XP_747922.1 ubiquinol-cytochrome C reductase complex core protein 2 XP_752718.1 ubiquinol-cytochrome C reductase complex subunit UcrQ XP_754751.1 eukaryotic translation initiation factor 3 subunit 2i XP_750679.1 DUF500 and SH3 domain protein


CSP vs. control fold change 11.7 9.4 5.9 4.6 4.2 4.1 4.0 3.8 3.6 3.5 3.5 3.5 3.4 3.4 3.4 3.3 3.2 3.1 3.0 3.0 3.0 3.0 3.0 2.9 2.9 2.8 2.8 2.8 2.8 2.7 2.7 2.7 2.7

CSP vs. control log2 fold change 3.5 3.2 2.6 2.2 2.1 2.0 2.0 1.9 1.9 1.8 1.8 1.8 1.8 1.8 1.7 1.7 1.7 1.6 1.6 1.6 1.6 1.6 1.6 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.4 1.4 1.4

p 4.2E-12 2.8E-11 5.3E-09 1.1E-07 3.9E-07 5.6E-07 8.1E-07 1.3E-06 2.9E-06 4.2E-06 4.9E-06 5.3E-06 6.3E-06 6.5E-06 8.6E-06 1.1E-05 1.4E-05 2.9E-05 3.2E-05 3.3E-05 3.5E-05 4.5E-05 4.5E-05 6.4E-05 7.1E-05 8.6E-05 1.1E-04 1.2E-04 1.2E-04 1.3E-04 1.7E-04 1.8E-04 1.9E-04

Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

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70995090 70981416 70991843 70999023 70995028 70995343 70987264 70991681 146323719 70999514 71002728 70993746 71002010 70993400 70997740 70993696 70998594 70984362 70987006 70989661 70986989 146322523 70985058 70993656 70983638 146324195 70989001 70992789 70993290 71000586

XP_752311.1 ribosomal protein S23 (S12) XP_731490.1 FAD binding domain protein XP_750770.1 NADH-ubiquinone oxidoreductase 304 kDa subunit precursor XP_754233.1 NADH-ubiquinone oxidoreductase B18 subunit XP_752280.1 conserved hypothetical protein XP_752429.1 S-adenosylmethionine synthetase XP_749110.1 PCI domain protein XP_750689.1 cell division control protein 2 kinase XP_752147.2 ubiquinol-cytochrome c reductase complex 14 kDa protein XP_754476.1 arginase XP_756045.1 cell division control protein Cdc48 XP_751720.1 acetyl-coenzyme A synthetase FacA XP_755686.1 translation elongation factor EF-2 subunit XP_751547.1 calnexin XP_753605.1 iron-sulfur protein subunit of succinate dehydrogenase Sdh2 XP_751695.1 saccharopine dehydrogenase Lys9 XP_754019.1 succinyl-CoA synthetase alpha subunit XP_747695.1 nascent polypeptide-associated complex (NAC) subunit XP_748988.1 CTP synthase XP_749680.1 mitochondrial F1F0 ATP synthase subunit F (Atp17) XP_748980.1 40S ribosomal protein S13 XP_750491.2 mitochondrial GTP/GDP transporter Ggc1 XP_748035.1 septin AspC XP_751675.1 conserved hypothetical protein XP_747346.1 mitochondrial ATPase subunit ATP4 XP_001481515.1 ssDNA binding protein Ssb3 XP_749350.1 40S ribosomal protein S10a XP_751243.1 conserved hypothetical protein XP_751492.1 actin-related protein ArpA XP_754976.1 Rho GTPase Rac


2.6 2.5 2.5 2.5 2.4 2.4 2.3 2.3 2.3 2.3 2.3 2.3 2.2 2.2 2.2 2.2 2.2 2.2 2.2 2.1 2.1 2.1 2.1 2.1 2.0 2.0 0.5 0.4 0.4 0.4

1.4 1.3 1.3 1.3 1.3 1.3 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.0 1.0 1.0 1.0 -1.0 -1.2 -1.2 -1.4

2.9E-04 3.7E-04 4.1E-04 4.5E-04 6.1E-04 7.7E-04 9.4E-04 1.1E-03 1.1E-03 1.3E-03 1.3E-03 1.4E-03 1.7E-03 1.9E-03 1.9E-03 2.1E-03 2.1E-03 2.4E-03 2.5E-03 2.8E-03 3.5E-03 3.6E-03 4.0E-03 4.0E-03 5.1E-03 6.7E-03 7.0E-03 1.5E-03 1.4E-03 2.5E-04

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Table 2. Proteins exclusively co-immunoprecipitated with Calcineurin in MFG treatment exceeding the 2σ (95% probability) regulation confidence interval. Accession Number 70983245 70997461 71000828 146323801 70999388 70992657 70984619 70991302 70991445 70996212 71000275 70997834 70995930 71001294 146322483 70999940 70994984 70998911 70985200 70992635 71000066 71001286 70982294 70983436 70984557 70992559 146323687 146322509 70991353 146323147 70992917 70999438 70994192

Protein locus and description XP_747150.1 methyltransferase SirN-like XP_753478.1 26S proteasome regulatory subunit Mts4 XP_755095.1 SRP receptor beta subunit (Srp102) XP_751845.2 hypothetical protein AFUA_4G09810 XP_754413.1 ADP-ribosylation factor 6 XP_751177.1 cytosolic large ribosomal subunit protein L7A XP_747816.1 conserved hypothetical protein XP_750500.1 26S proteasome regulatory particle subunit Rpn8 XP_750571.1 proteasome regulatory particle subunit Rpt4 XP_752861.1 peptidase D XP_754832.1 succinate dehydrogenase subunit Sdh1 XP_753649.1 nitroreductase family protein XP_752720.1 transketolase TktA XP_755328.1 mitochondrial Hsp70 chaperone (Ssc70) XP_750349.2 ribosomal protein L16a XP_754687.1 serine hydroxymethyltransferase XP_752268.1 methionyl-tRNA synthetase XP_754177.1 Ketol-acid reductoisomerase XP_748106.1 vacuolar dynamin-like GTPase VpsA XP_751166.1 hypothetical protein AFUA_6G12880 XP_754750.1 C1 tetrahydrofolate synthase XP_755324.1 fatty acid activator Faa4 XP_746675.1 NIMA-interacting protein TinC XP_747245.1 conserved hypothetical protein XP_747785.1 acetylglutamate kinase XP_751128.1 AhpC/TSA family protein XP_752229.2 glutaredoxin Grx5 XP_750420.2 t-complex protein 1, eta subunit XP_750525.1 glucosamine-fructose-6-phosphate aminotransferase XP_748443.2 aspartic-type endopeptidase XP_751307.1 5-oxo-L-prolinase XP_754438.1 phosphoglucomutase PgmA XP_751943.1 DUF757 domain protein


MFG vs. control fold change 4.8 3.8 3.7 3.7 3.5 3.5 3.5 3.3 3.2 3.2 3.1 2.9 2.9 2.9 2.9 2.9 2.9 2.8 2.8 2.8 2.8 2.8 2.8 2.8 2.8 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.6

MFG vs. control log2 fold change 2.3 1.9 1.9 1.9 1.8 1.8 1.8 1.7 1.7 1.7 1.6 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.4 1.4 1.4 1.4 1.4 1.4 1.4 1.4

p 3.9E-06 7.2E-05 8.5E-05 9.6E-05 1.8E-04 2.0E-04 2.0E-04 3.4E-04 4.7E-04 5.9E-04 6.8E-04 1.3E-03 1.3E-03 1.4E-03 1.5E-03 1.5E-03 1.5E-03 1.7E-03 1.8E-03 2.0E-03 2.0E-03 2.1E-03 2.1E-03 2.3E-03 2.4E-03 2.5E-03 2.8E-03 3.1E-03 3.3E-03 3.3E-03 3.4E-03 3.4E-03 3.5E-03


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70995532 70990724 70984364 70986952 70997049 70993752 70995446 70989367 146324747 146322982 70999742 70999522 70998630 71001148 70998726 70996304 70985998 70998710 70989083 70995173 70984804 146324059 146323070 146322910 70991469 70993636 71000467 70993160 70983336 70992323 70997379 70986492 70983023 71001952 71001738 70991907 70999658 146322408

XP_752521.1 ethanolamine kinase XP_750211.1 alcohol dehydrogenase, zinc-containing XP_747696.1 ATP synthase D chain, mitochondrial XP_748962.1 electron transfer flavoprotein alpha subunit XP_753279.1 calcium binding protein Caleosin XP_751723.1 NADH-ubiquinone oxidoreductase, subunit F XP_752478.1 60S ribosomal protein L6 XP_749533.1 alpha,alpha-trehalose-phosphate synthase subunit XP_747279.2 oligopeptidase family protein XP_755704.2 NADH-ubiquinone oxidoreductase 49 kDa subunit XP_754588.1 acetolactate synthase, large subunit XP_754480.1 proteasome regulatory particle subunit Rpt3 XP_754037.1 BAR adaptor protein RVS161 XP_755255.1 60S ribosomal protein L10 XP_754085.1 40S ribosomal protein S3Ae XP_752907.1 DUF1014 domain protein XP_748502.1 F-box domain protein XP_754077.1 nucleosome assembly protein Nap1 XP_749391.1 Coatomer subunit gamma XP_752351.1 mitochondrial inner membrane translocase subunit TIM44 XP_747908.1 hypothetical protein AFUA_5G04350 XP_754050.2 homoserine kinase XP_755989.2 phospholipase D (PLD) XP_755431.2 ADP-ribosylation factor XP_750583.1 Rho GTPase Rho1 XP_751665.1 alpha-ketoglutarate dehydrogenase complex subunit Kgd1 XP_754922.1 40S ribosomal protein S4 XP_751428.1 Hsp70 family protein XP_747195.1 alanyl-tRNA synthetase XP_751010.1 ribosomal protein L26 XP_753438.1 Rab small monomeric GTPase Rab7 XP_748739.1 fatty acid synthase beta subunit XP_747039.1 bifunctional catalase-peroxidase Cat2 XP_755657.1 bifunctional tryptophan synthase TRPB XP_755550.1 lectin family integral membrane protein XP_750802.1 oxidoreductase, short-chain dehydrogenase/reductase family XP_754546.1 40S ribosomal protein S7e XP_750101.2 ATP synthase gamma chain, mitochondrial precursor


2.6 2.6 2.6 2.6 2.6 2.5 2.5 2.5 2.5 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.3 2.3 2.3 2.3 2.3 2.3 2.3 2.2 2.2 2.2 2.2 2.2 2.2 2.2 2.1 2.1

1.4 1.4 1.4 1.4 1.4 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1

3.8E-03 4.0E-03 4.0E-03 4.2E-03 4.8E-03 5.8E-03 6.8E-03 6.8E-03 7.1E-03 7.4E-03 7.5E-03 7.5E-03 7.6E-03 7.7E-03 8.3E-03 8.4E-03 8.5E-03 8.9E-03 9.0E-03 9.3E-03 9.5E-03 1.1E-02 1.1E-02 1.2E-02 1.2E-02 1.3E-02 1.3E-02 1.4E-02 1.5E-02 1.6E-02 1.8E-02 1.9E-02 2.0E-02 2.1E-02 2.1E-02 2.2E-02 2.3E-02 2.5E-02

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70986856 70990130 146324723 146322400 71001364 70995568 70987220 70985198 70985024 71002512 70994744 70992913 70995822 71000215 146323430 71001378

XP_748915.1 cytokinesis EF-hand protein Cdc4 XP_749914.1 kinesin family protein XP_747206.2 U5 snRNP component Snu114, putative XP_750089.2 60S ribosomal protein L12 XP_755363.1 40S ribosomal protein S17 XP_752539.1 ADP-ribosylation factor XP_749089.1 cyclopropane-fatty-acyl-phospholipid synthase XP_748105.1 vacuolar ATP synthase catalytic subunit A XP_748018.1 ubiquitin C-terminal hydrolase (HAUSP) XP_755937.1 t-complex protein 1, alpha subunit XP_752149.1 iron-sulfur cofactor synthesis protein (Isu1) XP_751305.1 5'-nucleotidase XP_752666.1 SCF ubiquitin ligase complex subunit CulA XP_754812.1 PX domain protein XP_754484.2 arsenite translocating ATPase ArsA XP_755370.1 iron-sulfur cluster assembly accessory protein Isa2


2.1 2.1 2.1 2.0 2.0 2.0 2.0 2.0 2.0 2.0 0.4 0.4 0.3 0.3 0.3 0.2

1.1 1.1 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 -1.4 -1.5 -1.6 -1.6 -1.7 -2.1

2.6E-02 2.8E-02 3.1E-02 3.3E-02 3.8E-02 3.8E-02 4.0E-02 4.2E-02 4.3E-02 4.4E-02 3.2E-02 2.5E-02 2.1E-02 2.0E-02 1.4E-02 5.8E-03


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Table 3. Proteins exclusively co-immunoprecipitated with Calcineurin in VOR treatment exceeding the 2σ (95% probability) regulation confidence interval. Accession Number 70994720 70989235 70998480 70984753 70994244 70991867 70985178 70993702 70986306

Protein locus and description XP_752137.1 14-alpha sterol demethylase Cyp51A XP_749467.1 conserved hypothetical protein XP_753962.1 DUF89 domain protein XP_747883.1 dienelactone hydrolase family protein XP_751969.1 mitochondrial peroxiredoxin Prx1 XP_750782.1 conserved hypothetical protein XP_748095.1 thiamine biosynthesis protein (Nmt1) XP_751698.1 fructose-1,6-bisphosphatase Fbp1 XP_748647.1 FAD binding monooxygenase


VOR vs. control fold change 3.2 3.0 2.0 0.5 0.5 0.3 0.3 0.3 0.3

VOR vs. control log2 fold change 1.7 1.6 1.0 -1.0 -1.1 -1.6 -1.6 -1.6 -2.0

p 1.5E-05 3.2E-05 5.5E-03 5.2E-03 2.5E-03 4.7E-05 3.9E-05 2.3E-05 1.0E-06

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Table of Contents (TOC) graphic For TOC only:


Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Figure 1

Liquid Culturing of Aspergillus fumigatus Strain Expressing CnaA-GFP Fusion Protein (treatment with/without CSP, MFG, VOR for 24 h)

Homogenization of Harvested Mycelia in Liquid Nitrogen

Protein Extraction

Protein Estimation and Normalization

GFP-Trap Affinity Purification

On Resin Proteolytic Digestion with Trypsin

Quantitative LC-MS ACS Paragon Plus Environment

Study Pool QC Sampling

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Figure 2

(B)

# proteins/bin

(A)

# proteins

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41




Figure 3

(A)

(B)

CSP

CTRL MFG

SPQC

VOR


VOR VOR MFG MFG CSP CSP

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

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Figure 4

(A)



Figure 4

(B)


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Figure 4

(C)



Figure 5


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Figure 6



TOC Figure


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Scanning Quadrupole Data Independent Acquisition â Part B

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