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ORIGINAL ARTICLE
Year : 2022  |  Volume : 12  |  Issue : 2  |  Page : 39-45

Metacaspase Deletion Increases Carbonylated and Tyrosine-Phosphorylated Proteins associated with Protein Synthesis and Carbohydrate Metabolism in Saccharomyces cerevisiae


1 Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA
2 Proteomics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA
3 Department of Food Science and Nutrition, College of Agriculture and Marine Sciences, Sultan Qaboos University, Seeb; Ageing and Dementia Research Group, Sultan Qaboos University, Seeb, Oman
4 Section of Molecular Pharmacology and Toxicology, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism (NIAAA), Bethesda, MD, USA
5 Neuroinflammation Group, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW, Australia

Date of Submission14-Apr-2021
Date of Decision16-Apr-2021
Date of Acceptance30-Jun-2021
Date of Web Publication10-May-2022

Correspondence Address:
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijnpnd.ijnpnd_18_21

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   Abstract 


Hydrogen peroxide (H2O2) is an oxidant which could induce posttranslational modifications of proteins (PTMPs) in cells. It is still unknown that carbonylated proteins (CPs) were accumulated in caspase-suppressed leukemia cells or caspase-deleted Saccharomyces cerevisiae (yeast). Hence, we aimed to identify CPs and elucidate the role of metacaspase in regulating PTMPs and identify/compare the differentially expressed PTMPs in Δyca1 mutant compared to wild type with/without H2O2 exposure by proteomics approach. We found that deletion of the metacaspase gene (MCG) in yeast resulted in accumulation of high amounts of PTMPs associated with protein synthesis and carbohydrate metabolism compared to H2O2, which suggests that MCG is involved in the regulation of PTMPs and it could protect yeast from oxidative stress.

Keywords: carbonylation, metacaspase, proteome, tyrosine phosphorylation, yeast


How to cite this article:
Khan MS, Morgan M, Essa M, Akbar M, Guillemin GJ, Song BJ. Metacaspase Deletion Increases Carbonylated and Tyrosine-Phosphorylated Proteins associated with Protein Synthesis and Carbohydrate Metabolism in Saccharomyces cerevisiae. Int J Nutr Pharmacol Neurol Dis 2022;12:39-45

How to cite this URL:
Khan MS, Morgan M, Essa M, Akbar M, Guillemin GJ, Song BJ. Metacaspase Deletion Increases Carbonylated and Tyrosine-Phosphorylated Proteins associated with Protein Synthesis and Carbohydrate Metabolism in Saccharomyces cerevisiae. Int J Nutr Pharmacol Neurol Dis [serial online] 2022 [cited 2022 May 27];12:39-45. Available from: https://www.ijnpnd.com/text.asp?2022/12/2/39/345013




   Introduction Top


Protein carbonylation is often used as a cellular marker for increased oxidative stress and has been predominantly studied in aging and age-related diseases.[2],[3][4] Carbonylation can occur on proline, histidine, lysine, arginine, and cysteine residues of proteins through direct oxidation by reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxideanion (O2-), hydroxyl ion (OH), and/or peroxynitrite (ONOO).[5],[6] Endogenously produced H2O2 is associated with several physiologic and biochemical processes such as oxidative stress, posttranslational modifications of proteins (PTMPs), and apoptosis, depending on its time of occurrence and amount. This molecule also serves as a second messenger of cellular signal transduction.[7],[8],[9],[10] Apoptosis has been shown to remove or eradicate unwanted and unhealthy cells and cellular debris from the body. More importantly, it is also implicated in signal transduction pathways in response to the transiently changing levels of H2O2, which is essentially required for the maintenance of cellular homeostasis.[7]

Caspases are present in two different forms: procaspase (inactive) and cleaved caspase (active) depending on the presence and absence of prodomain, respectively. [11] The caspases are closely related with oxidative stress and apoptosis and play an important role in accumulation of oxidized proteins. In fact, our previous report showed that caspase inhibition with a pan-caspase chemical inhibitor (z-VAD-fmk) led to suppression of apoptosis with the increased levels of oxidatively modified proteins in the absence or presence of H2O2 plus ferrous iron. [8] In addition, genetic deletion of metacaspase gene Δyca1 in yeast or treatment of the Δyca1 mutant cells with H2O2 significantly increased the levels of PTMP and 20S proteasome activity, which was shown to be associated with the degradation of oxidized proteins.[9] Mukherjee and colleagues have shown that exposure of Ustilago maydis to oxidative stress causes accumulation of insoluble intracellular protein aggregates and that deletion of metacaspase alters the virulence of the fungus. [12] It has been shown that yeast metacaspase is involved in the regulation of glycosylation and the apoptosis. It was demonstrated that deletion of metacaspase gene increased O-glycosylation proteins after farnesol challenge in Candida albicans. [13] Polyunsaturated fatty acids such as linoleic acid (C18:2, n-6) and g-linolenic acid (C18:3, n-6) overexpression in Saccharomyces cerevisiae (Polyunsaturated fatty acid (PUFA strain)) have been shown to have decreased viability in response to oxidative stress. The PUFA strain exhibited greater proteasome activity with high levels of ROS, lipid peroxides, and carbonylated proteins (CPs) during chronologic aging. However, these oxidative stress-related events can be reduced with the treatment of an antioxidant vitamin C. Deletion of yca1 gene decreased caspase-like activity along with the reduced oxidative stress, whereas the lifespan of S. cerevisiae is slightly prolonged in the PUFA-producing strain.[14]

Proteomics is a powerful tool for the identification and comparison of proteins that are altered or differentially regulated during normal development or under different conditions. Several proteomic studies have previously identified H2O2-induced PTMPs as potential biomarkers for oxidative stress.[15],[16] However, the altered pattern and identity of PTMPs in the absence of yca1 gene are largely unknown. In the present study, we hypothesized that deletion of yca1 gene from S. cerevisiae enhances the levels of PTMPs (e.g., carbonylation and tyrosine phosphorylation) when analyzing the same proteins, that are usually modified by H2O2-mediated oxidative stress in wild-type (WT) yeast. Identification and comparison of PTMPs from H2O2-induced WT S. cerevisiae and its Δyca1 mutant cells by employing different proteomic approaches may provide important insights into the understanding of regulation of the H2O2-induced oxidative stress and apoptosis. Therefore, this study was aimed to investigate the oxidatively modified proteins in WT yeast and yca1 gene-deleted mutant[8] with or without exposure to H2O2 by using two-dimensional differential gel electrophoresis (2D-DIGE) followed by mass-spectral analyses to identify and delineate their functional roles.


   Materials and Methods Top


Strains and growth conditions

Saccharomyces cerevisiae WT yeast (BY 4743) and the Δyca1 mutant (homozygous diploid) cells, obtained from Open Biosystems (www.openbiosystems.com), were grown separately on yeast extract/peptone/dextrose medium containing 1% bactoyeast extract, 2% bactotryptone, and 2% glucose, whereas 200 μg/mL Geneticin antibiotic was added to the medium used for growth of Δyca1 cells. Both strains were grown to early stationary phase at 30°C. Cell suspensions with an OD600 of 2.0 were added to 100 mL fresh media containing various concentrations of H2O2, and the yeast strains were allowed to grow for additional 4 hours at 30°C[15].

Two-dimensional differential gel electrophoresis

Yeast proteins were prepared by disrupting the cells with acid-washed glass beads in a MiniBeads beater (BioSpec, Bartlesville, OK, USA). Salts and lipids were removed by chloroform/methanol precipitation. The resulting pellets were dried and resuspended in 100 μL of a lysis buffer containing 15 mM Tris-HCl, pH 8.5, 7 M urea, 2 M thiourea, and 4% (CHAPS (3-[13-Cholamidopropyl)dimethyl ammonio]-1-propanesulfonate hydrate0) CHAPS. Fifty micrograms of protein samples from each of the WT and Δyca1 mutant cells treated with and without H2O2 were labeled on the lysine residues with Cy3 and Cy5 dyes, respectively. As an internal standard, 50 μg protein mixtures consisting of 12.5 μg protein from each of the four samples (i.e., WT with and without H2O2 and Δyca1 mutant with and without H2O2) were labeled with Cy2 dye as described previously.[16] First, first dimension isoelectric focusing (1D-E) was carried out using the immobilized pH gradient (IPG) 24 cm Immobiline Dry Strip, pH 3 to 10 nonlinear (GE Healthcare, Chicago, IL, USA) for a total of 63 kV-h (Ettan, IPGphor; GE Healthcare). The strips were then placed onto an Ettan DALT-12 electrophoresis unit (GE Healthcare), and the proteins were separated in the second dimension (2D-E) on a 10% to 15% sodium dodecyl sulfate-polyacrylamide gel (Jule Inc., Milford, CT, USA) at room temperature for additional 16 hours under a constant voltage (105 V). Image analysis on the resulting 2D gels was performed using cross-stain analysis function with Progenesis Discovery software (Nonlinear Dynamics, Durham, NC, USA).

Conventional one (1D-E) − and two (2D-E)-dimensional electrophoreses

The CPs were analyzed by 1D-E and 2D-E, as described previously. [9],[18] Briefly, isoelectric focusing (IEF) for 2D-E was performed using 7 cm IPG Immobiline Dry strip (pH 3–10 nonlinear; Invitrogen, Carlsbad, CA, USA) according to the manufacturer”s protocol. Following IEF, each IPG strip was incubated in 10 mM 2,4-dinitrophenyl hydrazine (DNPH) in 2 M HCl for 15 minutes in the dark, equilibrated with 1% (dithiothreitol) DTT (w/v) for 15 minutes and finally equilibrated with 4% iodoacetamide for 15 minutes before being subjected to 2D-E. Two identical gels were run in parallel: one was used for Coomassie Brilliant Blue staining for protein identification by mass spectrometry (MS) and the other for immunoblotting with anti-DNP antibody. In-gel protein digestion with trypsin and spotting on matrix-assisted laser desorption ionization (MALDI) plates was performed as described previously.[16]

Peptides were analyzed using the Proteomics Analyzer (ABI 4700 MALDI-TOF/TOF, Applied Biosystems, Waltham, MA, USA). The first full scan MS spectra were followed by the second phase MS/MS spectra analyses. Protein identification was carried out using the search engine MASCOT (Matrix Science, Boston, MA, USA).

Immunoprecipitation of tyrosine-phosphorylated proteins

For immunoprecipitation, 1 mL aliquots of cell extracts solubilized with the immunoprecipitation buffer [20 mM Tris-base (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% NP-40, 10% glycerol, 1 mM Na3VO4, and protease inhibitor cocktail] were incubated with 15 μL of antiphosphotyrosine-specific antibody (Millipore, Billerica, MA, USA) overnight at 4°C on a constant rotator. Twenty microliters of magnetic Dyna beads M380 coated with sheep antirabbit secondary antibody (Invitrogen) were then added to the microtube containing the antigen–antibody complex, and the solution was incubated for an additional 1 hour. Immunocomplexed beads were magnetically separated with Dynal Magnetic Particle Concentrator (Invitrogen) and washed twice with the immunoprecipitation buffer. Following the removal of the supernatant from the last wash, the beads were resuspended in 30 μL of NuPage LDS buffer and heated at 100°C for 5 minutes. The supernatant of the solution containing tyrosine-phosphorylated proteins was applied to 1D-E NuPage gels (4–20%). One gel was used for Coomassie Brilliant Blue staining for protein identification and the other for immunoblotting using antiphosphotyrosine-specific antibody. For protein identification, protein spots were manually excised from the 1D-E gels. In-gel protein digestion with trypsin and peptide analysis was performed as mentioned earlier under the electrophoresis section.

Immunoblot analysis

Immunoblot analyses of protein carbonyls on 1D-E and 2D-E gels were performed as described previously.[9],[10] The DNPH-bound proteins were detected by using rabbit anti-DNPH (DAKO, Santa Clara, CA, USA) as a primary antibody (1:2000 dilutions). Phosphotyrosine-specific primary antibody (1:1000) (Millipore) was used to detect tyrosine-phosphorylated proteins using the secondary alkaline phosphatase-conjugated goat antirabbit antibody (Santa Cruz Biotech, Santa Cruz, CA, USA). The images were captured with X-ray films by enhanced chemiluminescence (Bio-Rad Laboratories, Hercules, CA, USA).


   Results Top


Differential expression and PTMPs in H2O2-exposed WT and Δyca1 mutant cells

To determine the levels of differential expression and altered PTMPs in WT and Δyca1 mutant with and without H2O2, we performed a comparative proteomic analysis using 2D-DIGE. [Figure 1] shows the overlay 2D protein profiles of WT and the Δyca1 mutant from untreated [Figure 1]a] and H2O2-treated [Figure 1]b] yeast cells. Analysis of the scans (the Cy3 scan for the WT and Cy5 scan of the Δyca1 mutant) did not reveal any significant difference in the levels of relative protein expression. However, the combined scans (overlay) did reveal significant changes with broadening of spot intensity, which we assigned as differential PTMPs [[Figure 1]a,b].
Figure 1 Comparison of two-dimensional (2D) differential gel electrophoresis patterns of differentially expressed modified proteins. (a,b) Wild-type and Δyca1 mutant cells treated without (a) or with (b) 0.5 mM H2O2. Wild-type and Δyca1 mutant cell extracts were labeled with Cy3 and Cy5 fluorescent dyes, respectively, mixed together and separated on 2D gels. Circled spots signify posttranslational modifications of proteins, which give rise to more diffuse isoelectric focusing points and molecular weights. Spots with yellow color refer to unmodified proteins. Green dots on the image are reference markers. H2O2, hydrogen peroxide; WT, wild type.

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Deletion of metacaspase enhances accumulation of carbonylated proteins

To investigate the role of metacaspase in oxidative stress, we studied whether the lack of metacaspase in cells can increase carbonylation of the same proteins due to the fluctuation/dysregulation of H2O2 levels. Therefore, we sought to identify the proteins with PTMPs and compare their levels in WT and Δyca1 mutant cells in the absence or presence of H2O2 treatment. The results obtained from 1D and 2D immunoblot analyses using the primary anti-DNPH antibody are shown in [Figure 2](a,b). Eight highly CPs in H2O2-treated and Δyca1 mutant cells were identified compared to those from nontreated WT cells. Interestingly, the protein carbonylation was much greater in Δyca1 mutant cells with and without H2O2 when compared with the corresponding H2O2-treated and/or nontreated WT cells, respectively. Although the WT cells treated with H2O2 showed the same number of CPs, their intensities were much lower than either H2O2-treated or nontreated Δyca1 mutant cells. These CPs were: heat shock protein 71 (HSP71), pyruvate decarboxylase isozyme 1 (PDC1), pyruvate kinase 1 (PK1), eukaryotic elongation factor 1A (eEF1A), d-glycerate hydrolyase 2 (ENO2), alcohol dehydrogenase 1 (ADH1), glyceraldehyde-3-phosphate dehydrogenase 3 (GAPDH3), and peroxiredoxin 1 (TSA1). These CPs are designated with numbers 1 to 8, respectively, in [Figure 2](a,b) and listed in [Table 1]. In fact, several of the eight CPs appeared only in the immunoblots of the Δyca1 mutant cells regardless of H2O2 treatment. Proteins HSP71 (spot# 1), ADH1 (spot# 6), and peroxiredoxin 1 (spot# 8) did not appear in the 2D immunoblots of WT cells without H2O2 exposure. It is of interest to note that when the immunoblot data from [Figure 2](b) was superimposed on a grid graph and aligned to the defined spots, huge upward (molecular weight) and slightly leftward (acidic pH) shifts in the CPs were observed, when compared with their respective control counterparts [[Figure 2]c].
Deletion of metacaspase enhances protein carbonylation in Saccharomyces cerevisiae. One-dimensional (a) and two-dimensional (b) immunoblot analyses of carbonylated proteins using 2,4-dinitrophenyl antibody. Proteins were identified by MALDI-TOF/TOF MS by excising protein spots manually from the second 2D gel run in parallel, stained with Coomassie Brilliant Blue-R250 as described in “Materials and Methods.” Differential migration patterns of carbonylated proteins in terms of IEF and molecular weight shift are mapped in grid graphs and compared (c). H2O2, hydrogen peroxide; WT, wild type.

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Table 1 Intensity of spots of carbonylated proteins

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Deletion of metacaspase and H2O2 stress increase tyrosine phosphorylation

To investigate the extent of phosphorylation of proteins on tyrosine residues, a pivotal posttranslational modification for signal transduction, whole cell extracts from WT and Δyca1 mutant cells in the presence or absence of H2O2 were immunoprecipitated using an antiphosphotyrosine antibody followed by immunoblot analysis with the antiphosphotyrosine antibody [[Figure 3]a]. The tyrosine-phosphorylated proteins obtained by immunoprecipitation were run on a second gel and stained with Coomassie Brilliant Blue to excise comparable spots for identification by MS and comparison with immunoblot data [[Figure 3]b]. Both immunoblot and Coomassie-stained gel patterns revealed increased protein tyrosine phosphorylation in H2O2-treated and Δyca1 mutant cells when compared with those from nontreated WT. Eight tyrosine-phosphorylated protein bands are shown in [Figure 3](a,b). The increases in tyrosine phosphorylation occurred in proteins involved in carbohydrate metabolism and protein synthesis. The data revealed that deletion of metacaspase also increases tyrosine phosphorylation in a similar manner that a high level of H2O2 does during oxidative stress.
Figure 3 Effect of metacaspase deletion and H2O2 stress on tyrosine phosphorylation in Saccharomyces cerevisiae. Coomassie staining (a) and immunoblot analysis (b). Proteins were identified by MALDI-TOF/TOF MS by excising protein spots manually from the second 1D gel run in parallel, as described in “Materials and Methods.”; H2O2, hydrogen peroxide; WT, wild type.

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   Discussion Top


Our previous reports revealed increased oxidatively modified proteins in caspase-deficient yeast, although their identities are unknown. Therefore, this study was conducted to identify the cabonylated or tyrosine-phosphorylated proteins and delineate their functional roles in WT and metacaspase-deficient yeast. The major new findings of this study show that (i) the yeast cells, which lack metacaspase, mimic H2O2-like oxidative stress and have significant shifts in IEF point and molecular weight of at least eight proteins due to PTMs, including carbonylation and tyrosine phosphorylation; (ii) the PTMPs are associated with the biologic process of protein synthesis and carbohydrate metabolism; and (iii) the proteins with increased tyrosine phosphorylation are mostly related to protein synthesis. The identification and comparison of PTMPs, elevated during H2O2-mediated oxidative stress and/or deletion of metacaspase, a critical component of apoptosis process, could help understand the causes and pathophysiologic consequences associated with cellular perturbation, loss of functional activity and signal transduction of proteins. This may provide an insight to establish a functional and regulatory relationship between H2O2- and metacaspase-related PTMs of many cellular proteins.

It is of interest to know how deletion of yca1 gene mimics H2O2-related oxidative stress and PTMPs. In several studies, it has been demonstrated that either the induction of programmed cell death or elimination of apoptotic machinery (abrogation or deletion of caspase/metacaspase) by chemical or genetics means, leads to perturbation of cell integrity, resulting in high levels of endogenous H2O2, which can subsequently cause various PTMs even in the absence of exogenous H2O2. [9, 10, 19, 20] Proteomic analyses of WT and Δyca1 mutant cells with and without H2O2 allowed us to identify and compare differential levels of PTMs of many cellular proteins. These PTMPs are identified with regards to IEF point and molecular weight by discriminating with shifts in pI and change in protein mobility, respectively, between the WT and Δyca1 mutant cells. Limited lysine labeling of proteins with Cy3 and Cy5 dyes allow us for reliable comparison of the two samples utilizing an internal standard, labeled with Cy2 dye, for normalizing the relative abundance of each protein.[16],[17] Comparison of the overlay of 2D-DIGE images of the protein profiles of the WT and Δyca1 mutant cells labeled with Cy3 and Cy5, respectively, revealed detectable alterations in IEF point and molecular weight of proteins. The shifts in the location of protein spots of HSP71, PDC1, PK1, eEF1A, ENO2, ADH1, GAPDH3, and TSA1 on 2D gels strongly suggested that these proteins are posttranslationally modified in Δyca1 mutant cells regardless of H2O2 exposure. However, our previously published data of genomic analyses showed that none of the aforementioned proteins were altered on transcriptional level in WT or Δyca1 mutant cells exposed to H2O2.

It is well-established that increased protein carbonylation is observed in the WT cells treated with H2O2 compared to untreated.[8],[9],[10],[21] Analyses of the images and identification of PTMPs in Δyca1 mutant cells with and without H2O2 suggest that HSP71, PDC1, PK1, eEF1A, ENO2, ADH1, GAPDH3, and TSA1 are highly carbonylated. Interestingly, these proteins exactly match the proteins that were also identified in cells treated with H2O2 and are found to be involved in the carbohydrate metabolism and protein synthesis.[17],[22],[23],[24],[25],[26],[27],[28],[29] To demonstrate a better analysis of the pI shifts and mobility changes of PTMPs, the CPs were mapped on grid graphs. Based on apparent molecular weight and slow protein migration pattern, these proteins are most likely posttranslationally modified as a result of reaction with reducing sugars.[30] However, the molecular weights of PTMPs in 2D-DIGE [[Figure 1]a,b] did not change much when compared with conventional 2D-E [[Figure 2]a,c]. Therefore, we postulate a potential reason for this contrasting result. Under oxidative stress proteins are not only carbonylated and phosphorylated, but they also become targets of a number of other posttranslational modifications such as oxidation, methylation, ubiquitylation, sumoylation, adduct formation, and acetylation that specifically target and compete for the lysine residues of target proteins.[9],[31][32],[33] In addition to the above-mentioned PTMs, propionylation and butyrylation, novel PTMs, of lysine residues have also recently been discovered in histones.[34] Hence, the lysine residues in PTMPs could be overloaded and may not be fully available to interact with fluorescent Cy dyes. As a result, some CPs are probably left undetected in our DIGE analyses. In addition, some of the proteins that are present in small quantities in yeast cells might not be identified by the current DIGE followed by mass-spectral analyses. Furthermore, other types of PTM such as oxidation, S-nitrosylation, adduct formation, nitration and phosphorylation on cysteine, tyrosine and other amino acids can also take place and thus affect the pI shifts in the Δyca1 mutant cells.[31] Phosphorylation has been extensively studied in the regulation of cellular processes including apoptosis and signal transduction pathways.[35] In S. cerevisiae more proteins are phosphorylated at Ser/Thr residues than Tyr (tyrosine). Modesti and colleagues have reported the existence of atyrosine-phosphorylation signaling system in S. cerevisiae. [36] They analyzed the cell lysates of S. cerevisiae using antiphosphotyrosine antibodies, which revealed >140 spots on 2D electrophoresis and indicated that tyrosine phosphorylation in budding yeast is an important process than previously suspected. Consequently, tyrosine phosphorylation in yeast may be an essential part of the cellular homeostasis process.[36],[37][38],[39][40] The tyrosine-phosphorylated proteins identified in the current study were similar to those reported in the earlier study,

[36] which suggested that they may play a major role in carbohydrate metabolism and protein synthesis.

In conclusion, the identification of posttranslational modifications of several proteins could provide significant insights into the functional and regulatory aspects of the pivotal molecules in protein synthesis and carbohydrate metabolism. Our results suggest that metacaspase is involved in the regulation of PTMPs and its presence protects S. cerevisiae from the oxidative stress through the reduced accumulation of CPs. Taken together, the findings suggest that these processes of apoptosis and oxidative stress may be interlinked and evidently integral for the maintenance of cellular integrity and homeostasis.

Acknowledgment

This research is dedicated to the memory of my mentor, the late Dr Earl R. Stadtman, in recognition of his pioneering studies that led to the contribution to the area of protein oxidation.

Financial support and sponsorship

This research was supported by the Intramural Research Program of the National Heart, Lung and Blood Institute, National Institutes of Health.

Conflicts of interest

There are no conflicts of interest.



 
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