Glutaredoxin Deletion Shortens Chronological Life span in

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Glutaredoxin Deletion Shortens Chronological Life span in Saccharomyces Cerevisiae via ROS Mediated Ras/PKA Activation Yan Liu, Fan Yang, Siying Li, Junbiao Dai, and Haiteng Deng J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00012 • Publication Date (Web): 23 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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Journal of Proteome Research

Glutaredoxin Deletion Shortens Chronological Life span in Saccharomyces Cerevisiae via ROS Mediated Ras/PKA Activation Yan Liu1, Fan Yang1, Siying Li2, Junbiao Dai3, 4*, Haiteng Deng1, 4* 1.

MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing, China, 100084

2.

Department of Biochemistry, University of California, Davis, USA, CA95616

3.

Center for Synthetic Genomics, Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China, 518055

4.

Centre for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing, China, 100084

*

Corresponding author:

Haiteng Deng, Ph.D. School of Life Sciences, Tsinghua University, Beijing, 100084 China, Tel: 8610-62790498 Fax: 8610-62797154

Junbiao Dai, Ph.D. Center for Synthetic Genomics, Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China, 518055 Tel: 86755 86585244 Email: [email protected]

1

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ABSTRACT Glutaredoxins (GRXs), small redox proteins that use reduced glutathione (GSH) as an electron donor, are key components of the cellular antioxidant system. In this study, we used Saccharomyces cerevisiae as a model system to investigate the effects of GRX deletion on yeast chronological life span (CLS). Deletion of either Grx1 or Grx2 shortened yeast CLS. Quantitative proteomics revealed that GRX deletion decreased expression of stress-response proteins, leading to increased cellular reactive oxygen species (ROS) accumulation and, subsequently, intracellular acidification. This activated the Ras/protein kinase A (PKA) signaling pathway. Genetic and biochemical analyses demonstrated that Ras/PKA activation decreased stress resistance and increased biosynthesis, requiring yeast cells to grow under unfavorable conditions and resulting in a shortened CLS. Our results provided new insights into mechanisms underlying exacerbation of the aging process by oxidative stress.

KEY WORDS: Glutaredoxin; aging; Ras/PKA; ROS; intracellular acidification; proteomics

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INTRODUCTION Since Denham Harman proposed the "free-radical theory of aging" in the mid-1950s,1 reactive oxygen species (ROS) have been closely associated with aging and aging-related diseases, such as carcinogenesis, neurodegeneration, atherosclerosis and diabetes.2-6 ROS are either radical or non-radical oxygen species, are formed by partial reduction of oxygen and include hydrogen peroxide (H2O2), superoxide anion (O2-) and hydroxyl radical (HO•).6 Intracellular ROS are mainly produced by oxidative phosphorylation in mitochondria or are generated by reactions between endogenous species and exogenous xenobiotic compounds. When cellular antioxidant defense systems are attenuated or ROS levels overwhelm the antioxidant capacity of the cell, oxidative stress occurs. This directly or indirectly causes damage to biomolecules including proteins, nucleic acids and lipids.6 As organisms grow larger and older, accumulation of ROS-induced damage leads to aging-related diseases.6-7 Glutaredoxins (GRXs) play important roles in antioxidant systems. GRXs were first described, forty years ago, as glutathione-dependent reductase responsible for disulfides formed by ribonucleotide reductase (RNR) in Escherichia coli.8-10 They are small redox proteins that use reduced glutathione (GSH) as an electron donor and they form the GRX system, together with NADPH, GSH and glutathione reductase, which transfers electrons from NADPH to GRX via GSH.11 GRXs belong to the thioredoxin (TRX) superfamily, sharing a similar active site motif (Cys–X–X–Cys).11-12 GRXs catalyze reduction of proteins and low molecular weight mixed disulfides with GSH and, under oxidative stress, catalyze formation of mixed disulfides with oxidized glutathione (GSSG). This can protect proteins from irreversible oxidation.13 In addition to their antioxidant functions, GRXs also participate in other biological processes, such as acting as the electron donor for RNR,8,

10

catalyzing protein

deglutathionylation and participating in iron metabolism.11, 14-15 Budding yeast is one of the most important model organisms used in aging research.16 Saccharomyces cerevisiae contains two classical dithiol GRXs, Grx1 and Grx2, which share 40%–52% identity with bacterial GRXs and 61%–76% similarity to mammalian GRXs.17 There are five monothiol GRXs, Grx3 to Grx7,

18-19

and a 3



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recently identified dithiol Grx8 that shares 30% and 23% sequence identity with yeast Grx1 and Grx2.20-21 Like all classical GRXs, Grx1 and Grx2 in yeast contain two highly conserved redox-active cysteine residues. Yeast Grx2 has two isoforms which localize in cytosol and mitochondria, while Grx1 is fully cytosolic.22 Yeast GRXs act as antioxidants and are responsible for protecting cells from ROS-induced damage. Both grx1 and grx2 mutants are sensitive to H2O2 while the grx1 mutant is also sensitive to O2-. Overexpression of GRX increased resistance to oxidative stress.17 Yeast GRXs differ from those in mammals, in which the mammalian GRXs catalyze RNR reduction whereas yeast TRXs perform this reaction.23 Previous studies showed that both expression level and activity of Grx1 decreased with aging; and more importantly, mitochondrial GRXs play an essential role in aging24-25 In the present study, we investigated the relationship between GRX deletion and yeast chronological life span (CLS), finding that GRX deletion shortened yeast CLS. We demonstrated that GRX deletion elevated intracellular ROS levels, which further induced cellular acidification and activated the Ras/protein kinase A (PKA) pathway. These effects led to acceleration of the aging process. Based on these results, we propose that GRXs contribute to maintaining yeast CLS and that ROS can accelerate aging in multiple ways.

EXPERIMENTAL METHODS Yeast Strains, Plasmids Construction and Media All strains used in this study were derivatives of BY4741 (a derivative of S288C, MATa, his3△1; leu2△0; met15△0; ura3△0). Knockout strains were obtained from YKO collection (MATa), with auxotrophic mark verification and PCR identification to be the correct deletion strains. Overexpression plasmids were constructed by integrating TDH3 promoter, target gene open reading frame (ORF) and ADH1 terminator together into pRS416 vector. The overexpression strains were generated by transforming relevant overexpression plasmids into the corresponding yeasts. YPD medium contained 1% bacto-yeast extract, 2% bacto-peptone,

2%

dextrose,

2%

bacto-agar (in solid media). YPGal medium contained 1% bacto-yeast extract, 2% 4


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bacto-peptone,

2% galactose, 2% bacto-agar (in solid media). Synthetic complete

(SC) medium contained 0.17% (w/v) yeast nitrogen base without amino acids and ammonium sulfate (YNB, USBio), 0.5% (w/v) ammonium sulfate, 2% (w/v) glucose or galactose, and 2% (w/v) bacto-agar (in solid media), supplemented with 2% (w/v) amino acids drop-out powder. Yeast Transformation Procedure Yeast transformation was performed using the lithium acetate (LiAc) method. Briefly, yeasts were incubated until the OD600 value was between 0.6 and 1.0, and the yeast cells were pelleted, washed with ddH2O and 0.1 M LiAc/TE buffer, and re-suspended in 0.1 M LiAc/TE buffer. 20 µl cell suspension was mixed with 2 µl plasmid or 10 µl linearized DNA fragment and 85.2 µl transformation solution containing 62.4 µl 50% PEG, 8.22 µl 1 M LiAc, 9.58 µl DMSO, 5 µl 10mg / ml ssDNA, and incubated for 30 min at 30°C. Then the mixture was placed in 42°C water bath for 15 min. The cells were pelleted, re-suspended in 5 mM CaCl2 solution, and plated into the selective plates. Yeast CLS Assay Yeasts were grown in SC medium supplemented with a 3-fold excess of histidine, leucine, methionine and uracil to avoid possible artifacts due to auxotrophic deficiencies of the strains. Yeast CLS was measured as previously described.26 Briefly, overnight culture was diluted into 20 ml fresh SC medium until the OD600 value was 0.1 (in a 100 ml conical flask). Then cultures were maintained at 30°C with shaking (200 rpm) to ensure aeration, and this day was considered as day 0. At day 3, aliquots from the cultures were properly diluted and plated onto YPD plates. The YPD plates were culture at 30 °C for 2–3 days, and then the Colony Forming Units (CFUs) were counted. The viability at day 3 was considered as 100% survival. Every 2 days, aliquots were properly diluted, plated onto YPD plates and the CFUs were countered to calculate the survival rate. The survival rate was continuingly monitored with CLS assay, and the maximum survival time was recorded when survival rate reached 1%. The maximum survival times for GRX deleted strains, wildtype strains and grx1△ with Pde1 overexpression strains were compared and analyzed. The CLS assay was 5



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carried out in biological triplicates. Serial Dilution Test Serial dilution test was conducted by diluting high concentration culture to low concentration with ddH2O in a gradual manner and drop them onto solid plates. The OD600 of the first concentration was 1, and then we diluted cultures by 10-fold sequentially. The heat resistance tests were conducted by incubating the plates in 39°C for heat shock test or 30°C as a control, with glucose as carbon source. The galactose consumption tests were conducted by incubating the plates in 30°C, with galactose as carbon source or glucose as carbon source as a control. The growth rates and colony sizes were compared among GRX deleted strains, wildtype strains and other strains. All serial dilution tests were conducted in biological duplicates. Quantitative Proteomic Analysis by 2D LC-MS/MS Yeasts grew for 64 hours from OD600=0.1 in YPD liquid media before harvest. The quantitative proteomic analysis was conducted as previously described.27 Briefly, proteins were extracted from yeasts with 8 M urea, then 200 µg proteins were reduced and alkylated. Next the proteins were digested with trypsin (Promega, Fitchburg, WI) at 37°C overnight. Tryptic peptides were desalted and labeled with the tandem mass tag (TMTsixplexTM, Thermo Waltham, MA) according to manufacturer’s protocol. For TMT labeling, peptides from three individual grx1△ strains were labeled using TMT6-129, TMT6-130 and TMT6-131 reagents while peptides from three individual wild type strains were labeled using TMT6-126, TMT6-127 and TMT6-128 reagents, and then labeled peptides were mixed in one sample. Moreover, peptides from three individual grx2△ strains were labeled using TMT6-129, TMT6-130 and TMT6-131 reagents while peptides from three individual wild type strains were labeled using TMT6-126, TMT6-127 and TMT6-128 reagents and then they were mixed in one sample. Samples were desalted and separated by two dimensional HPLC system. At the first step, reversed phase separation was performed on a Dionex UltiMate 3000 (Thermo Scientific) HPLC system at pH 10.0 by using an Xbridge Peptide BEH C18 Column (300 Å, 5 µm, 4.6 mm × 250 mm, Waters Scientific). Mobile phase A consisted of 2% acetonitrile; mobile phase B consisted of 98% acetonitrile; and both 6


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mobile phases were adjusted to pH 10.0. The flow rate was set at 1 mL / min. The gradient of mobile phase was set as follows: 5% - 8% mobile phase B for 5 min, 8% -18% mobile phase B for 35 min, 18% - 32% mobile phase B for 22 min, 32% - 95% mobile phase B for 2 min, 95% mobile phase B for 4 min, 95% - 5% mobile phase B for 4 min. 47 fractions were collected, combined into 12 fractions after rotary evaporation, and then resuspended in 0.1% formic acid for next separation. For LC-MS/MS analysis, the TMT-labeled peptides were separated by an EASY - nLC 1000 system (Thermo Scientific) using a homemade fused silica capillary column (75 µm ×150 mm, Upchurch, WA) packed with C-18 resin (300 Å, 5 µm, Varian, MA). Mobile phase A consisted of 0.1% formic acid while mobile phase B consisted of 100% acetonitrile with 0.1% formic acid. The flow rate was set at 0.25 µl / min. The gradient of mobile phase was set as follows: 3% - 8% mobile phase B for 5 min, 8% -28% mobile phase B for 102 min, 28% -80% mobile phase B for 3 min, 80% mobile phase B for 10 min. An Orbitrap FusionTM TribridTM (Thermo Scientific) mass spectrometer was operated in data-dependent acquisition mode. Precursor isolation mode was quadrupole isolation. Mass window for precursor ion selection was set at 1.6 m/z. Intensity threshold for triggering MS2 was set at 1.0×104. Mass resolution for MS1 was set at 120,000 and for tandem-MS was set at 15,000. Charge states of ions for screening were set at 2-7. Normalized collision energy was set at 38% (HCD). Dynamic exclusion time was set at 20 s. Scan strategy was data dependent mode with top speed. The MS/MS spectra from each LC-MS/MS run were searched against the Uniprot Saccharomyces cerevisiae database (release date of July, 14, 2017, 6645 sequences) using the SEQUEST searching engine of Proteome Discoverer 1.4 (PD 1.4) software. Precursor ion mass tolerance was 20 ppm and product ion mass tolerance was 0.02 Da. TMTsixplex (N-terminal and K) and carbamidomethylation (C) was set as the fixed modifications; the oxidation (M) was set as variable modification. Full tryptic specificity was required. Common contaminants were included in the database searches. The peptide false discovery rate (FDR) was estimated using target decoy 7



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function provided by PD 1.4, and the cutoff score was accepted as 1% based on the decoy database. Relative protein quantification was performed using PD 1.4 software according to manufacturer’s instructions on the intensity of six TMT reporter ions per peptide. Protein ratios were calculated as the median of all peptide hits belonging to a protein. Peptides only assigned to a given protein group were considered as unique while shared peptides were not used for quantification. Quantitation was carried out only for proteins with two or more unique peptide matches. If all six reporter ions were missing, the corresponding MS/MS spectrum was always excluded from the protein quantification. If one or more reporters were missing, with which the calculated ratios were either zero or infinity, the extremely high ratios were replaced by 100 and the extremely low ratios were replaced by 0.01. Quantification was normalized on protein median. Filter based on variability of reporter ion ratios for peptides assigned to a protein was set to be less than 50%. The mean ratios used for relative quantification were the mean of protein ratios from three biological repeats while the protein ratio of each biological repeat was the mean of protein abundance ratios of one GRX deletion strain against each of the three wildtype strains. The p-value was calculated using the protein ratios from three biological repeats with two-tailed unpaired t test with the standard of less than 0.05. Quantitative proteomic analysis compared protein abundances between GRX deleted strains and wildtype strain and was carried out in biological

triplicates.

The

proteomics

data

have

been

deposited

to

the

ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009699.28 Username: [email protected]. Password: Haj9cS4Q. Bioinformatics Analysis The differentially expressed proteins (DEPs) involved in GRX deletion were analyzed using Database for Annotation, Visualization, and Integrated Discovery (DAVID), Panther and Gene Ontology enRIchment anaLysis and visuaLizAtion tool (GORILLA).29-32

Some

of

the

pathway

information

was

obtained

from

Saccharomyces Genome Database (SGD). Intracellular ROS Measurement 8


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The intracellular ROS were measured by CellROX® Deep Red Reagents (Invitrogen, Grand Island, NY) following manufacturer’s instructions. Briefly, overnight culture was diluted into fresh medium until the OD600 value was 0.1 and continued culturing for 24 hours. Next, yeast culture was diluted to SC medium until OD600 value was 1 and added 5µM CellROX® Deep Red probe and then incubated at 30°C for 30 minutes in the dark. After that, the yeasts were analyzed on a BD FACS Aria Ⅱ Flow Cytometer (Becton Dickinson, NJ). The mean signal intensities of the fluorescence emission were compared between GRX deleted strains and wildtype strains. Intracellular ROS measurement was carried out in biological triplicates. Intracellular pH Measurement The intracellular pH was measured using ratiometric pHluorin.33-34 The ratiometric pHluorin ORF was synthesized and codon optimized (QINGLAN BIOTECH), and then integrated into pRS416 with TDH3 promoter and ADH1 terminator. The measurement was carried out as previously described.34 Briefly, overnight SC medium culture was diluted into fresh SC medium until the OD600 value was 0.1 and continued culturing for 24 hours. Then the yeasts were centrifuged and resuspended in PBS containing 100 µg/ml digitonin. After 10 min cells were washed with PBS and transferred to CELLSTAR black polystyrene clear-bottom 96-well microtitre plates (Greiner Bio-One) at an OD600 value of 0.5 in citric acid / Na2HPO4 buffers of pH values ranging from 5.0 to 8.0 to prepare for the standard curve plotting. The experimental groups were suspended in citric acid / Na2HPO4 buffer with wanted pH at the OD600 value of 0.5 and transferred to microtitre plates. Fluorescence emission was measured at 512 nm using a VARIOSKAN FLASH (Thermo Scientific) providing excitation bands of 12 nm centered around 390 and 470 nm. Background fluorescence of the corresponding strains containing pRS416 was subtracted from the measurements. The ratio of emission intensity of excitation at 390 and 470 nm was calculated (R390/470) and plotted against the corresponding buffer pH to make standard curves. Intracellular pH values were calculated according to standard curves and compared between GRX deleted strains and wildtype strains. Intracellular pH measurement was carried out in biological triplicates. 9



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Statistical Method GraphPad Prism 7.00 software was used for statistical analysis. Significant differences were determined by Student’s t test. Multiple comparisons were performed using ANOVA. P values of 1.3 or < 0.77) in proteins with 2 or more unique peptides, we identified 466 DEPs in the BY4741 and grx1△ groups, of which 234 proteins were upregulated and 232 were downregulated (Tables S1 and S2). We identified 364 DEPs in the BY4741 and grx2△ 10


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groups, with 175 proteins upregulated and 189 downregulated (Tables S3 and S4). Panther bioinformatics was employed to understand the biological relevance of the DEPs,30 with results showing that the majority of DEPs participated in metabolic processes and cellular processes (Figure 2). There were differences in DEP patterns between grx1△ and grx2△ strains. The grx2△ strain exhibited more upregulated proteins involved in cell communication as well as generation of precursor and energy, and fewer upregulated proteins associated with cell cycle and sulfur compound metabolic process as compared to grx1△ strain. The grx1△ strain exhibited downregulated proteins associated with sulfur compound metabolic process, vitamin metabolic process, reproduction and growth. We also compared the differences in DEPs between grx1△ and grx2△ strains and found that they shared a large part of DEPs. We suggested that differences in DEPs between grx1△ and grx2△ strains were due to the fact that Grx1 and Grx2 localized in different organelles (Figure S1). Using GORILLA, we found that antioxidant and stress resistance proteins Hyr1, Sod2, Trr2, Gpx1, Gtt1, Tsa2, Tps1 and Hsp104 were downregulated.31-32 This was contrary to our expectation that GRX deletion would upregulate other antioxidant and anti-stress proteins. After entry into the stationary phase, yeast must lower their metabolic rates and activate stress resistance pathways to survive under a suboptimal environment.35 The DEPs were analyzed and their associated pathways were mapped via KEGG (http://www.kegg.jp/kegg/pathway.html) and the results showed that biosynthetic processes were upregulated in GRX deleted strains (Figure S2). GRX Deletion Enhanced Ras/PKA Activity GORILLA analysis further showed that GRX deletion activated the Ras/PKA signaling pathway (Figure S3). The transcription factors Msn2 and Msn4 are downstream target genes of PKA, regulating transcription of genes controlled by stress response elements (STRE).36-38 Quantitative proteomic analysis showed that GRX1 or GRX2 deletion repressed many STRE-targeted genes (Figure S3). This suggested that enhanced Ras/PKA activity prevented Msn2 and Msn4 from entering the cell nucleus to activate transcription of STRE-controlled genes.39-41 To verify enhanced Ras/PKA activity, we examined strains with several phenotypes 11



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affected by the Ras/PKA signal pathway. As shown in Figure 3a, GRX deleted strains, compared with BY4741, were extremely sensitive to heat stress. Next we performed a serial dilution test (Figure 3b), finding that GRX deleted strains grew slower on galactose than did wildtype strains. Then we analyzed the DEPs involved in the trehalose and glycogen synthesis pathways, finding that nearly all associated proteins were downregulated in grx1△ and grx2△ strains (Figure 3c).

42-43

Moreover, our

quantitative proteomic results showed that, in the grx1△ strain, genes related to ribosome and amino acid biosynthesis were upregulated (Figure S2). The effect was similar, to a lesser extent, in the grx2△ strain. Taken together, we demonstrated that Ras/PKA activity was enhanced in GRX deleted strains. These findings were consistent with previous studies showing that decreased Ras/PKA activity enhanced stress resistance and lengthened yeast CLS, while constitutive Ras/PKA activation shortened yeast CLS.44-46 Thus, our results suggested that enhanced Ras/PKA activity in GRX deleted strains contributed to the shortening of yeast life span. Decreased Ras/PKA Activity in GRX Deleted Strains Prolonged Yeast CLS To further investigate the relationship between enhanced Ras/PKA activity and shortened CLS in GRX deleted strains, we decreased Ras/PKA activity by overexpression of Pde1, Pde2 and Bcy1. By examining heat sensitivity of these strains, we showed that overexpression of Pde1 and Bcy1 conferred heat resistance in both grx1△ and grx2△, while Pde2 overexpression conferred heat resistance in grx2△ (Figure 4a). Moreover, overexpressing Pde1, Pde2 and Bcy1 improved galactose usage in GRX deleted strains (Figure 4a). Next, we performed a CLS experiment using BY4741, grx1△ and grx1△ overexpressing Pde1 (grx1△+Pde1). We found that the decreased Ras/PKA activity in grx1△+Pde1 prolonged life span (Figure 4b). In summary, we demonstrated that decreased Ras/PKA activity contributed to the prolonged CLS in GRX deleted strains. GRX Deletion Increased Cellular ROS Levels, Leading to Intracellular Acidification that Activated the Ras/PKA Pathway Yeast GRXs act as antioxidants and protect cells against ROS.17, 47-48 GRX deletion 12


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increased intracellular ROS levels, as measured with the CellROX® Deep Red kit (Figure 5a). It is known that intracellular acidification causes an intensive and long

lasting increase in cAMP levels, thus activating the Ras/PKA pathway.49-50 To explore whether GRX deletion promoted intracellular acidification, we measured the intracellular pH of GRX deleted strains and BY4741 using pHluorin, a green fluorescent protein (GFP) variant for sensing pH in vivo.33-34 As shown in Figure 5b, intracellular pH values in GRX deleted strains were significantly lower than in BY4741, by approximately 0.4 to 0.5 units, showing that GRX deletion caused intracellular acidification. Next, we investigated whether there was a relationship between cellular ROS levels and intracellular pH. We treated BY4741 cells with 0.2 M H2O2 to increase intracellular ROS levels and simultaneously measured changes in intracellular pH. Upon H2O2 treatment, intracellular pH was continuously monitored, using citric acid/Na2HPO4 buffer, pH 4.6, as an in vivo acidification control.50 The intracellular pH was significantly decreased and the final cytoplasmic pH was below 6.0 (Figure 5c). Intracellular pH was decreased by about 0.5 unit at 10 min, and by 1.0 unit at 100 min. From these results, we demonstrated that elevated ROS levels caused intracellular acidification in a rapid and continuous manner.

DISCUSSION It has been amply documented that ROS contributes to aging and aging-related diseases,1-6 while GRXs are a vital component of the cellular antioxidant system.11 In our study, we used S. cerevisiae to investigate the effects of GRX deletion on yeast life span. We found that GRX deletion shortened yeast CLS. By analyzing DEPs in GRX deleted strains and BY4741, we found that Ras/PKA activity was enhanced in the GRX deleted strains, an effect known to be involved in regulating yeast CLS.44-46 Ras/PKA mediates stress resistance, metabolism and cell proliferation in yeast. Yeast PKA is a heterotetramer, consisting of two catalytic subunits and two regulatory subunits, and is responsible for regulating transcription of diverse downstream genes. PKA was shown to promote cell growth and cell cycle progression as well as to 13



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repress gluconeogenesis and mobilization of glycogen and trehalose.51-55 Ras/PKA activation increases cell susceptibility to stress through Msn2/Msn4-mediated STRE transcription.36-38 As shown in Figure 3a, GRX deleted strains were sensitive to heat stress. Also, enhanced Ras/PKA activity led to poor growth on non-fermentable and weakly fermentable carbon sources, for example galactose, via downregulation of Pgm2, the final enzyme of galactose metabolism.37,

50, 56-57

We found that GRX

deleted strains grew slower on galactose than wild type strain (Figure 3b). Another important consequence of high Ras/PKA activity is that it impairs storage of carbohydrates, like trehalose and glycogen, partly because PKA downregulates metabolic enzymes involved in synthesis of these carbohydrates through STRE elements.37, 56 We found that most proteins associated with trehalose and glycogen synthesis were downregulated in GRX deleted strains (Figure 3c). This is in contradictory to the previous results, in which Grx2 deletion increased the expression of genes involved in glycogen and trehalose biosynthetic pathways.58 We suggest that this discrepancy is caused by the difference of the growth phases when cells were harvested since glucose availability affects important signaling pathways.59 In McDonagh’s work, cells were harvested at exponential phase while in the present work cells were harvested at stationary phase. Moreover, constitutively-activated PKA increased transcription of genes encoded for ribosomal proteins to promote ribosome biosynthesis.42-43 As shown in Figure S2, genes related to ribosome and amino acid biosynthesis were upregulated in grx1△ strain while the effect was similar in grx2△ strain, to a lesser extent. We demonstrated that GRX deletion activated the Ras/PKA pathway. Next, we decreased Ras/PKA activity in GRX deleted strains by overexpression of Pde1, Pde2 and Bcy1. Pde1 is the low-affinity cAMP phosphodiesterase, responsible for degrading glucose or acidification-induced cAMP accumulation.60 Pde2 is the high-affinity cAMP phosphodiesterase, controlling basal cAMP levels in the cell.60 Bcy1 is the regulatory subunit of cAMP-dependent PKA, inhibiting PKA activity by forming an inactive heterotetrameric complex with its catalytic subunits.61 We found that overexpression of Pde1 and Bcy1 conferred heat resistance in both grx1△ and 14


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grx2△, while Pde2 overexpression conferred heat resistance in grx2△ (Figure 4a). We suggest this difference is caused by the fact that Grx1 is a cytosolic protein while Grx2 has two isoforms which localize in cytosol and mitochondria.22 Overexpressing Pde1, Pde2 and Bcy1 improved galactose usage in GRX deleted strains (Figure 4a). Moreover, we demonstrated that decreased Ras/PKA activity prolonged CLS in grx1△ (Figure 4b). As expected, cellular ROS levels in GRX deleted strains were significantly higher than in BY4741 (Figure 5a). PKA activation can be caused either by addition of glucose to yeast growing on a non-fermentable carbon source or by intracellular acidification.49-50 Because glucose supplementation was not used in our experiments, the enhanced Ras/PKA activity we observed was likely caused by intracellular acidification. Indeed, the intracellular pH values in grx1△ and grx2△ strains were significantly lower than in BY4741. We also verified that H2O2 treatment decreased intracellular pH (Figure 5c). Those results supported our proposal regarding the effects of GRX deletion on yeast CLS (Figure 6). GRX deletion caused increased ROS levels, which induced intracellular acidification, thereby, enhancing Ras/PKA activity. Consequently, Ras/PKA activation increased biosynthesis and decreased stress resistance through STRE elements.36-38 Under stress conditions, in which nutrition was exhausted and metabolic wastes were accumulated, yeast tends to lower their metabolic rates and activate stress resistance pathways.35 However, we found that GRX deleted strains exhibited increased biosynthesis (Figure S2). For example, Bat1, which involved in branched-chain amino acid biosynthesis and was highly expressed during exponential phase and repressed during stationary phase.62 Furthermore, GRX deleted strains exhibited decreased stress resistance, in which a series of stress-resistance proteins, like Sod2, Trr2, Gpx1 and Hsp104 were down-regulated, leading to shortening of yeast CLS. The increased intracellular ROS level and acidification will also contribute to the shortening of CLS in GRX deleted yeasts. Specifically, we found that GRX deletion downregulated the stress resistance proteins Hyr1, Sod2, Trr2, Gpx1, Gtt1, Tsa2, Tps1 and Hsp104. Of these, Hyr1, Sod2 15



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and Gpx1 are known antioxidant proteins.63-65 These findings were contrary to our assumption, based on common knowledge, that GRX deletion would upregulate other antioxidant and anti-stress proteins. We hypothesize that attenuated stress response, resulting from GRX deletion, would exacerbate oxidative stress. Ras/PKA activation may downregulate stress resistance genes through STRE elements and, subsequently, lead to decreased heat resistance of GRX deleted strains (Figure 3a). In this system, heat resistance was restored by decreasing Ras/PKA activity (Figure 4a). Previous studies showed that GRX1 activity decreased with aging, whereas Grx2 was increased in the matrix of mitochondria with the major portion in an inactive state as a dimeric iron–sulfur cluster complex in mammalian cells.25 Mammalian Grx1 is mainly cytosolic and also exists in the mitochondrial intermembrane space, while mammalian Grx2 localizes in the mitochondrial matrix.66 In mammalian cells, mitochondria are an essential organelle for initiation and activation of apoptosis,67 and the age-associated diminution of Grx1 may contribute to the increased susceptibility to apoptosis.24 In yeast, however, Grx1 is fully cytosolic while Grx2 localizes in both cytosol and mitochondria.22 We suggest that ROS induced oxidative stress is a key factor responsible for shortening CLS in GRX deletion strains. There are some other interesting findings in our quantitative proteomic results. Among the stress resistance proteins, Sod2, Trr2, Gpx1 and Gtt1 are mitochondrial proteins, which were downregulated in both grx1△ and grx2△.63, 68-70 The degree of downregulation for Sod2, Gpx2 and Gtt1 was similar in grx1△ and grx2△, whereas Trr2 downregulation was larger in grx1△ compared to grx2△. The mitochondrial peroxiredoxin counterpart, Prx1, was downregulated in grx1△ but not in grx2△.71 Given the fact that Grx1 is cytosolic while Grx2 localizes in both cytosol and mitochondria,22 we suggest that the differences in DEPs between grx1△ and grx2△ are relevant to their localization. Mitochondrial translocator Atm1 was upregulated in both grx1△ and grx2△, and the degree of upregulation was larger in grx1△ compared to grx2△.72 Tsa2 is an antioxidant protein, but it also plays an important role in maintaining genome stability while Hyr1 can sense cellular hydroperoxide to activate transcription factor Yap1.73-74 Both proteins were downregulated in both grx1△ and 16


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grx2△ strains. The degree of downregulation for Tsa2 was larger in grx1△ than that in grx2△ while the degree of downregulation for Hyr1 was similar in grx1△ and grx2△ strains. Grx5, a mitochondrial protein involved in iron metabolism and iron-sulfur cluster assembly, was upregulated in both grx1△ and grx2△ strains, which suggests GRX deletion may affect sulfur metabolism.75

CONCLUSIONS Taken together, our results showed that GRX deletion shortened yeast CLS. GRX deletion increased ROS levels, which promoted intracellular acidification, leading to Ras/PKA activation, decreased stress resistance and increased biosynthesis. These results demonstrated that the interplay between GRXs and ROS can accelerate the aging process.

SUPPORTING INFORMATION Supplemental Figures Figure S1. Venn diagrams showing differential behaviors of DEPs between grx1△ and grx2△. Figure S2. Biosynthesis related proteins were up-regulated in grx1△, analyzed and mapped by KEGG (http://www.kegg.jp/kegg/pathway.html). Figure S3. DEPs in grx1△ involved in Ras/PKA signal pathway. Supplemental Tables Table S1. The list of upregulated proteins in grx1△. Table S2. The list of downregulated proteins in grx1△. Table S3. The list of upregulated proteins in grx2△. Table S4. The list of downregulated proteins in grx2△.

AUTHOR INFORMATION Corresponding Author *Haiteng Deng, School of Life Sciences, Tsinghua University, Beijing, 100084 China, Tel: 8610-62790498; Fax: 8610-62797154; E-mail: [email protected] 17



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*Junbiao Dai, Center for Synthetic Genomics, Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China, 518055, Tel: 86755 86585244, Email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Conflicts of Interest The authors declare no conflict of interest. Grant Support This work was supported in part by the National Key Research and Development Program of China (Grant 2017YFA0505103), the Chinese Ministry of Science and Technology 2014CBA02005 (H.T.D) and the Science and Technology Pillar Program of Sichuan (2016JZ0015).

ACKNOWLEDGEMENT We thank the Protein Chemistry Facility at the Center for Biomedical Analysis of Tsinghua University for sample analysis. We thank the Cell Biological Facility at the Center for Biomedical Analysis of Tsinghua University for FACS analysis. We thank the Shared Instrument Facility at the Center for Biomedical Analysis of Tsinghua University for instrumentation. We thank Jeremy Allen, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

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FIGURE LEGENDS

Figure 1. Measurement of CLS of BY4741, grx1△ and grx2△. GRX deletion led to shortened CLS. Error bars represent ±SD.

Figure 2. Functional classification of DEPs involved in biological processes between BY4741 and GRX deletion strains with PANTHER. (a) Functional classification of up-regulated DEPs involved in biological processes between BY4741 and grx1△ as well as between BY4741 and grx2△. The analysis was conducted by PANTHER (http://www.pantherdb.org) for functional classification. (b) Functional classification of down-regulated DEPs involved in biological processes between BY4741 and grx1△ as well as between BY4741 and grx2△. The analysis was conducted by PANTHER for functional classification.

Figure 3. Phenotypic tests of enhanced Ras/PKA activity. (a) Serial dilution test for heat sensitivity. The GRX deletion strains were very sensitive to heat stress. (b) Serial dilution test for galactose usage. The GRX deletion strains showed a decreased galactose usage ability. (c) DEPs in grx1△ involved in trehalose and glycogen metabolism, showed that trehalose and glycogen synthesis processes were down-regulated. Blue color represented down-regulation, black color represented constant, yellow color represented not being found.

Figure 4. Phenotypic rescues after Ras/PKA down-regulation. (a) Serial dilution test for heat sensitivity and galactose usage after Ras/PKA down-regulation. Results showed that GRX deletion strains increased their heat resistance and galactose usage ability after Ras/PKA being down-regulated. pRS416, empty vector as a control. Pde1, low-affinity cAMP phosphodiesterase; Pde2, high-affinity cAMP phosphodiesterase; Bcy1, regulatory subunit of PKA; Pgm2, Phosphoglucomutase. (b) CLS of BY4741+pRS416 (BY4741 with control plasmid 26


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pRS416), grx1△+pRS416 (grx1△ with control plasmid pRS416) and grx1△+Pde1 (grx1△ with overexpressed Pde1), showed that grx1△ had a much longer CLS with down-regulated Ras/PKA activity. Error bars represent ±SD.

Figure 5. GRX deletion led to increased intracellular ROS level and intracellular acidification. (a) Intracellular ROS levels of BY4741, grx1△, and grx2△. (b) Intracellular pH values of BY4741, grx1△, and grx2△. (c) Intracellular pH value changes of BY4741 treated by 0.2 M H2O2 or pH4.6 citric acid /Na2HPO4 buffer and the untreated control plotted against treatment time. *p

Glutaredoxin Deletion Shortens Chronological Life span in

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