Frederico Marianetti Soriani, Marcia Regina Kress ... - DAGZ - Boku

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Functional characterization of the Aspergillus nidulans methionine sulfoxide 

reductases (msrA and msrB) 

Frederico Marianetti Soriani a , Marcia Regina Kress a , Paula Fagundes de Gouvêa a , Iran Malavazi d , 

Marcela Savoldi a , Andreas Gallmetzer c , Joseph Strauss c , Maria Helena S. Goldman b , 

Gustavo Henrique Goldman a, * 

a 

Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café S/N, CEP 14040-903, 

Ribeirão Preto, São Paulo, Brazil 

b 

Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil 

c 

Fungal Genomics Unit, Austrian Research Centers and BOKU University Vienna, Austria 

d 

Departamento de Genética e Evolução, Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos, Brazil 

article info 

Article history: 

Received 27 November 2008 

Accepted 27 January 2009 

Available online 5 February 2009 

Keywords: 

Aspergillus nidulans 

Oxidative stress 

Methionine sulfoxide reductases 

1. Introduction 

abstract 

Reactive oxygen species (ROS), such as oxygen ions, free radicals 

and peroxides are highly reactive due to the presence of unpaired 

valence shell electrons. ROS are produced by oxygen 

metabolism and have important roles in cell signaling. However, 

if the cells are subjected to environmental stress the ROS levels 

can increase dramatically, which can result in significant damage 

to cell structures. This phenomenon is described as oxidative 

stress. Oxidative damage to proteins and other biomolecules by 

ROS has been implicated in a variety of diseases and the aging process 

(Davies, 2005; Petropoulos and Friguet, 2005; Keating, 2008; 

Muller et al., 2007). Side chains of amino acids and the peptide 

backbone of proteins can be targeted and reversibly or irreversibly 

modified by oxidation (Kim and Gladyshev, 2007; Oien and Moskovitz, 

2008). Protein-bound methionine is the most vulnerable target 

to oxidation by ROS, resulting in the formation of methionine 

sulfoxide [Met(O)] residues. This modification can be repaired by 

methionine sulfoxide reductase (Msr), which catalyzes the thiore- 

* Corresponding author. Fax: +55 16 36024280. 

E-mail address: ggoldman@usp.br (G.H. Goldman). 

1087-1845/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. 

doi:10.1016/j.fgb.2009.01.004 

Fungal Genetics and Biology 46 (2009) 410–417 

Contents lists available at ScienceDirect 

Fungal Genetics and Biology 

journal homepage: www.elsevier.com/locate/yfgbi 

Proteins are subject to modification by reactive oxygen species (ROS), and oxidation of specific amino 

acid residues can impair their biological function, leading to an alteration in cellular homeostasis. Sulfur-containing 

amino acids as methionine are the most vulnerable to oxidation by ROS, resulting in 

the formation of methionine sulfoxide [Met(O)] residues. This modification can be repaired by methionine 

sulfoxide reductases (Msr). Two distinct classes of these enzymes, MsrA and MsrB, which selectively 

reduce the two methionine sulfoxide epimers, methionine-S-sulfoxide and methionine-R-sulfoxide, 

respectively, are found in virtually all organisms. Here, we describe the homologs of methionine sulfoxide 

reductases, msrA and msrB, in the filamentous fungus Aspergillus nidulans. Both single and double inactivation 

mutants were viable, but more sensitive to oxidative stress agents as hydrogen peroxide, paraquat, 

and ultraviolet light. These strains also accumulated more carbonylated proteins when exposed to hydrogen 

peroxide indicating that MsrA and MsrB are active players in the protection of the cellular proteins 

from oxidative stress damage. 

Ó 2009 Elsevier Inc. All rights reserved. 

doxin-dependent reduction of free and protein-bound Met(O) to 

methionine (Moskovitz et al., 1996, 1997, 1998). Msr genes are 

found in most organisms and are divided into two distinct enzyme 

families: (i) MsrA is specific for the S-form of [Met(O)] and (ii) MsrB 

can only reduce the R-form (Kim and Gladyshev, 2007; Weissbach 

et al., 2005). Msr genes are found in most organisms from bacteria 

to humans, even in the species that live under anaerobic conditions, 

but are absent in many hyperthermophiles and intracellular 

parasites (Delaye et al., 2007). 

The filamentous fungus Aspergillus nidulans has been used as a 

model system to understand metabolic regulation, cell cycle, 

development, and DNA damage response (reviewed by Goldman 

et al., 2002; Harris and Momany, 2004; Osmani and Mirabito, 

2004; and Goldman and Kafer, 2004). Here, we describe the methionine 

sulfoxide reductase (msrA and msrB) homologs in A. nidulans. 

These genes are the first Msr homologs described in filamentous 

fungi. Both single and double msr inactivation mutants were viable, 

but more sensitive to hydrogen peroxide, paraquat, and ultraviolet 

light. These strains also accumulated more carbonylated proteins 

when exposed to hydrogen peroxide indicating that MsrA and 

MsrB are important in the protection of the cellular proteins from 

oxidative damage.

2. Materials and methods 

2.1. Strains, media, and culture methods 

A. nidulans strains used are GR5 (pyroA4 wA1 pyrG89), UI224 

(pyrG89; argB2; yA1), TNO2a3 (pyroA4 pyrG89 DnkuA::argB + ), 

PFG8 (DmsrA::pyrG + pyrG89; argB2, pyroA4), FMS3 (pyrG89; 

argB2, pyroA4, DmsrB::pyroA + ), and FMS4 (pyrG89; argB2, pyroA4, 

DmsrA::pyrG + , DmsrB::pyroA + ). The media used were of two basic 

types, i.e., complete and minimal. The complete media comprised 

the following three variants: YAG (2% w/v glucose, 0.5% w/v yeast 

extract, 2% w/v agar, trace elements), YUU (YAG supplemented 

with 1.2 g/l [each] of uracil and uridine), and liquid YG or YG + UU 

medium with the same composition (but without agar). The minimal 

media were a modified minimal medium (MM; 1% w/v glu- 


F.M. Soriani et al. / Fungal Genetics and Biology 46 (2009) 410–417 411 

cose, original high-nitrate salts, trace elements, 2% w/v agar, pH 

6.5). Chlorate toxicity tests were performed as described earlier 

on 1% glucose minimal medium supplemented with 10 mM of 

ammonium tartrate, arginine, hypoxanthine, proline, urea, nitrate 

or nitrite as sole nitrogen source in the presence or absence of 

100 mM sodium chlorate. 

For the oxidative stress viability assay (Noventa-Jordão et al., 

1999), 1 10 6 conidia/ml were incubated in YG + UU for 5 h at 

30 °C in a reciprocal shaker (250 rpm). After this period, either 

50 mM of hydrogen peroxide or 10 mM of paraquat were added 

to the germlings which were then further incubated for 120 min 

at the same conditions. Conidia were conveniently diluted and plated 

in YAG + UU plates. Plates were incubated at 37 °C for 48 h. Viability 

was determined as the percentage of colonies on treated 

plates compared to untreated controls. Statistical differences were 

Fig. 1. Schematic illustration of the mrsA and mrsB deletion strategy: (A) genomic DNA from both wild type and DmrsA strains was isolated and cleaved with the enzyme 

EcoRV; a 2.0-kb DNA fragment 5 0 -flanking the mrsA ORF was used as a hybridization probe. This fragment recognizes two DNA bands (about 2.2- and 2.1-kb) in the wild type 

strain and also two DNA bands in the DmrsA mutant strain (about 1.1- and 2.1-kb) as shown in the Southern blot analysis and (B) genomic DNA from both wild type and 

DmrsB strains was isolated and cleaved with the enzyme EcoRV; a 2.0-kb DNA fragment 5 0 -flanking the mrsB ORF was used as a hybridization probe. This fragment recognizes 

a single DNA band (about 2.3-kb) in the wild type strain and also a single DNA band (about 3.8-kb) in the DmrsB mutant as shown in the Southern blot analysis.

determined by One-Way analysis of variance (ANOVA) followed by 

Newman-Keuls Multiple Comparison Test, using GraphPad Prism 

statistical software (GraphPad Software, Inc., version 5, 2003). 

2.2. Molecular techniques 

Standard genetic techniques for A. nidulans were used for all 

strain constructions (Kafer, 1977). DNA and RNA analyzes were 

performed as described by Sambrook and Russell (2001). For PCR 

experiments standard protocols were applied using a PTC100 96well 

thermal cycler (MJ Research, Watertown, MA) for reaction 

cycles. 

To characterize the function of A. nidulans MsrA and MsrB, ORF 

deletions were generated using a fusion PCR-based approach 

(Kuwayama et al., 2002). Supplementary Table S1 shows the oligonucleotide 

primers used in this work as well as the strategies for 

gene deletions. For all constructions, about 2 kb regions on either 

side of the ORFs were selected for primer design. Either Aspergillus 

fumigatus pyrG or pyroA genes were used as selection markers for 

auxotrophy. All genomic fragments were PCR amplified from genomic 

DNA from A. nidulans GR5 strain while the markers were 

amplified from either pCDA21 plasmid (Chaveroche et al., 2000; 

for pyrG) orA. fumigatus CEA17 (for pyroA). The cassettes were 

PCR amplified from these plasmids using high-fidelity Taq-polymerase 

(Invitrogen) and used for A. nidulans transformation 

(Osmani et al., 1987). 

2.3. RNA isolation 

For the real-time RT-PCR, 1.0 10 9 conidia/ml of A. nidulans 

wild type (GR5) strain were used to inoculate 50 ml of pre-warmed 

liquid cultures (YG) in 250-ml Erlenmeyer flasks that were incu- 


412 F.M. Soriani et al. / Fungal Genetics and Biology 46 (2009) 410–417 

Fig. 2. The DmrsA and DmrsB mutant strains have decreased survival in the 

presence of oxidative stressing agents. Viability was determined as the percentage 

of colonies on treated plates compared to untreated controls. The results were 

expressed by the average of four independent experiments and means ± standard 

deviation are shown. The DmrsA, DmrsB, and DmrsA DmrsB strains were significantly 

different from the wild type (p < 0.01). 

bated in a reciprocal shaker (250 rpm) at 37 °C for 12 h. After this 

period, the germlings were exposed or not to the corresponding 

stressing agent for different periods of time at 37 °C, and were harvested 

by centrifugation, washed with distilled water and frozen in 

liquid nitrogen. For total RNA isolation, the germlings were disrupted 

by grinding in liquid nitrogen and total RNA was extracted 

with Trizol reagent (Invitrogen, USA). Ten micrograms of RNA from 

each treatment were then fractionated in 2.2 M formaldehyde, 

1.2% w/v agarose gel, stained with ethidium bromide, and then 

visualized with UV light. The presence of intact 25S and 17S ribosomal 

RNA bands was used as a criterion to assess the integrity of 

the RNA. RNAse-free DNAse treatment was done as previously described 

(Semighini et al., 2002). 

2.4. Real-time PCR reactions 

All the PCR and RT-PCR reactions were performed using an ABI 

7500 Fast Real-Time PCR System (Applied Biosystems, USA). Taq- 

Man TM Universal PCR Master Mix Kit (Applied Biosystems, USA) 

was used for PCR reactions. The reactions and calculations were 

performed according to Semighini et al. (2002). The primers and 

Lux TM fluorescent probes (Invitrogen) used in this work are: MSRA 

(5 0 -CGATAAACAGCGTCGCCGAATTAT[FAM]G-3 0 ), MSRA1 (5 0 -CTC 

ACCAACGACGGGTCAAAG-3 0 ), MSRB 5 0 - CGGACCGAGTCACAAGTT- 

CA AGTC[FAM]G-3 0 ), MSRB1 (5 0 -GCTCCGGGAATTGAATCAAAGTA- 

3 0 ), TUBC (5 0 -CACTTTATGCCGTCGCCGAAAG[FAM]G-3 0 ), and TUB1 

(5 0 -GCAGAATGTCTCGTCCGAATG-3 0 ). FAM: 6-carboxyfluorescein. 

2.5. Detection of protein oxidation 

Oxidative modification of proteins by oxygen free radicals was 

monitored by Western blot analysis of carbonyl groups using the 

OxyBlot TM Protein Oxidation Detection Kit (Chemicon Ò International, 

Inc.). Briefly, the cultures of mutant and wild type strains 

were grown for 12 h at 37 °C in an orbital shaker (150 rpm). After 

that, the cultures were exposed to 10 mM H2O2 for 20 min. Both 

treated and control cultures (no H 2O 2 added) had the total proteins 

extracted by homogenizing the mycelia using liquid nitrogen in a 

buffer containing 50 mM Tris–HCl, pH 7.4, 1 mM EGTA, 0.2% Triton 

X-100, 1 mM benzamidine and 10 mg ml-1 each of leupeptin, pepstatin 

and aprotinine. The homogenates were clarified by centrifugation 

at 20,800g for 60 min at 4 °C. Total protein concentrations 

were determined by the Bradford method (Bradford, 1976) and 

20 lg were submitted to derivatization with dinitrophenylhydrazine 

(DNPH). About 10 lg of proteins were loaded into a 12% (w/ 

v) SDS-PAGE gel and electroblotted to a nitrocellulose membrane. 

The membrane was incubated with the antibody anti-DNP moiety 

of the proteins and the immunoblot was detected by chemiluminescent 

reaction. 

3. Results 

3.1. Identification of A. nidulans methionine sulfoxide reductases 

A BLASTp search of the A. nidulans genome database (http:// 

www.broad.mit.edu/annotation/fungi/aspergillus/) using Homo 

sapiens MRSA and MRSB as queries revealed two single open 

reading frames with significant similarity. The potential homologs, 

AN10562.3 and AN1932.3, are predicted 153- and 213amino 

acid proteins that possess identity to the H. sapiens 

MSRA and MSRB (e-values 2e 31 and 8e 16; 60% and 54% 

similarity and 42% and 39% identity, respectively). A phylogenetic 

analysis was performed to determine the relationship of 

A. nidulans MsrA and MsrB to MRSA and MRSB homologs in several 

different organisms (Supplementary Figure 1). Interestingly,

the fungal MsrAs and MsrBs form a distinct clade. To characterize 

the function of A. nidulans MsrA and MsrB, ORF deletions 

were generated using a fusion PCR-based approach (Supplementary 

Table 1). Protoplasts of A. nidulans strain TNO2a3 were 

transformed using the fusion PCR product, and several transformants 

were obtained by their ability to grow in selective medium. 

The candidates for msrA deletion were able to grow in 

YAG culture medium, in the absence of uridine and uracil, 

and for the msrB deletion candidates in MM + UU without pyridoxine. 

Allelic replacement of msrA and msrB was verified in 

several transformants by Southern blot analysis (data not 

shown). One transformant for each gene was crossed with 

UI224 strain in order to eliminate the DnkuA mutation; accordingly, 

both deletion strains are wild type for this locus. The segregation 

ratios for either DmsrA or DmsrB mutations were 

approximately 50% suggesting that there are no additional 

mutations in the segregants (data not shown). The allelic 

replacement of msrA and msrB was confirmed by Southern blot 

analysis in several segregants from each cross (one representative 

of each deletion is shown in Fig. 1A and B), thereby generating 

the DmsrA and DmsrB strains. A double deletion mutant 

was generated by genetic cross of DmsrA and DmsrB strains. Finally, 

both single and double mutants were viable, indicating 

that msrA and msrB are not essential genes in A. nidulans. 



3.2. Phenotypic characterization of the DmsrA, DmsrB, and DmsrA 

DmsrB mutant strains 

Two approaches were used as initial tests to investigate the 

MsrA and MsrB function: (i) we examined the survival of the mutant 

strains to some oxidative stressing agents, and (ii) we tested 

how the mRNA accumulation of the msrA and msrB genes is affected 

by these agents by using real-time RT-PCR. In the first test, 

the survival was evaluated in the presence of H2O2 and paraquat. 

The wild type survival was not affected by these oxidative stressing 

agents under the tested conditions (Fig. 2A and B). However, the 

DmrsA, DmrsB, and double DmrsA DmrsB mutant strains had about 

30%, 3% and 3% survival after exposure to H2O2, respectively 

(Fig. 2A and B). Furthermore, the DmrsA, DmrsB, and double DmrsA 

DmrsB mutant strains had about 40–45% survival after exposure to 

paraquat (Fig. 2A and B). These results suggest that msrA and msrB 

are epistatic during exposure to paraquat. In contrast, msrB has a 

more important role repairing the damage caused by H2O2. The 

strains were also subjected to oxidative stress imposed by 

100 mM chlorate, which is known to act as strong oxidizing agent 

and is toxic to cells due to its conversion to chlorite (Cove, 1976). 

Interestingly, the stress imposed by chlorate treatment did not result 

in significantly different growth phenotypes between wild 

type and msr single or double mutants and the induction of the 

Fig. 3. Transcript accumulation in mrsA and mrsB mRNA levels in response to oxidative stressing agents. Mycelia were grown either in the absence or in the presence of any 

oxidative stressing agent. Real-time RT-PCR was used to quantitate the mRNA. The measured quantity of the mrsA and mrsB mRNA in each of the treated samples was 

normalized using the CT-values obtained for the tubC RNA amplifications run in the same plate. The relative quantitation of msrA, msrB and tubulin gene expression was 

determined by a standard curve (i.e., C T-values plotted against logarithm of the DNA copy number). Results of four sets of experiments were combined for each 

determination; means ± standard deviation are shown.

nitrate reductase encoding niaD gene was not altered in the msr 

mutant strains (data not shown).To accomplish the second test, 

A. nidulans wild type was grown in the absence of any drug, transferred 

to a specific concentration of H 2O 2 or paraquat for 20 or 

60 min, respectively, and analyzed for the msrA and msrB mRNA 

accumulation (Fig. 3A and B). The msrA mRNA accumulation was 

increased after 20 min growth in the presence of 5, 25, and 

100 mM H 2O 2 about 50, 150, and 80 times, respectively, while in 

the presence of 5 and 10 mM paraquat for 60 min about 200 and 

70 times, respectively (Fig. 3A). In contrast, the msrB mRNA accumulation 

displayed much lower levels of mRNA accumulation; in 

the presence of 5, 25, and 100 mM H 2O 2 for 20 min about 1.25, 

3.25, and 2 times, respectively, while in the presence of 5 and 

10 mM paraquat for 60 min about 7 and 2.5 times, respectively 

(Fig. 3B). We cannot provide a reasonable explanation why higher 

ROS concentrations showed decreased msrA and msrB mRNA accumulation 

(Fig. 3). 



The msrB gene has a higher mRNA accumulation in both control 

and oxidative stressing treatments than the msrB gene (Fig. 3). 

However the cell survival during exposure to hydrogen peroxide 

is lower in DmsrB than in the DmsrA mutant and comparable during 

exposure to paraquat (see Fig. 2). Taken together, these results 

strongly indicate that the A. nidulans Msr genes are involved in the 

repair of cell damage caused by oxidative stress. Furthermore, it 

does not seem to occur a direct correlation between msrA and msrB 

mRNA accumulation and cell survival in the presence of oxidative 

stressing agents. 

3.3. Msr inactivation mutant strains accumulate more oxidized 

proteins 

Since the A. nidulans Msr null mutants were more sensitive to 

oxidative stressing agents, we investigated the carbonylated proteins 

within A. nidulans wild type and mutant strains exposed to 

Fig. 4. Pattern of oxidatively damaged proteins in the wild type, DmrsA, DmrsB, DmrsA DmrsB mutant strains exposed to H 2O 2. In each strain, the left and right panels show 

the Coomassie staining of a polyacrylamide gel and the same proteins blotted onto a nitrocellulose membrane and probed by the antibody anti-DNP moiety of the proteins 

(the immunoblot was detected by chemiluminescent reaction), respectively. Seven different protein bands were selected for densitometric analysis using the Image J program 

(available at http://www.rsbweb.nih.gov/ij/download.html). The table shows the absolute values of the area for each protein band analyzed. C = control (without H 2O 2) and 

HP = 10 mM H 2O 2 for 20 min.

H2O2 and monitored them by Western blot analysis of carbonyl 

groups using the OxyBlot TM Protein Oxidation Detection Kit. The increased 

concentration of carbonyl groups introduced into proteins 

by oxidative reactions with H 2O 2 reflects the inability to efficiently 

repair proteins damaged by oxidative stress. The membrane was 

incubated with the antibody to the DNP moiety of the proteins 

and the immunoblot was detected by chemiluminescent reaction. 

The assay of protein carbonyl content is particularly useful since 

this modification reports relatively accurately on the fraction of 

oxidatively damaged protein with impaired function in total protein 

samples (Requena et al., 2001). Note that oxidized methionine 

does not contribute to the protein carbonyl signal, so determination 

of protein carbonyls provided independent corroboration of 

protein oxidation. The intensity of the bands in the wild type exposed 

or not to H2O2 was comparable while an increase in protein 

carbonylation was evident in the msrA, msrB, and msrA msrB mutants 

(arrows indicate the increase in the concentration of some 

protein bands in the H2O2 treatment compared to the non-treated 

sample; Fig. 4). These bands were quantified by densitometry and 

they were increased in the mutant strains about two to three-times 

(see Table in Fig. 4). These results suggest that methionine sulfoxide 

reductases in A. nidulans are important for repairing [Met(O)] 

residues. 

3.4. Msr inactivation mutant strains are more sensitive to UV light 

Recently, it was shown that the peptide methionine-S-sulfoxide 

reductase is expressed in human epidermis and upregulated by 

Fig. 5. The mrsA–B deletion strains are more sensitive to UV light. Quiescent (A) and 

germinating (B) conidiospores from wild type, DmrsA, DmrsB, DmrsA DmrsB mutant 

strains were exposed to UV light, and viability was scored after exposure to this 

DNA damaging agent. Viability was determined as the percentage of colonies on 

treated plates compared to untreated controls. The results were expressed by the 

average of four independent experiments and means ± standard deviation are 

shown. Statistical differences were determined by One-Way analysis of variance 

(ANOVA) followed using GraphPad Prism statistical software (GraphPad Software, 

Inc., version 3, 2003). 



UVA (ultraviolet A) radiation (Ogawa et al., 2006; Schallreuter 

et al., 2006; Picot et al., 2007). To investigate a possible role of A. 

nidulans in UV-sensitivity, quiescent and germinating conidia from 

wild type and mutant strains were exposed to different UV light 

dosages. Quiescent conidia from wild type, DmsrA, and DmsrA 

DmsrB conidiospores showed the same UV light-sensitivity; however, 

DmsrB conidiospores are more sensitive to UV light 

(Fig. 5A). In contrast, DmsrA and DmsrB mutations are epistatic 

since DmsrA, DmsrB, and DmsrA DmsrB germinating conidiospores 

(i.e., mitotically active cells) exhibited increased and similar sensitivity 

to UV irradiation (Fig. 5B). Interestingly, we also observed increased 

msrA and msrB mRNA accumulation in the presence of 

some DNA damaging agents, such as methyl methane sulfonate, 

and bleomycin (Fig. 6). However, the growth of the deletion mutant 

strains was not affected by DNA damaging agents (data not 

shown). These results indicate that msrA and msrB genes are 

important for the response to UV light, and are possibly involved 

in the DNA damage response in A. nidulans. 

4. Discussion 

During aerobic metabolism ROS generated by cells can attack 

and modify reversibly or irreversibly side chains of amino acid residues 

and the peptidic backbone of proteins (for reviews, see Stadtman 

and Levine, 2003 and Stadtman, 2006). Sulfur-containing 

amino acids, cysteine and methionine, are the most susceptible 

to oxidation. Free and protein-bound methionines can be oxidized 

by ROS, generating a diastereomeric mixture of S- and R-forms of 

methionine sulfoxide due to the chiral nature of sulfur in methionine 

sulfoxide (Jacob et al., 2003; Kim and Gladyshev, 2007). Formation 

of methionine sulfoxide may lead to a significant change 

in protein structure and function (Tsvetkov et al., 2005). Additionally, 

formation of methionine sulfoxide might be again targeted by 

ROS to produce methionine sulfone or radicals and to propagate 

oxidative damage (Nakao et al., 2003). Cells evolved a mechanism 

to reverse methionine oxidation with a repair system that supports 

methionine sulfoxide reduction via the enzymes methionine sulfoxide 

reductases (Weissbach et al., 2002, 2005; Moskovitz, 

2005a; Cabreiro et al., 2006; Kim and Gladyshev, 2007). We present 

the characterization of an A. nidulans methionine sulfoxide 

reductase encoding-genes msrA and msrB. Several lines of evidence 

support our assumption that MsrA–B are homologs of the methionine 

sulfoxide reductases: (i) reciprocal Blast analysis with MsrA– 

B, or homologous proteins from H. sapiens or other fungi, such as S. 

cerevisiae or S. pombe, consistently identify A. nidulans proteins encoded 

by the ORFs AN10562.3 and AN1932.3; (ii) phenotypic analysis 

identifies that strains with these genes inactivated are more 

sensitive upon exposure to oxidative stressing conditions; and 

(iii) there is an increase in the concentration of oxidized proteins 

in the msr null mutant strains when compared with the wild type 

strain. 

Several biological processes are related to reversible oxidation 

of methionine residues, such as oxidative stress, aging and neurodegenerative 

diseases (Hoshi and Heinemann, 2001; Hou et al., 

2002; Cabreiro et al., 2006; Moskovitz, 2005b; Friguet, 2006; 

Petropoulos and Friguet, 2005). We have observed that A. nidulans 

Msr null mutants are more sensitive to oxidative stressing agents, 

suggesting they could be involved in the repair of cell damage 

caused by oxidative stress. This sensitivity was reflected by the 

accumulation of more oxidized proteins, as observed by an increase 

of carbonylated proteins. MrsA protects cells against oxidative 

stress in several microorganisms, including S. cerevisiae, 

Streptomyces aureus, Neisseria gonorrhoeae and Helicobacter pylori 

(Moskovitz et al., 1998; Skaar et al., 2002; Singh and Moskovitz, 

2003; Alamuri and Maier, 2004). MsrA was also found to play an 

important role in oxidative stress in the viability of lens cells

(Kantorow et al., 2004; Marchetti et al., 2006), retinal pigmented 

cells (Sreekumar et al., 2005), and human fibroblasts (Picot et al., 

2005). Another interesting feature of the MsrA is its up-regulation 

in human skin in response to UV irradiation and hydrogen peroxide, 

suggesting a role of MsrA in photoprotection in epidermis 

(Ogawa et al., 2006; Schallreuter et al., 2006; Picot et al., 2007). 

We extended these studies by showing that Msr null mutants are 

not only sensitive to UV light but also have their transcripts accumulated 

in the presence of DNA damaging agents. This last aspect 

raises the interesting possibility that Msr genes are activated upon 

the DNA damage response. 

Our results strongly indicate that A. nidulans msrA and msrB are 

involved in the repair of oxidatively damaged proteins. Interestingly, 

both genes seem to act together to repair protein damage, 

as seen in the assays of paraquat viability and UV-sensitivity of 

germinating conidia, or more specifically, as for example in the 

H 2O 2 viability and UV-sensitivity of quiescent conidia. This is the 

first step towards the functional characterization of msr genes in 

filamentous fungi. Considering the good qualities of A. nidulans as 

a model system, the A. nidulans Msr provide a great opportunity 

to study the function of these enzymes. 



Fig. 6. Fold increase in mrsA (A) and mrsB (B) mRNA levels in response to DNA damaging agents. Mycelia were grown either in the absence or in the presence of any DNA 

damaging agent. Real-time RT-PCR was used to quantitate the mRNA. The measured quantity of the mrsA and mrsB mRNA in each of the treated samples was normalized 

using the C T-values obtained for the tubC RNA amplifications run in the same plate. The relative quantitation of msrA, msrB and tubulin gene expression was determined by a 

standard curve (i.e., C T-values plotted against logarithm of the DNA copy number). Results of four sets of experiments were combined for each determination; 

means ± standard deviation are shown. The values represent the fold-change in gene expression compared to the wild type control grown without any drug (represented 

absolutely as 1.00). CPT (camptothecin), MMS (methyl methane sulfonate), BLEO (bleomycin), and 4-NQO (4-nitrroquinoline oxide). 

Acknowledgments 

This research was supported by the Fundação de Amparo à Pesquisa 

do Estado de São Paulo (FAPESP), Conselho Nacional de 

Desenvolvimento Científico e Tecnológico (CNPq), Brazil, and John 

Simon Guggenheim Memorial Foundation, USA. Work in Vienna 

was supported by Grant P20630 of the Austrian Science Fund 

FWF to J.S. We would like to thank André Oliveira Mota Júnior, 

Charley Staats and Joel Fernandes Lima for helping in the phylogenetic 

trees and in the UV-sensitivity assays, respectively. 

Appendix A. Supplementary data 

Supplementary data associated with this article can be found, in 

the online version, at doi:10.1016/j.fgb.2009.01.004. 

References 

Alamuri, P., Maier, R.J., 2004. Methionine sulfoxide reductase is an important 

antioxidant enzyme in the gastric pathogen Heclicobacter pylorii. Mol. Microbiol. 

53, 1397–1406.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of 

microgram quantities of protein utilizing the principle of protein–dye binding. 

Anal. Biochem. 72, 248–254. 

Chaveroche, M.K., Ghigo, J.M., d’Enfert, C., 2000. A rapid method for efficient gene 

replacement in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res. 

28, E97–E104. 

Cabreiro, F., Picot, C.R., Friguet, B., Petropoulos, I., 2006. Methionine sulfoxide 

reductases: relevance to aging and protection against oxidative stress. Ann. N.Y. 

Acad. Sci. 1067, 37–44. 

Cove, D., 1976. Chlorate toxicity in Aspergillus nidulans. Studies of mutants altered in 

nitrate assimilation. Mol. Gen. Genet. 146, 147–159. 

Davies, M.J., 2005. The oxidative environment and protein damage. Biochim. 

Biophys. Acta 1703, 93–109. 

Delaye, L., Becerra, A., Orgel, L., Lazcano, A., 2007. Molecular evolution of 

peptides methionine sulfoxide reductases (MsrA and MsrB): on the early 

development of a mechanism that protects against oxidative damage. J. Mol. 

Evol. 64, 15–32. 

Friguet, B., 2006. Oxidized protein degradation and repair in ageing and oxidative 

stress. FEBS Lett. 580, 2910–2916. 

Goldman, G.H., Kafer, E., 2004. Aspergillus nidulans as a model system to characterize 

the DNA damage response in eukaryotes. Fungal Genet. Biol. 41, 428–442. 

Goldman, G.H., McGuire, S.L., Harris, S.D., 2002. The DNA damage response in 

filamentous fungi. Fungal Genet. Biol. 35, 183–195. 

Harris, S.D., Momany, M., 2004. Polarity in filamentous fungi: moving beyond the 

yeast paradigm. Fungal Genet. Biol. 41, 31–400. 

Hoshi, T., Heinemann, S., 2001. Regulation of cell function by methionine oxidation 

and reduction. J. Physiol. 531, 1–11. 

Hou, L., Kang, I., Marchant, R.E., Zagorski, M.G., 2002. Methionine 35 oxidation 

reduces fibril assembly of the amyloid abeta-(1-42) peptide of Alzheimer’s 

disease. J. Biol. Chem. 277, 40173–40176. 

Jacob, C., Giles, G.I., Giles, N.M., Sies, H., 2003. Sulfur and selenium: the role of 

oxidation state in protein structure and function. Angew. Chem. Intl. Ed. Engl. 

42, 4742–4758. 

Kantorow, M., Hawse, J.R., Cowell, T.L., Benhamed, S., Pizarro, G.O., Reddy, V.N., 

Hejtmancik, J.F., 2004. Methionine sulfoxide reductase A is important for lens 

cell viability and resistance to oxidative stress. Proc. Natl. Acad. Sci. USA 101, 

9654–9659. 

Kafer, E., 1977. Meiotic and mitotic recombination in Aspergilllus and its 

chromosomal aberrations. Adv. Genet. 19, 33–131. 

Keating, D.J., 2008. Mitochondrial dysfunction, oxidative stress, regulation of 

exocytosis and their relevance to neurodegenerative diseases. J. Neurochem. 

104, 298–305. 

Kim, H-Y., Gladyshev, V.N., 2007. Methionine sulfoxide reductases: selenoprotein 

forms and roles in antioxidant protein repair in mammals. Biochem. J. 407, 321– 

329. 

Kuwayama, H., Obara, S., Morio, T., Katoh, M., Urushihara, H., Tanaka, Y., 2002. PCRmediated 

generation of a gene disruption construct without the use of DNA 

ligase and plasmid vectors. Nuclei Acids Res. 30, e2. 

Marchetti, M.A., Lee, W., Cowell, T.L., Wells, T.M., Weissbach, H., Kantorow, M., 

2006. Silencing of the methionine sulfoxide reductase A gene results in loss of 

mitochondrial membrane potential and increased ROS production in human 

lens cells. Exp. Eye Res. 83, 1281–1286. 

Muller, F.L., Lustgarten, M.S., Jang, Y., Richardson, A., Van Remmen, H., 2007. Trends 

in oxidative aging theories. Free Radical Biol. Med. Aug. 43, 477–503. 

Moskovitz, J.H., Weissbach, N., Brot, N., 1996. Cloning the expression of a 

mammalian gene involved in the reduction of methionine sulfoxide residues 

in proteins. Proc. Natl. Acad. Sci. USA 93, 2095–2099. 

Moskovitz, J., Berlett, B.S., Poston, J.M., Stadtman, E.R., 1997. The yeast peptidemethionine 

sulfoxide reductase functions as an antioxidant in vivo. Proc. Natl. 

Acad. Sci. USA 94, 9585–9589. 

Moskovitz, J., Flescher, E., Berlett, B.S., Azare, J., Poston, J.M., Stadtman, E.R., 1998. 

Overexpression of peptide-methionine sulfoxide reductase in Saccharomyces 

cerevisiae and human T cells provides them with high resistance to oxidative 

stress. Proc. Natl. Acad. Sci. USA 95, 14071–14705. 

Moskovitz, J., 2005a. Methionine sulfoxide reductases: ubiquitous enzymes 

involved in antioxidant defense, protein regulation, and prevention of agingassociated 

diseases. Biochim. Biophys. Acta 1703, 213–219. 



Moskovitz, J., 2005b. Roles of methionine sulfoxide reductases in antioxidant 

defense, protein regulation and survival. Curr. Pharm. Des. 11, 1451–1457. 

Nakao, L.S., Iwai, L.K., Kalil, J., Augusto, O., 2003. Radical production from free and 

peptide-bound methionine sulfoxide oxidation by peroxynitrite and hydrogen 

peroxide/iron(II). FEBS Lett. 547, 87–91. 

Noventa-Jordão, M.A., do Nascimento, A.M., Goldman, M.H., Terenzi, H.F., Goldman, 

G.H., 1999. Molecular characterization of ubiquitin genes from Aspergillus 

nidulans: mRNA expression on different stress and growth conditions. Biochim. 

Biophys. Acta 1490, 237–244. 

Ogawa, F., Cander, C.S., Hansel, A., Oehrl, W., Kasperczyk, H., Elsner, P., Shimizu, K., 

Heinemann, S.H., Thiele, J.J., 2006. The repair enzyme peptide methionine-Ssulfoxide 

reductase is expressed in human epidermis and upregulated by UVA 

radiation. J. Invest. Dermatol. 126, 1128–1134. 

Oien, D.B., Moskovitz, J., 2008. Substrates of the methionine sulfoxide reductase 

system and their physiological relevance. Curr. Top. Dev. Biol. 80, 93–133. 

Osmani, S.A., May, G.S., Morris, N.R., 1987. Regulation of the mRNA levels of nimA, a 

gene required for the G2-M transition in Aspergillus nidulans. J. Cell. Biol. 104, 

1495–1504. 

Osmani, S.A., Mirabito, P.M., 2004. The early impact of genetics on our 

understanding of cell cycle regulation in Aspergillus nidulans. Fungal Genet. 

Biol. 41, 401–410. 

Petropoulos, I., Friguet, B., 2005. Protein maintenance in aging and replicative 

senescence: a role for peptide methionine sulfoxide reductases. Biochim. 

Biophys. Acta 1703, 261–266. 

Picot, C.R., Petropoulos, I., Perichon, M., Moreau, M., Nizard, C., Friguet, B., 2005. 

Overexpression of MsrA protects WI-38 SV40 human fibroblasts against H2O2mediated 

oxidative stress. Free Radical Biol. Med. 3, 1332–1341. 

Picot, C.R., Moreau, M., Juan, M., Noblesse, E., Nizard, C., Petropoulos, I., Friguet, B., 

2007. Impairment of methionine sulfoxide reductase during UV irradiation and 

photoaging. Exp. Gerontol. 42, 859–863. 

Requena, J.R., Chao, C.C., Levine, R.L., Stadtman, E.R., 2001. Glutamic acid 

aminoadipic semialdehydes are the main carbonyl products of metalcatalyzed 

oxidation of proteins. Proc. Natl. Acad. Sci. USA 98, 69–74. 

Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, third ed. 

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 

Semighini, C.P., Marins, M., Goldman, M.H.S., Goldman, G.H., 2002. Quantitative 

analysis of the relative transcript levels of ABC transporter Atr genes in 

Aspergillus nidulans by real-time reverse transcription-PCR assay. Appl. Environ. 

Microbiol. 68, 1351–1357. 

Schallreuter, K.U., Rubsam, K., Chavan, B., Zothner, C., Gillbro, J.M., Spencer, J.D., 

Wood, J.M., 2006. Functioning methionine sulfoxide reductases A and B are 

present in human epidermal melanocytes in the cytosol and in the nucleus. 

Biochem. Biophys. Res. Commun. 342, 145–152. 

Skaar, E.P., Tobiason, D.M., Quick, J., Judd, R.C., Weissbach, H., Etienne, F., Brot, N., 

Seiferts, H.S., 2002. The outer membrane localization of the Neisseria 

gonorrhoeae MsrA/B is involved in survival against reactive oxygen species. 

Proc. Natl. Acad. USA 99, 10108–10113. 

Singh, V.K., Moskovitz, J., 2003. Multiple methionine sulfoxide reductase in 

Staphylococcus aureus: expression of activity and roles in tolerance of 

oxidative stress. Microbiology 149, 2739–2747. 

Sreekumar, P.G., Kannan, R., Yaung, J., Spee, C.K., Ryan, S.J., Hinton, D.R., 2005. 

Protection from oxidative stress by methionine sulfoxide reductases in RPE 

cells. Biochem. Biophys. Res. Commun. 334, 245–253. 

Stadtman, E.R., 2006. Protein oxidation and aging. Free Radical Res. 40, 1250–1258. 

Stadtman, E.R., Levine, R.L., 2003. Free radical-mediated oxidation of free amino 

acids and amino acid residues in proteins. Amino Acids 25, 207–218. 

Tsvetkov, P.O., Ezraty, B., Mitchell, J.K., Devred, F., Peyrot, V., Derrick, P.J., Barras, F., 

Makarov, A.A., Lafitte, D., 2005. Calorimetry and mass spectrometry study of 

oxidized calmodulin interaction with target and differential repair by 

methionine sulfoxide reductases. Biochimie 87, 473–480. 

Weissbach, H., Etienne, F., Hoshi, T., Heinemann, S.H., Lowther, W.T., Matthews, B., 

St John, G., Nathan, C., Brot, N., 2002. Peptide methionine sulfoxide reductase: 

structure, mechanism of action, and biological function. Arch. Biochem. 

Biophys. 37, 172–178. 

Weissbach, H., Resnick, L., Brot, N., 2005. Methionine sulfoxide reductases: history 

and cellular role in protecting against oxidative damage. Biochim. Biophys. Acta 

1703, 203–212.

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