Critical Review

Protein Oxidation, Repair Mechanisms and Proteolysis inSaccharomyces cerevisiae

Vitor Costa, Alexandre Quintanilha and Pedro Moradas-FerreiraIBMC, Instituto de Biologia Molecular e Celular, Porto, Portugal; and ICBAS, Instituto de Ciencias Biomedicas AbelSalazar, Departamento de Biologia Molecular, Universidade do Porto, Porto, Portugal

Summary

Reactive oxygen species, generated as normal by-products of

aerobic metabolism or due to cellular stress, oxidize molecules and

cause cell death by apoptosis. The accumulation of oxidized proteins

is a hallmark of aging and a number of aging diseases. Oxidation

can impair protein function as the proteins are unfolded leading to

an increase of protein hydrophobicity and often resulting in the

formation of toxic aggregates. The yeast Saccharomyces cerevisiaehas been used as a eukaryotic model system to analyze the mole-

cular mechanisms of oxidative stress protection. This paper reviews

how the identification in yeast of specific damaged proteins has

provided new insights into mechanisms of cytotoxicity and high-

lights the role of repair and degradative processes, including

vacuolar/lysosomal and proteasomal proteolysis, in housekeeping

after protein oxidative damage.

IUBMB Life, 59: 293–298, 2007

Keywords Saccharomyces cerevisiae; protein oxidative damage;reactive oxygen species; aging process.

INTRODUCTION

The yeast Saccharomyces cerevisiae has been a useful

eukaryote model for studying oxidative stress responses.

Indeed, yeast can be easily manipulated by adjusting environ-

mental conditions, has a well defined genome, and is a gene-

tically tractable organism. Genomics and proteomics have

emerged as key methodologies for high-throughput analysis of

cellular changes triggered by adverse conditions associated

with stress or inhibition of metabolic functions (1).

Cells when facing adverse environment conditions fight for

survival by developing a stress response that alters metabolic

fluxes. Several stress agents and diseases promote the

formation of reactive oxygen species (ROS), and often in

amounts exceeding the cell antioxidant capacity, a situation

designated as oxidative stress (2). Recent studies have shown

that oxidative stress induces cell death by apoptosis (3). To

avoid oxidative damages in proteins, lipids and DNA, cells

induce a system of both enzymatic and non-enzymatic

antioxidant defenses that scavenge ROS and repair or degrade

damaged molecules (Fig. 1). This response is triggered by

mechanisms sensing ROS and changes in the redox state,

including the Yap1 transcription factor, that increase oxida-

tive stress resistance (2, 4 – 6).

The progressive accumulation of oxidized and cross-linked

proteinacious materials has been implicated in the aging

process that is characterized by a gradual functional decline

and an increase in the probability of disease and death.

Chronological and replicative lifespan studies in yeast have

been used to model the aging effects of postmitotic tissues and

proliferating cells of higher organisms, respectively (7). Both

chronological and replicative aging show oxidative damage

markers (8 – 10). In postmitotic cells, damaged proteins cannot

be diluted; however, during replicative senescence, carbony-

lated proteins are retained in mother cells. Actin is required for

proper segregation of oxidized proteins during cytokinesis.

This asymmetric inheritance appears to be evolutionarily

conserved and contributes to free-radical defense and fitness of

daughter cells (11).

PROTEIN OXIDATIVE DAMAGES AND REPAIRMECHANISMS

In the presence of ROS, proteins can be damaged by direct

oxidation of their aminoacid residues (Fig. 2) and cofactors or

by secondary attack via lipid peroxidation end-products.

Regarding protein cofactors, the oxidation of 4Fe-4S clusters

Received 16 January 2007; accepted 16 January 2007Address correspondence to: Vıtor Costa, Instituto de Biologia

Molecular e Celular, Grupo de Microbiologia Celular e Aplicada, RuadoCampoAlegre, 823, 4150-180Porto, Portugal. Tel:þ351 22 6074961.Fax: þ351 22 6099157. E-mail: vcosta@ibmc.up.pt

IUBMBLife, 59(4 – 5): 293 – 298, April –May 2007

ISSN 1521-6543 print/ISSN 1521-6551 online � 2007 IUBMB

DOI: 10.1080/15216540701225958

by superoxide radicals in enzymes such as the mitochondrial

aconitase has major detrimental effects, due to the lost of

activity that impairs respiratory function. Furthermore, the

free iron released promotes the formation of highly reactive

hydroxyl radicals that propagate molecular damages (2).

Protein oxidation can lead to conformation changes associated

with the decrease in activity when target aminoacid residues

are at or close to active sites. Aminoacids prone to oxidation

include tryptophan, tyrosine, lysine, arginine, and proline.

Histidine, cysteine, and methionine are highly susceptible to

oxidation (12, 13). Proteomic studies of yeast cells have

significantly contributed to the identification of proteins

specifically oxidized, thus providing new insights into redox-

regulated physiological processes and into mechanisms of

cytotoxicity.

Oxidation of Sulfur-containing Aminoacids

Sulfur-containing aminoacids, such as methionine and

cysteine, function as antioxidants and are key components in

the regulation of cell metabolism. Protein methionine residues

are oxidized into methionine-S-sulfoxides (Met-S-SO) and

methionine-R-sulfoxides (Met-R-SO). Methionine sulfoxide

reductases (MsrA and MsrB) are able to reduce methionine

sulfoxides back to methionine. MsrA (¼ Mxr1 in yeast) acts

on Met-S-SO forms and plays a major role in oxidative stress

resistance and longevity. MsrB reduces Met-R-SO forms but

plays a minor role. These enzymes prevent the irreversible

oxidation of methionine residues into sulfone derivatives

(Met-SO2) and increase the scavenging efficiency of the system.

It has been proposed that the oxidation of surface exposed

methionines protects other essential residues from oxidative

damage (12, 14).

Interestingly, Mxr1 activity is regulated by the Gpx3

glutathione peroxidase. Gpx3 plays a major role in cellular

resistance to peroxides due to its redox signaling function. The

interaction between Gpx3 and Mxr1 under oxidative stress

may serve as an important and efficient regulatory link

between ROS detoxification enzymes and repairing proteins.

The Gpx3 Cys82 residue functions to assist the defense

mechanism of Mxr1 against oxidative stress whereas the Gpx3

Cys36 is the site of peroxide sensing. Under physiological

conditions, Cys82 of Gpx3 binds to Cys176 of Mxr1. Upon

oxidative stress, this disulfide bond is broken and Cys82 of

Gpx3 is able to bind to Cys36-SOH through a thiol-disulfide

exchange reaction. Concomitantly, Mxr1 is released and can

repair oxidized proteins. Mxr1 is also known to use an intra-

or inter-disulfide bond exchange mechanism such as Gpx3.

Cys176 of Mxr1 functions as a recycling cysteine during the

repairing process (15, 16).

Figure 2. Protein oxidation and repair mechanisms. Sulfur

containing aminoacids, such as methionine and cysteine, are

prone to oxidation. Methionine sulfoxide reductases (Mxr)

reduce methionine sulfoxides (Met-SO), preventing the irre-

versible oxidation of methionine residues into sulfone deriva-

tives (Met-SO2). Old yellow enzyme (Oye2) has been shown to

reduce cysteine disulfide bonds (Protein-(Cys-S)2) in actin.

Protein S-thiolation of cysteine sulfenic acid derivatives (Prot-

Cys-SOH) and its subsequent reduction by the Grx5

glutaredoxin restores protein thiols. Sulfiredoxin (Srx) can

specifically reduce sulfinic acid derivatives (Prot-Cys-SO2H) in

2-Cys peroxiredoxins. Protein sulfonic acids (Prot-Cys-SO3H)

and carbonyls (Prot-C¼O) cannot be repaired and have to be

targeted for degradation.

Figure 1. Protein oxidation and proteolysis of oxidized

proteins. The increased production of reactive oxygen species

(ROS) due to stress or associated with ageing and diseases

leads to the accumulation of oxidized proteins. Irreversibly

damaged proteins can be degraded by the 20S proteasome or

by vacuolar proteases. Extensively oxidized proteins cannot be

degraded and tend to cross-link forming aggregates that

impair the 20S proteasome and mitochondrial function,

thereby increasing ROS production.

294 COSTA ET AL.

The oxidation of protein cysteine thiol groups (��SH) can

generate thiyl radicals (��S.), disulfide bonds (��S��S��), as

well as sulfenic (��SOH), sulfinic (SO2H), and sulfonic

(7SO3H) acid derivatives. The formation of stable protein

disulfide bonds is rare in the cytoplasm due to its reducing

environment; however it has been described for a few proteins,

including the Yap1 transcription factor and actin. The

formation of disulfide bond in Yap1 is controlled by the

Gpx3 glutathione peroxidase by a mechanism similar to that

described for Mxr1. The Cys36 of Gpx3 can form an

intermolecular protein disulfide bond with Cys598 of Yap1.

This Gpx3-S-S-Yap1 intermediate undergoes a subsequent

intramolecular thiol-disulfide interchange involving Yap1

Cys303, generating oxidized Yap1 (Cys303 –Cys598) and re-

reduced Gpx3. Oxidized Yap1 accumulates in the nucleus

where it transcriptionally activates the expression of genes

associated with the oxidative stress response (6, 17).

In contrast, the formation of a disulphide bond between

actin Cys285 and Cys374 contributes to oxidative stress

sensitivity. Oxidation of yeast actin decreases its dynamics

causing depolarization of the mitochondrial membrane and an

increase in ROS production that contributes to cell death.

The disulfide bond in oxidized actin can be reduced by

the old yellow enzyme (Oye2), a FMN containing NADPH

oxidoreductase that interacts with actin and controls its redox

state. Thus, the accumulation of oxidized actin due to Oye2

deficiency causes cellular sensitivity to oxidative stress and

senescence (18, 19).

Using two distinct thiol-labeling approaches combined with

2D-gel electrophoresis, Le Moan et al. (20) showed that

cells growing in the presence of oxygen, in contrast with

anaerobically grown cells, contain a large number of proteins

with oxidized thiols. The majority of these proteins are

involved in carbohydrate metabolism and aminoacid bio-

synthesis, probably due to the presence of redox-reactive

catalytic or metal-coordinating cysteine residues. Treatment of

yeast cells with H2O2 does not lead to de novo protein thiol

oxidation, but rather increases the oxidation state of a specific

group of oxidized proteins. This high selectivity is conserved in

evolution and the main H2O2 target proteins are enzymes

involved in carbohydrate metabolism, including the key

glycolytic enzyme glyceraldeyde-3-phosphate dehydrogenase

(GAPDH), oxidative stress protection, such as the peroxi-

redoxins Tsa1 and Ahp1, protein translation and protein

folding (20). Notably, inactivation of the thioredoxin system

leads to specific increases in thiol oxidation of proteins

involved in H2O2 detoxification, indicating that thioredoxin

is important for thiol redox buffer of these antioxidant

defenses. Oxidation of peroxiredoxins probably reflects

enzyme cycling due to reduction of peroxides. Furthermore,

it was recently described that oxidized Tsa1 forms a high

molecular weight complex with chaperone activity that is

important for cellular protection. The switch between

thioredoxin-dependent peroxidase and chaperone functions

is linked to oxidation of the peroxidatic Cys47 residue.

Tsa1 deficiency causes the accumulation of aggregated

proteins, mainly ribosomal proteins, that increases by

oxidative stress. Thus, the Tsa1 complexes function to

chaperone misassembled ribosomal proteins, preventing their

aggregation and the consequent inhibition of translation

initiation (21).

The cysteine sulfinic and sulfonic acids are thought to be

irreversible forms of protein oxidation, although recent studies

have shown that sulfiredoxin can specifically reduce cysteine

sulfinic acid derivatives in 2-Cys peroxiredoxins (22, 23),

enzymes that are involved in peroxide detoxification. The

generation of these oxidized cysteines can be prevented by

formation of mixed disulfides between glutathione (GSH) and

thiyl or sulfenic acid forms. This modification is referred to as

S-thiolation and plays an important role in redox regula-

tion (24, 25). Protein S-thiolation is a reversible process and

these proteins are deglutathionylated by the monothiol

glutaredoxin Grx5 during recovery from oxidative stress,

thus restoring protein activity (26). S-thiolation has been

reported to occur in proteins such as protein translation

factors, ubiquitin-conjugating enzymes, proteasome and cha-

perones of the Hsp70 family (24, 25, 27). The S-thiolation of

translation factors may prevent the inhibition of protein

synthesis during oxidative stress or during cell recovery. The

protection of the 20S proteasome may prevent the inhibition

of proteolysis of irreversibly oxidized proteins (see below).

Chaperones recognize hydrophobic surfaces exposed as a

result of protein oxidation and may function to assist

protein refolding. If refolding is unsuccessful then chaperones

may assist in the unfolding of proteins that precedes

proteolysis.

Protein Carbonylation

Formation of protein carbonyl groups results from oxida-

tion of specific aminoacids (lysine, arginine, proline and

histidine) and protein backbone cleavage (at proline, gluta-

mate and aspartate residues). Protein carbonylation is

irreversible and is correlated with oxidative stress-induced cell

death. Indeed, we showed that yeast cells lacking the Yap1 or

Skn7 stress response regulators, which are very sensitive to

oxidative stress, accumulate increased levels of protein

carbonyls (28). Proteomic studies showed that the major

protein targets carbonylated belong to two important groups,

namely glycolytic enzymes such as GAPDH and molecular

chaperones such as Hsp60 (28, 29). Interestingly, these groups

of proteins are also carbonylated during exposure to the toxic

metal chromium and in aged cells (9, 30) and are also thiol

oxidized in H2O2 treated cells, as discussed above. It has been

proposed that the oxidative inactivation of GAPDH down

regulates glycolytic fluxes during oxidative stress, favoring

NADPH production via the pentose phosphate pathway (24).

The inactivation of molecular chaperones affects cell metabo-

lism since these proteins display multiple functions such as

PROTEIN OXIDATION IN S. CEREVISIAE 295

control of protein folding and membrane translocation.

Indeed, Hsp60 deficient cells are not viable and Hsp60

overexpression increases oxidative stress resistance (31).

Oxidized proteins are randomly distributed inside cells and

oxidized aminoacids seem to cluster at both contiguous and

discontinuous sites in protein structures (13). Oxidation at

discrete regions of a protein may be of critical importance in

disease. Indeed, oxidation of a stretch of 20 aminoacid

residues at the C-terminus of a-synuclein plays a role in

Parkinson’s disease (32).

Deficiency in superoxide dismutase (SOD) increases mito-

chondrial protein carbonylation in late stationary phase and

cells submitted to oxidative stress (33). Eukaryotic cells

express two forms of SOD: Cu,Zn-SOD (Sod1), present in

the cytosol and the mitochondrial intermembrane space, and

Mn-SOD (Sod2), located in the mitochondrial matrix. Both

SODs have both unique and overlapping functions in the

protection of mitochondrial proteins from oxidation.

Mn-SOD specifically protects six mitochondrial matrix

proteins, whereas CuZn-SOD and Mn-SOD are both required

to protect some mitochondrial proteins, including porin. Porin

is a key protein in mitochondria-mediated apoptosis and its

oxidation may promote efflux of potential apoptotic regula-

tors to the cytosol that trigger caspase activation and lead to

cell death. The inactivation of mitochondrial proteins may

relate to functional defects observed in amyotrophic lateral

sclerosis (ALS), a disease characterized by degeneration of

motor neurons and associated with mutations in CuZn-SOD.

The detrimental effects of mutated CuZn-SOD have been

associated to the production of peroxides by a toxic gain of

function or to the nitration of tyrosines on neurofilament

proteins leading to activation of programmed cell death

pathways (34).

TURNOVER OF IRREVERSIBLY OXIDIZED PROTEINS

Proteins irreversibly inactivated by formation of methio-

nine sulfones, cysteine sulfinic or sulfonic acids and carbonyl

derivatives cannot be repaired and have to be recognized and

degraded by cellular proteolytic processes. Failure to remove

early increases in protein oxidation may allow for oxidized

proteins to become more severely oxidized and/or cross-linked

(Fig. 1). These ‘indigestible’ protein aggregates are toxic and

have been linked to the initiation and progression of numerous

diseases and aging (10). These detrimental effects include

mitochondria damage, resulting in decreased ATP synthesis

and enhanced formation of ROS, and proteasome inhibition,

which impair degradation of oxidized proteins and further

promote cellular damage. Thus, the selective degradation of

oxidized proteins is of critical importance to maintain cellular

homeostasis. In yeast, our data shows that protein catabolism

is indeed the most significantly overrepresented function

upregulated after oxidative damage (35). Induced genes are

associated with both proteasomal and vacuolar (lysosomal in

higher eukaryotes) mediated proteolysis. The function of the

20S proteasome in the turnover of oxidized proteins is well

established whereas vacuole/lysosome function in this

process has emerged more recently. Mitochondria also contain

a proteolytic system that is responsible for removal of

misfolded or damaged proteins. In mammalian cells, the

ATP-dependent mitochondrial Lon protease is important for

degradation of oxiditively modified proteins (36). The role of

the yeast Lon protein (Pim1) in the degradation of

oxidized mitochondrial matrix proteins has not been

characterized.

Role of the Proteasome

The proteasomes are multicatalytic protease complexes that

play an important role in the degradation of unwanted

proteins, including normal, damaged, mutant or misfolded

proteins. It has been described that the 20S proteasome is

activated under oxidative stress conditions and is able

to degrade oxidized proteins in an ATP- and ubiquitin-

independent manner. In contrast, the ubiquitin-dependent 26S

proteasome as well as ubiquitin-activating and -conjugating

enzymes are very sensitive to direct oxidative inactivation, and

cells deficient in ubiquitin-conjugating activity are able to

degrade oxidized proteins at near normal rates (37, 38). The

key role of the 20S proteasome in the clearance of oxidized

proteins is supported by data showing that several genes

encoding 20S proteasome subunits are upregulated in cells

exposed to oxidative stressors and also during recovery after

oxidative damage (4, 35). Furthermore, yeast cells expressing

increased levels of proteasome activity are able to degrade

carbonylated proteins more efficiently than wild type cells and

display an enhanced lifespan (39).

Role of the Lysosome/Vacuole

The degradation of cytosolic components by lysosomes/

vacuole (autophagy) also plays a key role in the maintenance

of cellular homeostasis. In yeast, protein sorting, vacuole

function, and vacuolar acidification are core functions

required for broad resistance to oxidative stress (5). Consistent

with a role of vacuolar proteolysis in cellular homeostasis after

oxidative damage, our data shows that genes encoding

vacuolar proteases and genes associated with protein sorting

into the vacuole and vacuolar fusion are upregulated after

oxidative damage. In addition, the activity of vacuolar Pep4

aspartyl protease increases and its deficiency decreases the

capacity to remove oxidized proteins (35). Vacuolar proteo-

lysis also plays a major role in the turnover of proteins

oxidized by endogenously generated ROS. Indeed, Pep4

deficiency increases protein carbonyls in unstressed cells and

accelerates the progressive accumulation of oxidized proteins

during chronological aging, decreasing yeast lifespan (35).

Increased levels of the vacuolar protease Pep4 prevents the

accumulation of oxidized protein, although it does not

increase lifespan, suggesting survival of stationary cells is not

296 COSTA ET AL.

limited only by protein oxidation. In mammalian cells, a

chaperone-mediated autophagy pathway is activated by

oxidative stress and is important for the efficient removal of

oxidized cytosolic proteins by lysosomes. An Hsp70 family

chaperone recognizes oxidized proteins targets and mediates

its translocation into lysosomes for proteolysis (40). In yeast,

an Hsp70-dependent mechanism of transport into the vacuole

has been described for a few cytosolic proteins; however,

oxidized proteins may not be degraded by Pep4 in the vacuole

since cells treated with H2O2 release Pep4 into the cyto-

plasm (41).

FINAL REMARKS

The balance between synthesis, repair and degradation of

damaged proteins plays a major role in cell homeostasis.

Oxidation of aminoacid residues mediated by ROS affects the

biological function of proteins. Studies using Saccharomyces

cerevisiae have generated novel data on the identification of

proteins sensitive to oxidation and the characterization of

mechanisms important for protection and repair of damaged

proteins. Oxidation of aminoacids such as methionine and

cysteine and their reduction by methionine sulfoxide reduc-

tases and the S-thiolation/glutaredoxin system, respectively,

provides a ROS-scavenging protective mechanism that may

prevent the detrimental effects on metabolism due to irrever-

sible protein oxidative damage.

The discovery that oxidized proteins can be degraded by

alternative pathways recognized the involvement of the 20S

proteasome and vacuolar proteases in this process. At present

the mechanism underlying vacuolar proteases remains unclear,

namely if the proteolysis requires the transport of the oxidized

cytosolic proteins into the vacuole or alternatively the trans-

location of proteases from the vacuole into the cytosol.

Experimental data generated using yeast mutants indicates

that loss of cell vitality in cells challenged by adverse

environmental conditions and in aged cells is linked to the

accumulation of oxidized proteins. Failure either to prevent

protein oxidation in antioxidant deficient cells or to complete

housekeeping by repair or proteolysis leads to accelerated

aging. Conversely, increased levels of these protective mechan-

isms can increase oxidative stress resistance and lifespan.

Oxidative stress plays a role in diseases, such as Alzheimer,

Parkinson and ALS, and yeast has proven to be a valuable

experimental tool to elucidate disease-related events. Thus,

yeast-cell based assays can contribute to elucidate whether

oxidized protein repair mechanisms and proteolysis can rescue

these pathologies.

ACKNOWLEDGEMENTS

We thank by FCT, POCTI and FSE-FEDER for financial

support to our work and apologize to authors whose articles

could not be cited due to space limitations.

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