Redox proteomics and drug development - UCCS Home · Redox proteomics and drug development Angelo D'Alessandro, Sara Rinalducci, Lello Zolla⁎ Department of Environmental Sciences,

J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 2 5 7 5 – 2 5 9 5

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Invited Review

Redox proteomics and drug development

Angelo D'Alessandro, Sara Rinalducci, Lello Zolla⁎

Department of Environmental Sciences, University of Tuscia, Largo dell'Università, snc, 01100 Viterbo, Italy

A R T I C L E I N F O

⁎ Corresponding author. Tuscia University, LaE-mail address: [email protected] (L. Zolla).

1874-3919/$ – see front matter © 2011 Elsevidoi:10.1016/j.jprot.2011.01.001

A B S T R A C T

Article history:Received 15 October 2010Accepted 9 January 2011Available online 15 January 2011

As alterations of the redox homeostasis lie at the root of many pathophysiological processes inhuman health, redox proteomics holds the promise to shed further light on fundamentalbiological processes. In this review, themechanismsof reactiveoxygenspecies (ROS) andreactivenitrogen species (RNS) production are reviewed, mainly addressing those chemical phenomenawhich have already been associatedwith pathological conditions (of the central nervous system,cardiovascular system, or simply related to aging and altered-cell cycle regulation).From Alzheimer's to Parkinson's and Hungtinton's disease, from ageing to cancer, oxidativestress (OS) appears to represent a common trait in somany relevant biological aspects of humanhealth, that further investments in the field of redox proteomics ought to be mandatory.For the foreseeable future, redox proteomics will likely play a pivotal role in the quest for newtherapeutical targets and their validation, in the process of determining OS-triggered cellularalteration upon drug treatments and thus in the very heart of the design and testing of newdrugs and their metabolites against those pathologies relying on altered redox homeostasis.

© 2011 Elsevier B.V. All rights reserved.

Keywords:Redox proteomicsDrugCarbonylationNeurodegenerative diseasesNutraceutical

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25762. Redox proteomics: a brief look at the basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2576

2.1. Oxidation/nitrosylation of cysteine thiol groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25762.1.1. Gel-based approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25762.1.2. Quantitative gel-based redox proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25772.1.3. Shotgun proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25782.1.4. Quantitative redox proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2578

2.2. Analysis of nitrated peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25782.3. Carbonylations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2579

3. Human diseases and oxidative stress: early hints from proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . 25803.1. Nervous system and OS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2580

3.1.1. Alzheimer's, Parkinson's and Huntington's disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25813.1.2. Amyotrophic lateral sclerosis, multiple sclerosis and muscular dystrophy . . . . . . . . . . . . . . . 25823.1.3. Depression and behavioural disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2583

3.2. Vascular aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25833.3. Aging and cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2583

rgo dell'Università snc, 01100 Viterbo, Italy. Tel.: +39 0761 357 100; fax: +39 0761 357 630.

er B.V. All rights reserved.

http://dx.doi.org/10.1016/j.jprot.2011.01.001

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http://dx.doi.org/10.1016/j.jprot.2011.01.001

2576 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 2 5 7 5 – 2 5 9 5

4. Drugs and OS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25844.1. CNS: neurodegenerative diseases and mood/psychiatric disorders . . . . . . . . . . . . . . . . . . . . . . . . 2584

4.1.1. Treatment of neurodegenerative diseases through OS-related strategies . . . . . . . . . . . . . . . . 25844.1.2. Mood and psychiatric disorders and OS-targeting approaches . . . . . . . . . . . . . . . . . . . . . . 2587

4.2. Cardiovascular aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25874.3. Aging and metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25884.4. Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2589

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2590Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2590Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2590References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2590

25892590259025902590

1. Introduction

Redox proteomics is an emerging branch of proteomics aimedat investigating oxidative-stress induced modifications ofproteins. Oxidative injuries to proteins are produced bychemically reactive species. Modifications could addressoxygen species and thus generate Reactive Oxygen Species(ROS), such as hydroxyl, peroxide and superoxide radicals, orproduce mixed nitrogen-oxygen species (RNS), viz nitric oxide(NO) and peroxynitrite (ONOO−).

The ROS/RNS are inevitably generated in metabolic path-ways inall cells, and someof themmightplay important roles incell signalling [1,2]. However, excessive levels of ROS fromeitherthe environment or aberrations in electron transport canproduce such high levels of oxidative stress (OS) that largeamountsofproteins canbe irreparablyaltered [3].Under chronicOS, damaged proteins can even accumulate up to reach toxiclevels, often causing cell death as in a plethora of OS-associatedphysiological disorders and pathological diseases.

Among biomolecules, proteins aremajor targets of ROS/RNSthus complicating the whole proteome through side-chainmodifications and covalent changes which have repercussionson protein activity, unfolding, degradation, as well as in cellfunctioning [4,5].

Thus, protein-oriented investigations upon prolonged OS-exposure, either under physiological or pathological conditions,are gaining momentum.

2. Redox proteomics: a brief look at the basics

Redox proteomics aims at detecting and analyzing redox-basedchangeswithin the proteome both in redox signalling scenariosand in OS [5]. The interested reader is referred to Table 1 for arapid glimpse at the contents of this section, which is mainlyfocused on the assessment of oxidations/nitrosylations in thiolgroups through gel-based and shotgun proteomic approachesand nitration on tyrosines. The basics of carbonylation-target-ing redox proteomics are also briefly described.

2.1. Oxidation/nitrosylation of cysteine thiol groups

2.1.1. Gel-based approachesTo date, most of the proteomic studies of the oxidative stressresponse have used 2DE as a protein separation and quanti-

fication tool, coupled with mass spectrometry (MS) as aprotein identification tool. Despite its weaknesses in theseparation of certain protein categories (i.e. more hydrophobicand high molecular weight proteins, such as membraneproteins) and limitation in dynamic range, 2DE is still thebest separation tool when dealing with redox-based proteinchanges. ROS/RNS add different footprints in the cells in theform of covalent modifications to proteins, thus it is oftenpossible to reveal these changes by applying specific labellingfollowed by detection. A common strategy is to performwestern blot analysis of proteins separated by 2DE [6].

Amongst the many kinds of amino acid residues suscep-tible to oxidative stress, cysteine is by far one of the mostsensitive. Oxidation of its -SH groups can have functionalsignificance by regulating protein function and can be thetarget of oxidative insult as well. For this reason, severalexperimental approaches have been developed for thesystematic and exhaustive characterization of the so-calledthiol proteome. One major limit in such an analysis is thechemical labile nature of Cys redox modifications, thusbasically two critical steps are needed in analyzing the thiolproteome,which consist in a temporary trapping of free thiolsand their subsequent reduction. Different strategies exist forquenching the thiol groups, ranging from the simple TCA(trichloroacetic acid)-based acidification [7] to the use of cell-permeable Cys-specific reagents such as the alkylating agentsiodoacetamide (IAA) or N-ethylmaleimide (NEM) [8]. In thisapproach the subsequent use of more or less selectivereducing agents will allow detection of a specific form ofoxidation. For instance, cysteine residues in the sulfenic acidform are difficult to identify because of their unstablechemical nature, however this has been achieved by exclu-sive reduction of the sulfenic acid by sodiumarsenite [9], or byits reaction with specific chemicals such as dimedone [10].S-nitrosothiols are rather selectively reduced by ascorbate[11], whereas stronger reductants such as DTT reduce bothnitrosothiols and disulfides. However, detection of proteinS-nitrosylation is not easily performed with traditional gel-based methods such as immunoprecipitation and westernblot analysis where the S―NO bond is broken during theelectrophoresis step. On the other hand, commerciallyavailable anti-S-nitrosocysteine antibodies have beenapplied with good results only in immunohistochemistrystudies [12,13] and little in the context of proteomicsinvestigations.

Table 1 – Redox proteomics: a brief look at the basics.

Authors Ref Year Strategy/target

Gel-based approachesand enrichment strategies

Kim et al. [52] 2011 The method is based on fluorine-fluorine interaction affinity purificationfollowing chemical conversions of the nitro groups on yrosines to highlyfluorinated moieties

Sheehan [6] 2010 Immunoblots (Abs against carbonylated proteins, for example) of 2DEseparations

Lee et al. [51] 2009 Introduction of a metal chelating motif into the NO-modified tyrosineresidues followed by solid-phase extraction with Ni2+-nitrilotriacetic acid(NTA) magnetic agarose beads

Le Moan et al. [7] 2009 Thiol proteome investigation through trichloroacetic acid)-basedacidification quenching of thiol groups

Cox et al. [8] 2009 Thiol proteome investigation through alkylating agents iodacetamide (IAA)or N-ethylmaleimide (NEM) quenching of thiol groups

Gianazza et al. [24] 2009 Biotin-based strategies are largely used for detection of S-glutathionylationBruschi et al. [17] 2009 Redox-DIGE through the NEM or IAM derivatives of cyanine (Cy3, Cy5)Camerini et al. [23] 2007 This strategy is a further development of the biotin-switch where the molecule

used to specifically bind nitrosylated peptides has been replaced by a His-tagZhang et al. [50] 2007 Derivatization of nitrotyrosines with N-succinimidyl S-acetylthioacetate (SATA)

and subsequent enrichment by thiopropyl sepharose beadsRiederer et al. [20] 2007 Redox-DIGE through the NEM or IAM derivatives of DY-dyesPoole et al. [10] 2005 Sulfenic acid reaction with specific chemicals such as dimedoneHochgräfe et al. [13] 2005 Use of thiol-reactive reagents to reveal the extent of Cys oxidation by

2DE gels: BOD-IPY FL C1-IASaurin et al. [9] 2004 Exclusive reduction of the sulfenic acid by sodium arseniteYano [16] 2003 Use of thiol-reactive reagents to reveal the extent of Cys oxidation by

2DE gels: Cys-specific fluorescent reagent monobromobimaneNikov et al. [49] 2003 Enrichment of nitrated proteins through immunoprecipitation with

anti-nitroTyr antibodies followed by reduction to amino-Tyr with sodiumdithionite and subsequent biotin tagging

Baty et al. [14] 2002 Use of thiol-reactive reagents to reveal the extent of Cys oxidation by2DE gels: IAM-derivatives 5-iodoacetamidofluorescein

Jaffrey et al. [13] 2001 Upstream enrichment step for the oxidized protein-thiol fraction of theproteome using the biotin-switch method

Shotgun Chang et al. [29] 2010 Anionic affinity capture using poly-arginine-coated nanodiamonds ashigh-affinity probes

Amoresano et al. [53] 2007 Dansyl chloride labelling of nitration sites prior to analysis on a linear iontrap mass spectrometer combining a precursor ion scan and a MS3 step

Leitner et al. [27] 2006 Extraction and enrichment strategies for cysteinyl-peptide enrichmentprior to MS analysis

Dai et al. [28] 2005 Isolation of peptides containing oxidized cysteinesQuantitativeredox-proteomics

Chiappetta et al. [54] 2009 Quantitative identification of nitration sites based on the use of iTRAQreagents

Leichert et al. [33] 2008 OxICAT: proteins are denatured to gain access to all reduced cysteineswhich are irreversibly labelled with light ICAT. All reversible oxidativethiol modifications within the same sample are then reduced using thestrong thiol reductant Tris(2-carboxyethyl)phosphine (TCEP) and allnewly accessible cysteines are modified with heavy ICAT.

Mirzaei and Regnier [67] 2007 Dinitrophenylhydrazine (DNPH) or biotin hydrazide (BHZ) derivatization ofcarbonyls prior to affinity chromatography enrichment.

Sethuraman et al. [30] 2004 Isotope-coded affinity tag (ICAT) reagents to quantify oxidant-sensitiveprotein thiols

2577J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 2 5 7 5 – 2 5 9 5

2.1.2. Quantitative gel-based redox proteomicsA general workflow in redox proteomics consists of qualitativeidentification of oxidized proteins and subsequent quantitativedetermination of the extent of the redox status. Quantifying theredox state of a protein is surely more informative andrepresents the true challenge. Several thiol-reactive reagentshave beenused to reveal the extent of Cys oxidation by 2DE gels.They include, for example, the IAM-derivatives 5-iodoacetami-dofluorescein [14] and BOD-IPY FL C1-IA [15] or the Cys-specificfluorescent reagent monobromobimane [16]. An improvementof 2DE-based fluorescence analysis of the “redoxome” (redox-

proteome) has been obtained by applying the differential in gelelectrophoresis (DIGE) technique. This strategy uses a set offluorophores of similar molecular weights and chemical struc-tures that differ by their spectral features (absorption andemission wavelengths). Redox-DIGE has been performed usingthe NEM or IAM derivatives of cyanine (Cy3, Cy5) [17–19] andDY-dyes [20]. An important limitation of all the methodsdescribed above is that only the most abundant proteins areusually detected, often missing “fancy” proteins such astranscription factors and other regulatory proteins. One way tocircumvent this problem is to performan upstreamenrichment


step for the oxidized protein-thiol fraction of the proteomeusing the biotin-switch method originally developed by Jaffreyet al. [11]. Since then, several variants havebeendeveloped, alsowith chemical entities alternative to biotin as tag [e.g., 21–23].Biotin-based strategies are largely used for detection ofS-glutathionylation [24,25] and S-nitrosylation [21], two Cysmodifications which occur extensively in diseases character-ized by oxidative stress.

2.1.3. Shotgun proteomicsDue to the technical limitations of 2DE-based methods, inrecent years emphasis has gradually shifted towards gel-freetechnologies, such as shotgun-proteomics strategies. 2DE is,in fact, time- and labour-consuming with the major drawbackthat the extent of oxidation always corresponds to an averagecontribution of the Cys residues present in a polypeptide. Theshotgun-proteomics approach in its most general sense refersto the direct analysis by MS/MS of proteolyzed proteinmixtures to rapidly generate a global profile of the proteincomplement within themixture itself. This mixture, however,is generally highly complex. A solution to overcome thishurdle is represented by alternative sample preparationstrategies, which could be suitable to perform a preliminaryenrichment of peptides containing redox-modified cysteines.Although various extraction and enrichment strategies havebeen developed for cysteinyl-peptide enrichment prior to MSanalysis [27], only few methods are directed toward isolatingpeptides containing oxidized cysteines [28]. One of the mostrecent approaches designed for the specific enrichment ofsulfopeptides in tryptic digests is based on anionic affinitycapture using poly-arginine-coated nanodiamonds as high-affinity probes [28]. The method was applied to selectivelyconcentrate peptides containing Cys sulfonic acid from eithera highly dilute solution or a complex peptidemixture in whichthe abundance of the sulfonated analyte was as low as 0.02%.

2.1.4. Quantitative redox proteomicsThe most attractive redox proteomic techniques are thoserelated to quantification of changes in a thiol redox proteomeupon OS exposure. In this respect, Sethuraman et al. describedan interesting shotgun proteomic approach, which exploitsisotope-coded affinity tag (ICAT) reagents [29] to quantifyoxidant-sensitive protein thiols. ICAT reagents have beenextensively used in quantitative proteomics to evaluate theabundance of expressed proteins [30] and, more recently,successfully applied to redox proteomics. This technique usesa certain type of marker which consists of three different parts:(i) a thiol-reactive compound (an iodoacetamide analogue), (ii) alinker containing either heavy or light isotopes, and (iii) a biotintag for separation by avidin-coupled affinity chromatography.The principle of the ICAT approach in redox proteomics is thatonly free thiols are susceptible to modification by the IAMmoiety of the ICAT reagent. After exposing equivalent proteinsamples to either control or oxidant conditions in a non-reducing environment, they are differentially labelled with theheavy or light form of the ICAT. The protein samples aremixedand, after tryptic digestion, the labelled peptides are separatedby affinity chromatography. Finally, the captured peptides areanalyzed by LC-MS/MS for identification of the oxidant-sensi-tive cysteine thiols. Because oxidized cysteines are not suscep-

tible to labelling with ICAT reagent, the labelling intensitydecreases from the control to the oxidized sample [31,32]. Thisapproach is potentially more powerful than conventionalmethods, especially because the protein identification and thequantification of the extent of thiol oxidation are made inthe same analysis [32]. A remarkable development of thismethodologyhas been recently presentedand theadaptation ofICAT to redox proteomics has been definitively ratified with thecoinage of the term OxICAT [33]. In the first step of OxICAT,proteins are denatured to gain access to all reduced cysteineswhich are irreversibly labelled with light ICAT. All reversibleoxidative thiol modifications within the same sample are thenreduced using the strong thiol reductant Tris(2-carboxyethyl)phosphine (TCEP) and all newly accessible cysteinesare modified with heavy ICAT. Importantly, this proceduregenerates chemically identical proteins, which only differ in thespecific mass of their ICAT-label (9 Da additional mass perheavy ICAT) depending on their previous redox state. Peptidesthat contain originally only reduced cysteines are predicted toyield singlemasspeaks corresponding to the light ICAT-labelledform. Peptidescontainingoriginallyoxidizedcysteineswillhavemasses that are exactly 9 Da higher (or multiples thereof) thanthe corresponding light ICAT peptides, depending on thenumber of oxidized cysteines present. After trypsin digestionand affinity purification, the ICAT-labelled peptides are identi-fied by LC-MS/MS,which also establishes the ratio of oxidized toreduced (heavy to light) Cys residues according to the relativeMS intensities. As the extent of oxidation is given as an Ox/Redratio, absolute protein amounts are not considered, thereforeallowing cell extracts comparisons [33]. Contrary to theapproach of Sethuraman and coworkers, where relativechanges in the availability of free protein thiols are determinedin two separate samples, the OxICAT method provides theabsolute ratio of reduced to oxidized protein within a singlesample making this technique suitable to monitor oxidativethiol modifications in vivo. Interestingly, slight modifications ofthe basic scheme, i.e. substitution of the nonspecificthiol reductant TCEP with more specific reductants, such asglutaredoxin or ascorbate, may allow the use of OxICAT tospecifically detect glutathionylations or nitrosylations,respectively.

2.2. Analysis of nitrated peptides

Protein tyrosine nitration to form 3-nitrotyrosine (3-NT) is animportant post-translational modification which has widelyconsidered as a valid biomarker of protein RNS insult [34]occurring in a variety of diseases including cancers, neurode-generative and age-related disorders [35]. 3-NT is also arelatively stable modification, thus it can be suitably analyzedby different specific techniques. Methods for separation,detection, and quantification of 3-nitrotyrosine in biologicalsamples include immunochemical techniques using anti-3-nitrotyrosine antibodies, liquid and gas chromatography incombination with various detection systems [36,37]. However,with the introduction of MALDI and ESI as soft ionizationmethods formass spectrometry of biomolecules, nitration hasbeen also investigated directly at the protein level with theability to determine polypeptide modification sites [38–40].Indeed, application of mass spectrometry, in combination


with 2DE separation and western blot analysis [41] attempteda higher throughput characterization of protein targets fortyrosine nitration in cells and several tissues [42–45]. In thecase ofMS analysis of tyrosine-nitrated peptides, a characteristicmass increase of 45 Da corresponding to NO2-Tyr is observed byboth ESI and MALDI methods [44,46]. However, during MALDIanalysis of the nitrated peptides, a series of additional modifiedpeaks were observed as a consequence of photodecompositionreactions determining the formation of 3-NO-Tyr, 3-NHOH-Tyrand 3-NH2-Tyr adducts [47,48]. This means that MALDI mea-surements yield to significant underestimation of the modifica-tion extent, highlighting the unreliability of thismethodology forthe sensitive detection of nitrated products. On the contrary, thisphenomenon of artificial NO2-Tyr fragmentation was notobserved in ESI-MS measurements, allowing a complete evalu-ationof theproteinnitration level.Moreover, theuseofprecursorion scanning for the specific immonium ion at m/z 181.06 hasfound a broader application in the identification of nitratedpeptides during LC-ESI-MS/MS analysis of protein digests [47]. Atany rate, protein tyrosine nitration is typically a low-yieldprocess and though mass spectrometry represents a sensitiveanalytical method, proteome-wide analysis of nitrated proteins/peptides is still challenging due to the lack of effectiveenrichment methods prior to mass analysis. This is likely dueto the poor chemical reactivity of nitric oxide groups [34].However, the nitro groups can be converted to the morechemically reactive amine groups which can be hence used asa chemical handle to employ tagging groups in nitrated residues.The tagged peptides are then extracted and isolated to solidsupports and subsequently analyzed by MS which gives highlyenhanced signal corresponding to the analyte peptide. Toprovide some recent examples in this regard, Nikov et al.reported a method for the enrichment of nitro-Tyr-containingproteins based on a first immunoprecipitation with anti-nitrotyrosine antibodies followed by a reduction of nitro-Tyr toamino-Tyr with sodium dithionite and subsequent biotintagging. Protein sample was then proteolyzed, thereby theresulting biotinylated tryptic peptides were purified on astreptavidin affinity column and identified by MS [49]. Zhang etal. introduced an improved strategy where nitrotyrosines werefirstly derivatized into free sulfhydryl groups through N-succini-midyl S-acetylthioacetate (SATA) and sulfhydryl-containingpeptides were subsequently enrichedwith thiopropyl sepharosebeads [50]. Very recently, Lee and co-workers introduced a newefficient chemical approach for the enrichment of nitratedpeptides based on incorporation of a metal chelating motif intothe modified tyrosine residues [51]. This strategy comprised aseries of chemical modifications to convert the nitro groupson the tyrosine side chains to the metal chelating groups(bispyridinylated tyrosines) followed by solid-phase extractionwith Ni2+-nitrilotriacetic acid (NTA)magnetic agarose beads [51].Interestingly, the same research team developed anotherefficient enrichment method of nitrated peptides one year later[52]. The feasibility of this newapproachwas testedwith successon in vivo model systems, providing an alternative tool fornitroproteome profiling. The method is based on fluorine-fluorine interaction affinity purification following chemicalconversions of the nitro groups on tyrosines residues to highlyfluorinated moieties [52]. Alternatively, an innovative approachinvolving dansyl chloride labelling of nitration sites that rely on

the enormous potential of MSn analysis has been reported byAmoresano et al. [53]. Briefly, discrimination between nitro- andunmodified peptides is based on two instrumental selectivitycriteria obtained by combining a precursor ion scan and an MS3

analysis. Following MS/MS fragmentation, in fact, dansyl-derivatized peptides produce a stable fragment ion at m/z 170useful for aprecursor ion scan;moreover these speciesundergoaspecific and diagnostic transition from m/z 234 to 170 in MS3,effectively providing the second selectivity criterion [53]. Inter-estingly, taking advantage of the experience made with theabove strategy, the Amoresano's laboratory also developed ananalysis method for the simultaneous localisation and quanti-fication of 3-NT residues in proteins, by exploiting the potentialof iTRAQ reagents [54].

2.3. Carbonylations

After the reactions involving the sulphur-containing aminoacids, carbonylation is the most commonly occurring oxidativeprotein modification. Lysine, arginine, proline, and threonineside-chains canbe oxidatively converted to reactive aldehydeorketonegroups (carbonylation) causing inactivation, crosslinkingor breakdown of proteins [55,56]. 2-Amino-adipic semialdehyde(AAS) and gamma-glutamyl semialdehyde (GGS) are the mostabundant carbonyls in aged cells [57]. Multiple methods havebeen described for selection and recognition of carbonylatedproteins, all of which exploit the relatively unique property ofcarbonyl groups to form Schiff bases. Affinity chromatographycan be used to select oxidized proteins upon derivatization ofcarbonyls through dinitrophenylhydrazine (DNPH) or biotinhydrazide (BHZ). This protocol ends up greatly enrichingoxidized proteins or their proteolytic fragments in the process.

After a convenient method was developed for detectingprotein carbonyls on PVDF membrane [58], electrophoreticand proteomic analyses of carbonylated proteins in OS-relatedpathologies have been extensively carried out bymany groups[59–66]. As a rule of thumb, proteomic analysis of carbonylatedproteins through BHZ tagging have been achieved in threedifferent ways: (i) performing tryptic digest of biotinylatedsamples followed by affinity selection and RP-LC-MS/MSidentification; (ii) targeting native proteins with subsequentaffinity selection, proteolysis and RP-LC-MS/MS identification;(iii) further fractionating affinity selected biotinylated proteinsby LC before proteolysis and identification of peptidefragments. Interestingly, Mirzaei and Regnier [67] comparedthese three different strategies. They found that performingthe affinity selection and chromatography at the protein levelbefore proteolysis and MS protein identification is moreinformative, due to the possibility of detecting also cross-linked or truncated protein forms, thus expanding the level ofstructural discrimination on the separation side. This strategyis not restricted to liquid chromatography, as 2DE can also beused. Unfortunately, DNPH derivatization changes theisoelectric point of proteins and can lead to sample lossduring fractionation. One way to deal with this problem is bystarting the fractionation with isoelectric focusing, thenderivatizing with DNPH, and finally going to molecular weightseparations [68]. However, as previously mentioned above,limitations of 2DE are low sensitivity, poor reproducibility, andlimited dynamic range.


For a more detailed and exhaustive description of thetechniques above-mentioned, the reader is invited to refer tospecialized reviews [e.g. 24,26,69–71].

3. Human diseases and oxidative stress: earlyhints from proteomics

ROS represent a fundamental asset to immune cells in immunesystem responses against pathogens [72] and also play aphysiological key role in normal plant cell physiology bytriggering specific cascades [73]. Nonetheless, in humans OS isalso involved in many diseases (Table 2). Examples includeatherosclerosis/vascular aging [74], heart failure andmyocardialinfarction [75], nervous system diseases, such as Parkinson'sand Alzheimer's disease (AD) [76], muscular dystrophy [77],fragile X syndrome [78] and chronic fatigue syndrome [79], butshort-termOSmay also be important in prevention of aging [80]through the induction of a process named mitochondrialhormesis (or mitohormesis) [81]. In parallel, as treatments forthe diseases of youth and middle age have helped raise lifeexpectancy significantly, OS has been growingly tied to agingthrough the gradual decline of cognitive capacity [82]. As it hasprogressively emerged, the central nervous system appears tobe an eligible target for irreversible damage provoked by ROS.

In recent years, a role for redox proteomics has progres-sively emerged in delving into alterations redox poise whichtarget protein species in several diseases. However, onlypreliminary results are currently available, as it will bediscussed as follows.

3.1. Nervous system and OS

Due to its high rate of oxygen utilization, high content ofunsaturated lipids and relative lack of antioxidant enzymes,the brain is very vulnerable to free radical damage. Indeed, OS

Table 2 – Human health and oxidative stress.

Disease/pathological condition

Central nervous system Neurodegenerative diseases(Parkinson's, Alzheimer's,Huntington's diseases,amyotrophic lateral sclerosis)

AltereubiqupeptidtoxiciGSH oalteraPRDXinducdismu

Behavioural and mood disorders(depression, schizophrenia,psychosis, anxiety)

GSH-poxida

Vascular aging Atherosclerosis, vascularreperfusion injury, ischemia,hypoxic stress

Nitriclipoxymitocperoxof Mndysfuchain

Ageing and cancer Cellular/tissue/organ aging; cancer; Free rimpai

is associated with the onset and pathogenesis of severalprominent central nervous system disorders [83]. Along withneurotrophic support, a series of dramatically widespreadnervous system diseases, such as Alzheimer's, Parkinson'sand Huntington's disease are thought to be triggered byoxidative damage [84]. OS can cause down-regulation ofneurotrophic factors.While normal functioning of the nervoussystems involves a positive feedback loop between antioxi-dant processes and neurotrophic support, breakdown of thisfeedback loop ultimately leads to diseased states [84].

Amyotrophic lateral sclerosis rightfully belongs to the groupof the most relevant neurodegenerative syndromes worldwide.Again, the relationship between protein aggregation and themolecularevents leading toneurodegenerationhasnotyet beenexhaustively clarified, although it seems to involve mitochon-drial dysfunction and thus, oxidative damage resulting inmutated and/or misfolded proteins [85].

Accumulating data indicate that OS plays a major role in thepathogenesis of multiple sclerosis as well. ROS generated inexcess primarily by macrophages, have been implicated asmediators of demyelination andaxonal damage inbothmultiplesclerosis and experimental autoimmune encephalomyelitis [86].These preliminary observations made it desirable to pursuitantioxidant treatments against this disease, although theirefficacy has yet to be fully demonstrated [87].

OS to the central nervous system also leads to a wide arrayof behavioural disorders, such as anxiety [88], depression [89],bipolar disorders and schizophrenia [90].

An increasing number of individuals regularly consume adiet high in fat, with high-fat diet consumption known to besufficient topromotemetabolic dysfunctiondue to an increasedsusceptibility to OS, although the links between high-fat dietconsumption, aging and brain aging, in particular, are only nowbeginning to be elucidated. Analogous considerations could bemade for a wide array of metabolic disorders (type II diabetis,obesity, etc.).

Oxidative stress alterations References

d protein nitrosylation, carbonylationitination/degradation, (amyloid betae, glutamine synthase, etc.); nitric oxidety; DNA and lipid oxidative damage; MDA;xidation; Complex I electron transport/tion; oxidation of NADH;

[24,25,27,28] (AD)

1, 2, 6 and glutathione peroxidasestion; mitochondrial Cu,Zn-Superoxidetase 1 mutations;

[29,32,33] (PD)[38,39] (HD)[51,63] (ALS)

x, SOD, CAT activities; MDA; altered peripheraltive stress (erythrocytes, platelets, plasma);

[67,68,73–75]

oxide homeostasis; xanthine oxidase;genase; NADPH oxidase; alteredhondrial redox homeostasis;ynitrite-mediated nitration and inhibitionSOD; decline in GSH content; Nrf2/AREnction; dysfunctional electron transport; p66Shc

[78–81]

adical accumulation; aerobic glycolysis;red electron transport (mitochondria);

[126,129] (ageing)[88] (cancer)


3.1.1. Alzheimer's, Parkinson's and Huntington's disease

3.1.1.1. Alzheimer's disease. While life expectancy hasincreasedover the last century, cognitivedeclineandprogressivememory loss due to gradual neurodegeneration has emerged asone of the greatest health threats of old age, with nearly 50% ofadults over the age of 85 afflicted with AD. Developingtherapeutic interventions for such conditions demands a greaterunderstanding of the processes underlying normal and patho-logical brain ageing. In this respect, the emerging field ofneuroproteomics promises to provide powerful strategies forfurther characterizing neuronal dysfunction and cell lossassociated with neurodegenerative diseases [91]. Recentadvancements in the biology of ageing in model organisms,together with molecular and systems-level studies of the brain,are beginning to shed light on these mechanisms and theirpotential roles in cognitive decline [82].

Oxidative damage can lead to several events targeting, lipids,carbohydrates, DNA, RNA and proteins. As for the latter, the lossin specific protein function, abnormal protein clearance, deple-tion of the cellular redox balance end up interfering with the cellcycle, and, ultimately, lead to neuronal death. Protein carbonyls,a marker of protein oxidation, are increased in AD brain [92].Some preferential protein targets have been identified throughpreliminary immunochemistry investigations, such as creatinekinase BB and beta-actin [51,52], while others (glutaminesynthase, and ubiquitin carboxy-terminal hydrolase L-1) haverecently been added to the list through widespread redoxproteomics approaches (2DE, western blot through anti-gluta-mine synthase antibodies, MALDI-TOF) [92]. Along with thesemarkers, amyloid β-peptide (1-42) has been shown to induceOSand neurotoxicity in vitro and in vivo [93]. Indeed, AD patho-physiology is characterized by the presence of extracellularsenileplaques, intracellularneurofibrillary tangles, andsynapseloss, the formerbeing composed of an amyloidbetapeptide (Aβ)core surrounded by dystrophic neurites. Aβ peptide is a 39-43amino acid peptide that is derived from the proteolyticprocessing of amyloid precursor protein (APP), a ubiquitouslyexpressed transmembrane glycoprotein. Aβ is producedby proteolytic cleavage of APP at the amino terminus byβ-secretase and at the carboxy terminuswithin the lipid bilayerby γ-secretase. Cleavage by γ-secretase can occur at differentpositionswithin the carboxy terminus of the Aβ resulting in theproduction of Aβ peptides of varying length. Aβ1-40 and Aβ1-42constitute the majority of the Aβ peptide found in the humanbrain [93]. As a result of oxidative damage, also anomalousprotein nitrosylation (3-nitrotyrosines) at cysteine residues,carboxylations and altered composition of ubiquinatedproteins(mainly are glial fibrillary acidic and tau proteins in AD-affectedpatients) have been widely observed in AD [94,95]. Tau proteinsare also hyperphosphorylated in AD patients [95]. Theserecently gained insights might be important in providingpotential targets for drug therapy in AD.

3.1.1.2. Parkinson's disease. Parkinson's disease (PD)-affectedpatients suffer fromprogressive loss of dopaminergic neurons inbasal ganglia and the substantia nigra, accumulation of Lewybodies, as well asmorewidespread neuronal changes that causecomplex andvariablemotor andnon-motor symptoms.OSplaysa dramatic role in PD, in that it is involved in dopamine cell

degeneration and intimately linked to other components of thedegenerative process, such as mitochondrial dysfunction,excitotoxicity, nitric oxide toxicity, S-nitrosylation and inflam-mation. It is therefore difficult to determinewhether OS leads to,or is a consequence of these events [96]. Oxidative damage tolipids, proteins, and DNA occurs in PD, and toxic products ofoxidative damage, such as 4-hydroxynonenal (HNE), can reactwith proteins to impair cell viability. There is a convincingevidence for the involvement of nitric oxide that reacts withsuperoxide to produce peroxynitrite and ultimately hydroxylradicals production. The main concept involves the metabolismofdopamine,whichmightbe responsible for thehighbasal levelsof OS in substantia nigra. The degradation of dopamine bymonoamine oxidase to produce hydrogen peroxide (H2O2)further emphasized how OS might arise [97]. Enzymatic oxida-tionof dopamine toH2O2 caused increased formationof oxidizedglutathione (GSH), suggesting the occurrence of OS and impair-ment of a major antioxidant system.

Another mechanism which has recently been implicated askey to dopaminergic cell death in PD involves altered ubiquitina-tion and degradation of proteins [98]. OS can impair theseprocesses directly and products of oxidative damage, such asHNE, can damage the 26S proteasome. The ubiquitin-protea-some system and proteolytic stress underlie nigral pathology inboth familial andsporadic formsof the illness [99]. Accumulatingevidence suggests thatmutations in alpha-synuclein, that causethe protein tomisfold and resist proteasomal degradation, are atthe root of familial PD. Similarly, mutations in two enzymesinvolved in the normal function of the ubiquitin-proteasomesystem, parkin and ubiquitin C-terminal hydrolase L1, are alsoassociated with hereditary PD. Furthermore, structural andfunctional defects in 26/20S proteasomes with accumulationand aggregation of potentially cytotoxic abnormal proteins havebeen identified in the substantia nigra pars compacta of patientswith sporadic PD [99].

In PD, especially in sporadic PD, an increase in OS damageto mitochondrial components is often observed. This is linkedto systemic deficiency of the electron transport chain NADH-quinone oxidoreductase (Complex I) activity, and thus resultsin a reduced bioenergetic capacity, making it clear thatmitochondrial dysfunction lies at the root of PD [100]. Inmolecular terms, oxidation of the catechol ring of dopamineresults in the formation of ROS and the electron-deficientdopamine quinone. Exposure of mitochondria to oxidizeddopamine results in uncoupling of mitochondrial respirationand mitochondrial swelling. Electron-deficient dopaminequinones will readily bind to thiol groups on proteins, oftenresulting in inactivation of the protein function [97]. Inparallel, Bisaglia et al. have observed that the generation ofdopamine quinones in isolated respiring mitochondriatriggers the opening of the permeability transition pore,most probably by inducing oxidation of NADH [101], furtherimpairing the bioenergetic activity of mitochondria.

Concordingly, products of several PD-associated genes,including SNCA, Parkin, PINK1, DJ-1, LRRK2 and HTR2A, showa degree of localization to the mitochondria under certainconditions [78]. Genetic therapies aimed at restoring Complex Iactivity have recently been proposed. One of these approachesexploits the yeast alternative NADH dehydrogenase, the Ndi1protein, to reinstate the mitochondrial respiratory chain. The


benefits which stem from compensation for disabled complex Ithrough Ndi1 seem to include retardation of PD [102].

The role of proteomics in research on PD has recentlyexpanded, especially on animal models of PD [82]. The shifttowards a protein-oriented scenario mainly lies on theaccumulation of a plethora of data pinpointing an abnormalprocessing of the neuronal protein α-synuclein as a pivotalmechanism leading to aggregation, inclusions formation anddegeneration [103].

3.1.1.3. Huntington's disease. Huntington's disease (HD) is aneurodegenerative disorder, which is characterized by uncon-trolled choreiform movements, psychiatric symptoms andcognitive decline. Histopathological changes in HD brainsreveal considerable damage to basal ganglia, particularlyaffecting middle-sized spiny neurons from the caudate-puta-men region. Neurochemical changes are specifically oriented todeplete GABAergic and cholinergic systems, primarily causedby expansion of CAG repeats in exon 1 of the huntingtin gene,which initially affect the striatum and progressively the cortex.

Concomitant causes to the etiology of this disease includethe generation of excitotoxic events, the alteration in energymetabolism and mitochondrial dysfunction, the resulting OS,and htt aggregation [104]. These factors are strictlyinterconnected, making it difficult to understand whether OSrepresents a primary cause of neural death, or a physiologicalprocess along the way of neural death. For example, excito-toxicity involves a drastic increase in intracellular Ca2+

concentrations in response to an overexposure of neuronsexcitatory amino acids, such as glutamate and its analogues.Sudden and massive influx of Ca2+ into the neuronal cell maytrigger lethal metabolic pathways involving proteolyticenzymes (proteases and endonucleases), thus increasing theformation of ROS and RNS [105].

In a preliminary proteomic study Sorolla et al. [106] foundthat antioxidantdefenseproteins (e.g. peroxiredoxins1, 2, and6,as well as glutathione peroxidases 1 and 6) were stronglyinduced in striatum, but also detectable in cortex. The activitiesof other antioxidant enzymessuchasmitochondrial superoxidedismutase and catalase were also increased in HD against theirmatched controls. Aconitase, a protein involved in energymetabolism, showed decreased activities in striatum of HDpatients. Protein carbonyls, used as markers of OS, wereincreased in HD, and glial fibrillary acidic protein, aconitase,gamma-enolase, and creatine kinase B were identified as themain targets. Recently, the same group compared HD samplestomatched controls as to identify 13 differentially carbonylatedproteins, including enzymes involved in the glycolytic pathwayand mitochondrial proteins related to ATP production [107]. Inparticular, carbonylation of pyridoxal kinase and antiquitin 1,both involved in the metabolism of pyridoxal 5-phosphate, theactive form of vitamin B6, was linked by the Authors to thealterations in thesynthesisof glutathione,GABAanddopamine,neurotransmitters that play a key role in HD pathology.

3.1.2. Amyotrophic lateral sclerosis, multiple sclerosis andmuscular dystrophy

3.1.2.1. Amyotrophic lateral sclerosis. Along with Parkinson's,Alzheimer's and Huntington's diseases, amyotrophic lateral

sclerosis (ALS, also referred to as Lou Gehrig's disease) is aneurodegenerative disorder that results in loss of motorneurons, leading to a rapidly progressive form of muscleparalysis that is fatal [108]. There is no available cure andcurrent therapies only provideminimal benefit at best. The realproblem is the lack of in-depth knowledge of the molecularcauses of ALS, asmutations in the Cu,Zn-Superoxide dismutase1 (SOD1) gene account for only 20%of familial ALS.Nonetheless,recent evidence suggests that OS plays a central role in ALS aswell. In a recent study, Baillet et al. [109] examined OS markersof 31 patients suffering from ALS against 30 matched controls,either aiming at determining oxidation levels for lipids (mal-ondialdehyde — MDA) and proteins (plasma glutathione,carbonyls and thiols), and the activity of antioxidant enzymesi.e. erythrocyte Cu,Zn-SOD1, Glutathione peroxidase (GSH-Px)and catalase. MDA and thiols were significantly higher in ALSpatients versus control population. In parallel, a trend for anincrease in oxidized glutathione was noted in ALS patients.Moreover, univariate analysis showed that SOD activity wassignificantly decreased in ALS.

Analogously, Bonnefont-Rousselot et al. compared bloodsamples from 167 ALS patients against control subjects in orderto assess peripheral oxidation levels related to thisdisease [110].Significantly higher values of thiobarbituric acid-reactivesubstances and a significant enhancement of the erythrocyteSOD activity were observed in the ALS patients.

Anomalously mutated SOD1 proteins have been related toALS onset and mitochondrial dysfunction [111], which mightthus represent a converging point of multiple pathwaysunderlying ALS pathogenesis and progression.

Taken together, these observations emphasize the role ofOS in ALS. So far, proteomics application to ALS research hasbeen only limited to animal models [112]. Meanwhile, recentreviews highlighted an increased incidence of protein nitra-tion in a series of neurodegenerative diseases, including ALS,which mainly target MnSOD and neurofilament-L [113]. In thelight of these early results, experimentation on humansamples will represent a significant area of research for thenext few years.

3.1.2.2. Multiple sclerosis. Multiple sclerosis (MS, also knownas disseminated sclerosis or encephalomyelitis disseminata)is an inflammatory disease in which the fatty myelin sheathsaround the axons of the brain and spinal cord are damaged,leading to demyelination and scarring as well as a broadspectrum of signs and symptoms, viz hypoesthesia andparaesthesia, ataxia, dysarthria, dysphagia and many others.OS plays a major role in the pathogenesis of MS. ROS, leadingto OS, generated in excess primarily by macrophages, havebeen implicated as mediators of demyelination and axonaldamage in both MS and experimental autoimmune enceph-alomyelitis (EAE), its animal model. ROS cause damage tocardinal cellular components such as lipids, proteins andnucleic acids (e. g., RNA, DNA), resulting in cell death bynecrosis or apoptosis. In addition, weakened cellular antiox-idant defense systems in the central nervous system (CNS) inMS, and its vulnerability to ROS effects may increase damage.Thus, treatment with antioxidants might theoretically pre-vent propagation of tissue damage and improve both survivaland neurological outcome. Indeed, several experimental


studies have been performed to see whether dietary intake ofseveral antioxidants prevents or reduces the progression ofEAE. Although a few antioxidants showed some efficacy inthese studies, little information is available on the effect oftreatments with such compounds in patients with MS. Well-designed clinical studies using antioxidant intake, as well asinvestigations based on larger cohorts studied over a longerperiods of time, are needed in order to assess whetherantioxidant intake together with other conventional treat-ments, might be beneficial in treating MS [114].

3.1.3. Depression and behavioural disordersIf on the one hand OS has been increasingly related toneurodegenerative diseases, accumulating evidence suggestsfor a role of ROS and RNS in anxiety, depression, bipolardisorders and schizophrenia as well.

Anxiety is a normal emotional response to a threat orpotential threat, which becomes pathological when the fear isdisproportionate to the nature of the threat. Upon assessmentof GSH-Px SOD, CAT activities and MDA levels in 20 patientsaffected by psychiatric disorders against matched controls,Kuloglu et al. [115] established a link between OS and certainanxiety disorders (obsessive–compulsive disorder and panicdisorder), demonstrating that other systems, such as oxidativemetabolism, can affect the regulation of anxiety. Theseobservations were even more evident in patients affected bysocial phobia, while no relation between OS and post-traumatic stress disorders has been documented [116].

Conversely, there is mounting evidence of altered antiox-idant enzyme activities and increased levels of lipid peroxida-tion in schizophrenia [117,118]. Zhang et al. reported that theactivities of SOD andGSH-Pxwere decreased but levels ofMDAwere elevated in patients with a chronic form of schizophreniaas compared with normal controls [117]. SOD and GSH-Pxactivities were found to be significantly lower in paranoid andresidual subtypes compared to both disorganized subtype andthe control group. MDA levels were significantly higher in allsubtypes (paranoid, disorganized and residual groups) com-pared to the control group. Concomitantly, a decreased GSH:GSSG ratio was also found in the schizophrenic group, alongwith a negative correlation to age of both GSSG andglutathione reductase levels in schizophrenic patients, whencompared against matched controls [119].

Notably, a peripheral component to OS in psychiatricpatients has been growingly reported, starting from observa-tions of protein and lipid oxidation in erythrocytes [120],plasma [121] and platelets [122] of this category of patients.These observations are suggestive of a likely systemicoxidative unbalancewhich ultimately exerts itsmain negativeeffect, again, at the brain level.

Analogously, converging evidence suggests that systemicOS plays a critical role in the pathophysiology of depressivedisorder and associated medical co-morbidities. Indeed,nearly 40 studies have identified biomarkers of oxidativedamage in the venous peripheral blood of patients [123].Nonetheless, Teyssier et al. concluded that the pathogenic roleof the OS in the cerebral mechanism of depression cannot beinferred from the alteration of peripheral parameters, as nosignificant differences were observed in the levels of SOD1,SOD2, CAT, gluthatione peroxidase 1 (GPx1), 8-oxoguanine

DNA glycosylase, nei-like 1, methionine sulphoxide reductaseA, telomere repeat-binding factor 2 and C-FOS, when compar-ing prefrontal cortex of patients with depressive disorderagainst matched controls [123].

3.2. Vascular aging

Whereas the central nervous system represents the mostsusceptible target to OS, accumulation of OS in peripheralblood has deleterious effects on the cardiovascular system,proportionally to aging or pathophysiological conditions.

The large andmedium-sized arteries in elderly people showvarying degrees of intimal and medial change. The medialchange is known as age-related medial degeneration andsclerosis. The smoothmuscle cells in the inner half of the aorticmedia of elderly people degenerate andundergo apoptosis. Thiscauses degradation of elastin fibers and the accumulation ofcollagen fibers in themedia, but the inflammatory infiltrates arescarce [74]. Age-related decrease of elastin and its crosslinks,and an increase of collagen and its crosslink have beenpositively related to an increased likelihood of glycation(Maillard reaction) and glyco-oxidative reaction [74].

ROS accumulation under pathophysiological conditionsincreases the incidence of cardiovascular diseases. ROS arereleased from different sources, such as xanthine oxidase,lipoxygenase, NADPH oxidase, the uncoupling of nitric oxidesynthase and, in particular, mitochondria [124]. Endothelialdysfunction, characterized by a loss of nitric oxide (NO)bioactivity, occurs early on in the development of atheroscle-rosis, and determines future vascular complications.

Other factors triggering cardiovascular aging and athero-sclerosis seem to involve anomalous protein nitration, espe-cially of apolipoproteins (ApoB-100, APOE) or complement/inflammation components (C3, complement factor H; CD5,prostacyclin synthase), even if most of these preliminaryredox proteomics investigations have been performed onanimal models [113].

Although the molecular mechanisms responsible for mito-chondria-mediated disease processes are not clear, OS seems toplay an important role. In general, ROS are essential to cellfunction, but adequate levels of antioxidant defenses arerequired in order to avoid the harmful effects of excessive ROSproduction. Mitochondrial OS damage and dysfunction contrib-ute to a number of cell pathologies that manifest themselvesthrough a range of conditions [125]. Themolecularmechanismsresponsible for age-relatedmitochondrial OS in the vasculatureare multifaceted and likely involve cell-autonomous effects,including dysregulation of antioxidant defenses such as perox-ynitrite-mediated nitration and inhibition of MnSOD, decline inGSH content, Nrf2/ARE dysfunction, and a dysfunctionalelectron transport chain, as reviewed by Ungvari et al. [125].

Mitochondrial redox status and induction of apoptoticcascades appear to be regulated by p66Shc, the “longevitygene”, since it has been related to an augmented lifespan inmammals [126,127].

3.3. Aging and cancer

OSproducedbyROSactivitymightbe responsible for apparentlyantithetic biological processes, such as cellular aging and


cancer, the former leading to cellular senescence, while thelatter resulting in cellular immortalization.

Biological aging is a physiological process which representsa major risk factor in the development of neurodegenerativeand cardiovascular diseases, as well as cancer, in vertebrates[128].

During the last decades, the common view of ageing as astochastic process has been gradually replaced by the notionthat a tight regulatory system modulates the maximumlifespan of an organism, through a series of mechanismswhich involve ROS-triggered oxidative damage, and theircorrelation to life-span and incidence of age-related diseases[129,130].

While it was previously thought that a major role wasmerely played by telomere attrition [131], unbalanced regula-tion of mitochondrial ROS production appears to turn oncellular senescence programs through multifaceted mecha-nisms either involving p53 oncosuppressor or Ras oncogene,as elegantly schematized by Colavitti and Finkel [130].Nonetheless, both pathways are realistically intertwined, asit recently emerged that telomerases might also play atelomere-independent survival function. Telomerase is aribonucleoprotein that counteracts telomere shortening andcan immortalise human cells, while it has recently beenobserved that TERT, the catalytic subunit of human telomer-ase, protects human fibroblasts against OS [132].

Impaired metabolic regulation leading to mitochondria-triggered ROS production seemingly lies at the root ofneurodegenerative and cardiovascular diseases and aging,but also cancer and metabolic disorders.

Emerging evidence indicates that impaired cellular energymetabolism is the defining characteristic of nearly all cancers,regardless of cellular or tissue origin [133]. Nearly all cancersexpress aerobic glycolysis (the so-called “Warburg effect”[134]), regardless of their tissue or cellular origin. Aerobicglycolysis in cancer cells involves elevated glucose uptakewith lactic acid production in the presence of oxygen, due toimpaired oxidative phosphorylation. In parallel, numerousstudies show that, in tumour cells, mitochondria are struc-turally and functionally abnormal and incapable of generatingnormal levels of energy [135,136]. Lipid abnormalities (alteredlevels of cardiolipin) and impaired electron transport capacityultimately disrupt the regular flux of oxidative phosphoryla-tion and provoke accumulation of ROS, which gradually butinevitably leads to genomic instability and cancer progression,as Seyfried and Shelton [133] recently reviewed.

4. Drugs and OS

Due to accumulating evidence relating OS to a large number ofdiseases (see Section 3), there is a growing need for simple,convenient, and reliable markers for the assessment both invitro and in vivo of the metabolic/oxidative distress and of itsmodulation, if any, induced by the administration of pharma-ceutical products [137]. In a pharmacological perspective,drugs may be designed to target OS-specific biomarkers or toprevent their generation by aiming at blocking ROS sources;conversely, certain classes of drugs might be created to targetspecific tissues (in cancer, for example) and thereby exert a

pro-oxidant effect through local generation of ROS (Fig. 1). Inthis context, redox proteomics might result pivotal in high-lighting which are the main targets of protein oxidations andwhich biological pathways are involved or compromised bythese phenomena. While this application of proteomics todrug designing and development is but at its earliest phases,preliminary redox proteomics results have helped paving theway for further research in this field.

In the following sections a series of examples will beprovided, while referring to examples of human pathophysio-logical conditions involving OS (Section 3 and Table 3).

4.1. CNS: neurodegenerative diseases and mood/psychiatricdisorders

4.1.1. Treatment of neurodegenerative diseases throughOS-related strategiesAs it emerged from the previous section, although neurode-generative diseases such as AD, PD and HD each have distinctclinical symptoms and pathologies, they all share commonmechanisms such as protein aggregation, oxidative injury,inflammation, apoptosis, and mitochondrial injury thatcontribute to neuronal loss [138]. Therefore, in the treatmentof neurodegenerative diseases, neuroprotective agents whichtarget ROS sources and aim at preventing their generationrepresent one class of drug therapeutics of great interest in thepharmaceutical endeavour. Among them, the inhibitors oftype B monoamine oxidase, such as selegiline and rasagiline,are themost promising neuroprotective agents to date, in thatthey prevent ROS generation through themechanism reportedin Section 3.1.3 [139]. These inhibitors protect neuronal cellsagainst cell death induced in cellular and animal models. Theneuroprotective functions are ascribed to the stabilization ofmitochondria, the prevention of death signalling process andthe induction of pro-survival anti-apoptotic Bcl-2 proteinfamily and neurotrophic factors, thus counteracting mito-chondria-mediated apoptotic pathways. In cellular models,selegiline and rasagiline increased the amounts of differentneurotrophic factors classes, neurotrophins (nerve growthfactor, brain-derive neurotrophic factor, neurotrophin 3) andligands of glial cell line-derived neurotrophic factor. Studies innon-human primates and patients with PD confirmed furtherthe induction of these specified neurotrophic factors.

Alternatively, targeting specific receptors in order to inducethe cells to give an anti-oxidant/anti-apoptotic responsesmightrepresent an alternative (not yet definitive) clue to neurodegen-erative diseases. PPARgamma agonists, for example, have thepotential to modulate various signalling molecules/pathways,including matrix metalloproteinase-9, mitogen-activated pro-tein kinases, signal transducer and activator of transcription,mitochondrial uncoupling protein 2, mitoNEET expression,amyloid precursor protein degradation, beta-site amyloidprecursor protein cleaving enzyme 1 and Wnt signalling. In sodoing, PPARgamma agonists prevent mitochondria-triggeredoxidation and apoptosis [140]. Cigitazone, a PPARgammainducer, has been shown to up-regulate apolipoprotein E(ApoE)mRNA expression [141]. ApoE, the most prevalentcholesterol transport protein in the central nervous system,has also been shown to co-localize with amyloid deposits andneurofibrillary tangles in AD. This is relevant in that ApoE takes

Fig. 1 – A schematic representation of the drug intervention strategies to tackle human diseases in which oxidative stress hasproduced pathological alterations. Strategies include direct or indirect anti-oxidant activities (A), which implies that drugs aredesigned to directly scavenge free radicals or indirectly stop their production through the inhibition of pro-oxidant enzymes orthe induction of anti-oxidant enzyme expression (and/or catalyzing their activity). On the other hand, drugsmight be designedto stimulate targeted ROS/RNS production in diseased tissues (e.g. cancer tissues) (B). In this case, drugs are delivered to thetarget tissue by specific peptides (also called Trojan horse peptides). Their mechanism of action can involve either free radicalgeneration through drug active group metal-based catalysis (Fenton's reactions, for example) or rather trigger increased ofmitochondrial activity. Last box (C) shows a series of foods which have been demonstrated to contain molecules which displayan elevated anti-oxidant activity. These foods might contribute to restore the physiological status under prolonged oxidativestress, if introduced in a specific diet in parallel to drug treatment.


part in cholesterol catabolism (lipid oxidation and amyloiddeposition) andacetylcholinedysfunction, twoneuropathologiclandmarks in AD [142].

While a wide array of clinical trials are currently in progress,no effective therapy exists at themoment. Alternative strategieshave been suggested which aim at tackling OS by reducing ironlevels while not affecting basal iron metabolism, as iron playsvital roles in sustaining cellular function, other than beinginvolved in Fenton reactions from which most dangerous ROSare generated (HO). It has therefore been proposed that nutrition(low iron intake, diet restriction) and physical activity mightcontribute to fighting neurodegenerative diseases, by perhapspromoting neurogenesis (BDNF, NGF) or taking part in antioxi-dant/inflammation-related pathways (NF-kappa B pathways)[143]. In particular, nutrition might become relevant in ADpatients, who could potentially benefit from the antioxidantpotential of polyphenolic compounds obtained from dietarysources, such as anthocyanins from berries, catechins andtheaflavins from tea, curcumin from turmeric, resveratrol fromgrapes and peanuts, the dihydrochalcones aspalathin andnothofagin from rooibos and the xanthone mangiferin fromhoneybush [144]. Analogously, vitamin E supplementation hasbeen reported to be positively correlated with improved survival

in a cohort of AD patients [145], although further trials aremandatory.

Conversely, early clinical trials have hitherto failed todemonstrate a potentially positive correlation betweenomega-3 fatty acid supplementation and the management ofearly AD [146].

Pharmaceutical intervention involving drugs which directlyexert anti-oxidant activities includes coenzyme Q10 adminis-tration. CoenzymeQ(10) (CoQ(10)) is an essential cofactor in themitochondrial electron transport pathway, and is also a lipid-soluble antioxidant. CoQ(10) deficiency has been implicated inseveral clinical disorders, including, but not confined to, heartfailure, hypertension, Parkinson's disease andmalignancy. It isendogenously synthesised via the mevalonate pathway, andsome is obtained from the diet. The efficacy of CoQ(10)supplements in the treatment of neurodegenerative diseasesis currently under clinical evaluation [147].

Future strategies might involve cell-penetrating peptides(CPP), also called protein transduction domains, membrane-permeable peptides, or Trojan horse peptides, which havebeen used to promote intra-cellular delivery of heat shockproteins (HSPs) for re-folding of unfolded, misfolded ordenatured proteins in models for apoptosis, necrosis, OS,

Table 3 – Drugs and therapeutic strategies for diseases involving oxidative stress.

Strategy Drugs against disease

CNS CVD Ageing/cancer

Anti-oxidant Direct – Vitamin E [99]; –Beta blockers (carvedilol, atenolol, labetalol,metoprolol,pindolol, propranolol, sotalol, and timolol displayantioxidant activity by testing them against superoxideradical, hydrogen peroxide, hydroxyl radical,hypochlorous acid, peroxyl radical, nitric oxide, andperoxynitrate) [120]

– EUK8, EUK134 [131]: DRUGS– Coenzyme Q10 [101];

– Calcium antagonists (dihydropyridines (eg, nifedipine,nisoldipine)) [122];

– Zn(II)-glycine, and CoQ10 [141]– N-acetyl-cisteine [110]: – Vitamin E [142];– Lithium [113] – Free radical metal chelators (gold, iron,

cisplatinum, chromium, vanadium);

– Tetrathiomolybdate (copper blocker)

Indirect – Type B monoamine oxidase inhibitors (selegiline andrasagiline) [93];

– Angiotensin-converting enzyme (ACE) inhibitors,captopril and enalapril [123];

– Metformin (insulin-pathways) [131];

– PPARgamma agonists (cigitazone) [94]; – Sartans (losartan [128]);– Vitamin E, vitamin C, coenzyme Q, carotenoids,vitamin A, flavonoids, polyphenol, resveratrol,antioxidant from virgin olive oil and seleniumagainst doxorubicin (adriamicin) toxicity [146];

– Trojan horse peptides engineered to deliver HSPs inintracellular compartments [102]

– Statins (fluvastatin, atorvastatin) [127] – Bcl-2 antisense protein expression modulators(G3139), Bcl-2 antagonist Bax delivery through viralvectors, BH3 mimetic peptides [147];

– Fluoxetine and acetylsalicylic [105] or commonantidepressants sertraline, paroxetine, andescitalopram) [107];

– Honokiol and betulinic acid [148];– Phenotiazines (trifluoroperazine) and inhibition ofmitochondrial permeability [109] – Oxidative homeostasis (cisplatin; ethidium

ditercalinium, topoisomerase inhibitors) [148];Pro-oxidant Direct – Metal ion chelators bound to amyloid βeta peptide

[103]– Forcing mitochondrial respiration (lonidamine,oxamate, dichloroacetate) [148]

Indirect – Glycolysis inhibition [149]Nutraceuticals – Anthocyanins from berries, catechins and theaflavins

from tea, curcumin from turmeric, resveratrol fromgrapes and peanuts, the dihydrochalcones aspalathinand nothofagin from rooibos and the xanthonemangiferin from honeybush [98];

– Flavonoids and polyphenols [128]; – Ascorbic acid, alpha-tocopherol, carotenoids,polyphenols [141];

– Ginseng metabolites ((S)-protopanaxadiol (code nameS111)) [115];

– Resveratrol (wine, berries) [129] – Curcumin (hydrophobic polyphenol diferuloylmethane [145];

– Ascorbic acid (in parallel to fluoxetine, imipramine andbupropion) [117];

– resveratrol (wine) [145];

– Vitamin B-6, B-12, folate [118]– Polyphenols epicatechin and epigallocatechin-3-gallate from green tea [146];– Broccoli, red pepper, beans [158];– Aphanizomenon flos-aquae extracts [159]

(*CNS = Central Nervous System; CVD = Cardiovascular disease).(Antioxidant: Direct = ROS-scavenging; metal chelators; Indirect: mitochondria stabilization; co-factor in anti-oxidant radical scavenging enzymatic activity; targeting receptors upstream of anti-oxidantdefense pathways); (Pro-oxidant: Direct=stimulation of ROS generation through metal-based reactions, mitochondria stimulation, pro-apoptotic signalling; Indirect: stimulation of pro-oxidant signals,such as inhibition of mitochondrial apoptotic pathway blockade).

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neurodegenerative diseases, stroke, cystic fibrosis, smoothmuscle relaxation, myocardial injury, scar formation, andothers [148].

A completely opposite approachcouldbe basedonpromotingtargeted OS in the very regions which lie at the root of theneurodegenerative disorder, thus blocking its spread andlimiting thegravityandprogressionof thedisease in thepatients.In AD for example, metal ion chelators bound to amyloid βetapeptide can promote aggregation and OS, and can thus have alocal detrimental effect, while providing suitable targets towardsamyloid βeta peptide aggregates [149].

4.1.2. Mood and psychiatric disorders andOS-targeting approachesMood disorders such as bipolar disorder, major depressivedisorder and psychiatric disorders (schizophrenia) are com-monly treated with drugs targeting the proteins glycogensynthase kinase-3 (GSK-3) and protein kinase C (PKC), thepurinergic system, histone deacetylases (HDACs), the melato-nergic/serotoninergic system, the tachykinin neuropeptidessystem, the glutamatergic system, but also OS and bioenerge-tically-related molecules [150].

Most of the drugs which are routinely adopted for thetreatment of psychiatric disorders have not been at firstdesigned to tackle OS. Fluoxetine is one of these drugs. In thestudy by Galecki et al., anti-oxidant enzyme activities (CAT,SOD, etc.) and malondialdehyde levels were significantlyhigher in erythrocytes from 50 patients suffering from majordepressive disorders against their matched controls, althoughthese parameters were not significantly altered upon threemonths of successful fluoxetine treatment with remission ofdepression [151]. On the other hand, a combined treatmentwith fluoxetine and acetylsalicylic acid resulted in a reductionof enzymatic activity and total antioxidant status, suggestiveof a reduction in OS in treated patients [152]. These observa-tions are in agreementwith Cumurcu et al. [153], who reportedthat treatment with common antidepressants (sertraline,paroxetine, and escitalopram) reduced total oxidative statusand increased anti-oxidative capacity in treated patientsagainst non-treated patients.

A wide series of drugs primarily aimed at counteracting OShave been proposed over the last decades. Some drugs whichare routinely used in the treatment for schizophrenia,psychosis and anxiety, such as phenothiazines (PTZ), areknown toact throughstimulationofmitochondrial permeability,thus enhancing OS in target cells, although at the expenses ofincreased hepatotoxicity [154]. Conversely, trifluoperazine, apiperazinic PTZ derivative, has been reported to show antioxi-dant activity at relatively low concentrations, which has beenassociated with its inhibition of mitochondrial permeability[155].

Antioxidants, such as N-acetyl-cysteine, compounds thatmimic GPX activity, and zinc exhibit antidepressive effects[156]. In a recent study, Kumar and Garg [157] demonstratedthe efficacy of trazodone and imipramine in the treatment ofsleep deprivation-induced anxiety-like behavior and oxidativedamage in mice, through the restoration of reduced glutathi-one levels, catalase activity and attenuation of raised lipidperoxidation and nitrite concentrations as compared tountreated sleep-deprived animals.

Other drugs for the treatment of anxiety, such as venlafaxine,might be involved in NOmodulation, although themechanismsare not yet fully elucidated [158].

Another strategy involves metal ions, such as lithium,which is a mainstay in the acute and prophylactic treatmentof bipolar affective disorder through its role in the homeosta-sis of the glutathione system [159].

Major depression is characterized by significantly lowerplasma concentrations of a number of key antioxidants, suchas vitamin E, zinc ions and coenzyme Q10, and a lowered totalantioxidant status [157]. Therefore, a complementary ap-proach to drug treatments involves dietary supplementationwith antioxidant or pro-antioxidant biomolecules. CoQ10supplementation might represent a winning strategy notonly for the treatment of neurodegenerative diseases, assuggested in Section 3 [147], but also in chronic fatigue,depression and cardiovascular disorders [160].

An intestinal metabolite of ginseng, (S)-protopanaxadiol(code name S111), demonstrated antidepressant-like activityas potent as fluoxetine, while S111, but not fluoxetine,significantly reduced brain OS and down-regulated serumcorticosterone concentration in an animal model [161].However, dietary supplementation alone might not representan actual clue, since diet analyses of patients suffering frommajor depressive disorders showed that the dietary intake ofvitamin E was not related to plasma alpha-tocopherol levels,while 89% of the subjects met or exceeded the recommendedintake for vitamin E [162]. Nevertheless, administration ofascorbic acid to patients suffering from major depressivedisorders caused a synergistic antidepressant-like effect withconventional antidepressants (fluoxetine, imipramine andbupropion), through a mechanism which might involvemonoaminergic neurotransmission [163]. Finally, recentobservations hint at a long-term association of vitamin B-6,folate, and vitamin B-12 with depressive symptoms amongolder adults over time, and a high dietary intake of thesevitamins might play a protective role against depressivesymptoms [164].

4.2. Cardiovascular aging

A key role of OS is evident in the pathologic mechanisms ofendothelial dysfunction and associated cardiovascular diseases.Vascular enzymes such as NADPH oxidases, xanthine oxidase,and uncoupled endothelial nitric oxide synthase are involved inthe production of ROS. The question remains whether pharma-cologic approaches can effectively combat the excessive ROSproduction in the vasculature. Interestingly, registered cardio-vascular drugs can directly or indirectly act as antioxidants,thereby preventing the damaging effects of ROS, although theyhad not been purposely designed for that end [165].

Beta blockers (sometimes written as β-blocker) is a class ofdrugs used for various indications, but particularly for themanagement of cardiac arrhythmias, cardioprotection aftermyocardial infarction (heart attack), and hypertension. Asbeta adrenergic receptor antagonists, they diminish theeffects of epinephrine (adrenaline) and other stress hormones.Gomez et al. showed that carvedilol, atenolol, labetalol,metoprolol, pindolol, propranolol, sotalol, and timolol displayantioxidant activity by testing them against superoxide


radical, hydrogen peroxide, hydroxyl radical, hypochlorousacid, peroxyl radical, nitric oxide, and peroxynitrate, demon-strating their direct antioxidant capacity using ascorbic acid,lipoic acid, melatonin, rutin, and ebselen as positive controls[166]. Carvedilol, in particular, seems to inhibit the oxygen-free-radical-initiated lipid peroxidation and α-tocopheroldepletion in vitro in rat brain homogenate through a directscavenging activity and with an efficiency at least two ordersofmagnitude greater than that of all the other β-blockers [167].Carvedilol contains β- and α1-adrenoceptor-blocking pharma-cophore as well as a specific carbazole moiety to enhance itsantioxidant activity. Moreover, its metabolites are activeantioxidants as well [165].

Ca2+ antagonists, such as dihydropyridines (eg, nifedipine,nisoldipine) have been shown to directly reduce H2O2-induceddecreases in contractile function of rat hearts and low-densitylipoprotein oxidation [168].

Indirect antioxidant activity (pro-oxidant activity, sincethese drugs promote cellular anti-oxidant defenses) have beenobserved for two angiotensin-converting enzyme (ACE) inhi-bitors, captopril and enalapril, which feature sulfhydrylgroups [169]. The putative mechanism involves ACE inhibi-tion, which in turn is known to promote NADPH oxidase, thuspreventing O2•− radical generation [165]. A similar indirectantioxidant effect may be expected from the Ang II receptor-blockers, the “sartans”, although recent proteomics investiga-tions on platelets from moderately hypertensive patientsupon treatment with olmesartan medoxomil showed thatthe treatment did not modify the expression of someinflammation and oxidation-related protein markers (HSPs,antioxidant enzymes) [170]. On the other hand, losartanreduced the insulitis score and the intensity of nitrotyrosinestaining in a mouse model of diabetes [171].

Similarly, some of the mechanisms at the basis of thepleiotropic effects exerted by statins could be explained by anindirect antioxidant activity, through the inhibition of thecoupling of the AT1 receptor to the NADPH oxidase and thusprevention of radical production [172]. In this respect,fluvastatin and that of atorvastatin have been shown to playa strong anti-peroxyl radical activity [173].

Finally, food-derived compounds appear to be effectiveinhibitors of OS and preserve vascular function. For example,flavonoids, highly abundant polyphenols of fruits and vege-tables, appear to play beneficial cardiovascular effects, as anelevated intake of fruits has been correlated to decrease bloodpressure in humans [174]. Flavonoids effect on ET-1 synthesisor arginase inhibition increases the availability of L-arginine,which forms the rate-limiting factor for cellular NO• produc-tion. It has further been proposed that the prevailing mode ofaction of flavonoids is inhibition of NADPH oxidase, thuslowering the O2•− generation that leads to elevation of NO•

levels in the cell and thus vasodilatatory effects [175].Analogously, resveratrol from wine, peanuts and berries,

existing in both cis- and trans-isomeric forms, has beenshown to act moderately both as a direct and indirect anti-oxidant, through regeneration of alpha-tocopherol [175]. Forthese reasons, resveratrol is believed to decrease circulatinglow-density lipoprotein cholesterol levels and reduce thecardiovascular disease risk, although convincing evidence isstill lacking.

4.3. Aging and metabolism

Early attempts at antioxidant intervention as ameans to delayaging were initiated soon after the free radical theory of agingwas proposed. These attempts stemmed from the postulationof the free radical theory of ageing which posits thataccumulation of oxidative damage underlies the increasedcellular, tissue and organ dysfunction and failure associatedwith advanced age. However, these anti-oxidant interventionshave so far failed to extend life span in most cases [176,177].

A series of encouraging, albeit preliminary results havebeen reported in C. elegans and Drosophila through the use ofenzymatic synthetic drugs miming SOD and CAT activities,such as EUK-8 and EUK-134 [177]. However, while increasinganti-oxidant defenses in these organisms, the drugs did notproduce any significant increase of the lifespan.

The direct impact of antioxidant enzyme treatment on lifespan is even more controversial in mammalian models. Forinstance, transgenic mice that constitutively over-expresshuman CuZn-SOD did not live longer than control animals[178], while heterozygous mice with reduced MnSOD activityhavea life expectancy that is similar towild-typemice, althoughthese animals have increased oxidative damage to DNA [179]. Itis gradually becoming clear that antioxidant enzyme activitiesare not broadly correlated with longevity, as Page et al.concluded in their investigation on 14 mammalian and avianendotherm species with maximum life spans ranging from3 years to over 100 years [180]. Analogously, maintenance ofprotein homeostasis through up-regulation of protein repairingand recycling machinery (glutaredoxin, thioredoxin reductase,etc.) is not associated with extended life spans in 15 vertebratespecies [181]. Therefore, if free radicals are actually correlated toaging, a winning strategy should be targeted at preventing theirproduction rather than increasing defenses and repairingmechanisms against ROS-induced damages. TheMitochondrialFree Radical Theory of Aging (MFRTA) proposes that mitochon-drial free radicals, produced as by-products during normalmetabolism, are the major responsible of oxidative damage.According to MFRTA, the accumulation of these oxidativephenomena is the main driving force in the aging process.Although widely accepted, this theory remains unproven,because the evidence supporting it is largely correlative. Inthis view, restingmetabolismshould represent anadvantage onthe road to life span improvement. Nonetheless, moderateexercise produces changes in free radical production andoxidative damage without altering maximum life expectancy[182].

Ristow and Zarse recently reviewed accumulating evidencereporting that caloric restriction and specifically reducedglucose metabolism induces mitochondrial metabolism toextend life span in various model organisms, includingSaccharomyces cerevisiae, Drosophila melanogaster, Caenor-habditis elegans and possibly mice [71]. In conflict withHarman's free radical theory of aging, these effects may bedue to increased formation of ROS within the mitochondriacausing an adaptive response that results in increased stressresistance assumed to ultimately cause a long-term reductionof OS. Concordingly, it has been observed that hyperactivemice live longer than controls [183]. This type of retrograderesponse has been named mitochondrial hormesis or


mitohormesis, and may in addition be applicable to thehealth-promoting effects of physical exercise in humansand, hypothetically, impaired insulin/IGF-1-signalling inmodel organisms [71].

Recent findings shed further light on the strong linkagebetweenagingandmetabolism, openingbrandnewscenarios inthe field of drug discovery [184]. Metformin, a biguanide drugcommonly used to treat type-2 diabetes, has been noted toextend lifespan of nondiabeticmice andC. elegans [185]. Insulin-like signalling inC.elegansactivates the transcription factorSNK-1, which is known to defend against oxidative stress bymobilizing the conserved phase 2 detoxification response andit is thus referred to as the longevity-promoting factor [186].

While ageing per se still remains a controversial issue, goodresults have been obtained in the field of cosmesis, as far as skinaging is concerned. Recent advances in anti-oxidant drugdevelopmentagainst skinaginghavebeenextensively reviewed[187]. A role has been proposed for ascorbic acid, alpha-tocopherol, carotenoids, polyphenols and other substances,such as ergothioneine, Zn(II)-glycine, and CoQ10 in the treat-ment of skin aging [187]. In particular, topical application ofCoQ10 and antioxidants like alpha-glucosylrutin diminishedresistance in keratinocytes of old donors against UV irradiation(photoaging) either in vitro or in vivo study [188].

4.4. Cancer

Over the last decades, most of the efforts of the pharmaceu-tical establishment have focussed on designing and testingeffective drugs against cancer progression. As many noted[189], cancer shares some fundamental aspects with ageing.Cancer cells often display altered metabolism (aerobic glycol-ysis) and are rather prone to suffer from OS-induced injuries,often intertwined with the likelihood of accumulation ofgenetic mutations. These considerations have paved the wayfor the development of specific drugs aimed at preventing OS(in order to reduce the accumulation of genetic mutations),fighting it (to counteract ROS production induced by metabo-lization of chemotherapeutics or chelating radicals throughmetal-based drugs) or promoting it (thus exploiting ROSagainst OS-sensitive cancer cell populations).

Vitamin E (gamma-tocopherol in particular) belongs to thefirst group of biomolecules, as it represents a promising assetin prevention treatments for several cancers, including colon,prostate, mammary and lung tumorigenesis in animalmodels[190]. Analogously, curcumin, a hydrophobic polyphenolderived from rhizome (turmeric) of the herb Curcuma longacommonly called diferuloyl methane, may be used aschemopreventive or chemoprotectant, due to its antioxidantproperties. Besides, curcuminmay potentiate the anti-tumoureffect of gemcitabine [191].

Anthracyclines (doxorubicin, daunorubicin, epirubicin, andidarubicin) are currently the most effective group of anti-neoplastic drugs used in clinical practice. Of these, doxorubicin(also called adriamycin) is a key chemotherapeutic agent incancer treatment, although its use is limited as a consequence ofthe chronic and acute toxicity associated with this drug. Themolecularmechanisms of doxorubicin account for both the anti-cancer and the toxic side effects. Many antioxidants have beenassayed, with positive or negative results, to prevent the toxicity

of doxorubicin. In particular vitamin E, vitamin C, coenzyme Q,carotenoids, vitamin A, flavonoids, polyphenol, resveratrol,antioxidant fromvirgin olive oil and seleniumhave been assayedas to see whether (and to which extent) they might be used asantioxidant co-adiuvant in chemotherapeutic treatments [192].

An opposite strategy might involve drugs which aim atproducing ROS with specificity to the cancer tissue. Targetingcancer cell mitochondria, through Bcl-2 family members whichallow increasing mitochondrial membrane permeability inorder to have them generate suicidal signals, might reveal awinning strategy [193]. These strategies include Bcl-2 antisenseprotein expression modulators (G3139), Bcl-2 antagonist Baxdelivery through viral vectors, BH3 mimetic peptides (reviewedin [194]), which have been thought to trigger pro-apoptoticcascades mediated by cytochrome c release and ROS-induceddamage in target cells. Permeabilization of mitochondrialmembranes could be also achieved through honokiol andbetulinic acid [195], while other strategies target mitochondrialDNA in order to prevent expression of specific respiratoryproteins and thus alter the oxidative homeostasis (cisplatinum;ethidium ditercalinium, topoisomerase inhibitors) [194]. Anemerging field of research takes into account the quasi-exclusiveness of the Warburg effect metabolism in tumourcells and thus aims at manipulating metabolic pathways fortherapeutic purposes. Relevant studies include approaches ofglycolysis inhibition or forcing mitochondrial respiration (loni-damine, oxamate, dichloroacetate) [194,195].

Alternatively, mitochondria could be targeted as to preventangiogenesis which is necessary to sustain tumour growthand progression through nutritional and oxygen support [196].

Various metal complexes are currently used as therapeuticagents (e.g., Pt, Au, and Ru), due to their capacity for generatingradical species (Fentonchemistry) in the treatmentofmalignantdiseases, including several types of cancers [197,198]. Loweringthe metal concentrations might reduce the OS potential bylimiting metal-triggered ROS production and thus counteractmalignant transformation and tumour growth and progression.In this view, the potential role of proteomics in target discoveryand validation, metal-based drug design and development hasbeen reviewed byWang and Chiu [197]. Brewer's group tested adrug known as tetrathiomolybdate, which binds dietary copperbefore it can be absorbed by the body and thus reduces copper-triggered DNA and lipid peroxidation, in patients with differenttypes of metastatic cancer. The drug was observed to actthrough the inhibition of cytokines, NfKB-pathways andangiogenesis [199].

The non-metal element selenium has been observed toplay a protective role against several types of cancers, mainlyprostate cancer, through a likely pro-oxidative rather thananti-oxidant activity [200], although proper prospective stud-ies are currently under development.

Analogous considerations could bemade for some nutraceu-ticals, that is to say a food or food product that provides healthandmedical benefits, including the prevention and treatment ofdisease. Resveratrol from red wines [201], polyphenols epicate-chin and epigallocatechin-3-gallate from green tea [202] or oliveoil phenolic extracts [203] might hold relevant clinical implica-tions due to their likely pro-oxidant role in sensitizing cancercells specifically. Nonetheless, most of the nutraceuticals exerttheir activity mainly by counteracting OS and free radicals, such


as in the case ofmany compounds from vegetables (broccoli, redpepper and beans) [204], which might be helpful in theprevention of OS-induced damages caused by chemotherapictreatments (such as Aphanizomenon flos-aquae extracts atten-uating cisplatinum-induced renal dysfunction [205]).

5. Conclusions

Redox homeostasis maintenance and modulation is a funda-mental physiological process, which is compromised inseveral pathophysiological events of importance in humanhealth. In this context, redox proteomics holds the promise toshed further light on these pivotal biological processes. Whilepreliminary investigations have already contributed preciousdata, we are still far from looking at thewhole picture in its fullcomplexity. An increasing number of techniques are availablewhich aim at investigating, either qualitatively or quantita-tively, modifications to specific amino acids (cysteins,tyrosines, etc.) or specific groups (carbonylations, nitrosyla-tions, etc.). In this review, the mechanisms of ROS and RNSproduction have been reviewed, mainly addressing thosechemical phenomena which have already been associatedwith pathological conditions (of the central nervous system,cardiovascular system, or simply related to aging and altered-cell cycle regulation). In these fields, redox proteomics has yetto exhaustively contribute, although the increasing number oflaboratories committed in these research endeavours arguesfor big strides in the upcoming years. In particular, redoxproteomics might represent a valid tool to explore protein-targeting oxidative modifications prior to or upon drugadministration, which could greatly contribute either toaccumulate further basic knowledge or to produce thetranslational know-how which is necessary to speed thewhole process of drug designing and testing up. Nonetheless,only preliminary research has been so far performed, mainlylimited to animal models.

For the foreseeable future, redox (and quantitative [206])proteomics will likely play a pivotal role in the quest for newtherapeutical targets and their validation, in the process ofdetermining OS-triggered cellular alteration upon drug treat-ments and thus in the very heart of the design and testing ofnew drugs and their metabolites against those pathologiesrelying on altered redox homeostasis. Redox-proteomicscurrently represents a breakthrough into the virgin forest ofdrug discovery and development, which is, nonetheless, yet tobe fully explored. So far, therapeutic interventions have beenthought to counteract oxidative stress (both directly andindirectly) or rather to promote it, strictly targeting in situ thearea of pathological/diseased tissues. New redox-based drugs,like golden coins, have been sparsely found along the way.However the actual treasure trove has yet to be opened.

AbbreviationsAD Alzheimer's diseaseALS amyotrophic lateral sclerosis
BHZ biotin hydrazideCNS central nervous systemDNPH dinitrophenylhydrazineHD Huntington's disease
HSP heat shock proteinIAA iodoacetamideICAT isotope-coded affinity tagMDA malondialdehydeNEM N-ethylmaleimideNO nitric oxideOS oxidative stressPD Parkinson's diseasePTZ phenothiazinesRNS reactive nitrogen speciesROS reactive oxygen speciesSOD1 Superoxide dismutase 1TCA trichloroacetic acid

Acknowledgments

The authors were supported by funds from the researchprograms “GENZOOT” and “Nutrigenomica mediterranea: dallanutrizionemolecolare alla valorizzazione dei prodotti tipici delladieta mediterranea - NUME” (Italian Ministry of Agricultural,Food and Forestal Politics - MIPAF) and from the Italian NationalBlood Centre (Centro Nazionale Sangue – Istituto Superiore diSanità - Rome, Italy).

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Redox proteomics and drug development - UCCS Home · Redox proteomics and drug development Angelo D'Alessandro, Sara Rinalducci, Lello Zolla⁎ Department of Environmental Sciences,