The chemical foundations of nitroalkene fatty acid signaling through addition reactions with thiols

Accepted Manuscript The chemical foundations of nitroalkene fatty acid signaling through addition reactions with thiols Lucía Turell, Martina Steglich, Beatriz Alvarez PII:

S1089-8603(17)30305-1

DOI:

10.1016/j.niox.2018.03.014

Reference:

YNIOX 1767

To appear in:

Nitric Oxide

Received Date: 7 November 2017 Revised Date:

19 March 2018

Accepted Date: 21 March 2018

Please cite this article as: Lucí. Turell, M. Steglich, B. Alvarez, The chemical foundations of nitroalkene fatty acid signaling through addition reactions with thiols, Nitric Oxide (2018), doi: 10.1016/ j.niox.2018.03.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT The chemical foundations of nitroalkene fatty acid signaling through addition reactions with thiols

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Lucía Turella*, Martina Steglicha, Beatriz Alvareza

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Laboratorio de Enzimología, Facultad de Ciencias and Center for Free Radical and BiomedicalResearch, Universidad de la República, Montevideo, Uruguay

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To whom correspondence may be addressed: Laboratorio de Enzimología. Facultad de Ciencias. Iguá 4225, Montevideo, Uruguay 11400.Tel.: + (598) 2 525-0749; E-mail: [email protected]

ACCEPTED MANUSCRIPT Abstract

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Nitroalkene fatty acids can be formed in vivo and administered exogenously. They exert pleiotropic signaling actions with cytoprotective and antiinflammatory effects. The presence of the potent electron withdrawing nitro group confers electrophilicity to the adjacent β-carbon. Thiols (precisely, thiolates) are strong nucleophiles and can react with nitroalkene fatty acids through reversible Michael addition reactions. In addition, nitroalkene fatty acids can undergo several other processes including metabolic oxidation, reduction, esterification, nitric oxide release and partition into hydrophobic compartments. The signaling actions of nitroalkenes are mainly mediated by reactions with critical thiols in regulatory proteins. Thus, the thio-Michael addition reaction provides a framework for understanding the molecular basis of the biological effects of nitroalkene fatty acids at the crossroads of thiol signaling and electrophilic lipid signaling. In this review, we describe the reactions of nitroalkene fatty acids in biological contexts. We focus on the thio-Michael addition reaction and its mechanism, and extrapolate kinetic and thermodynamic considerations to in vivo settings.

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Keywords: Nitroalkene fatty acid, Michael addition, elimination, thiols, signaling

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Abbreviations: NO2-OA, nitro-oleic acid; NO2-LA, nitro-linoleic acid; NO2-AA, nitroarachidonic acid; NO2-CLA, nitro-conjugated linoleic acid; GSH, glutathione; NF-κB, nuclear factor κB; Keap1, Kelch-like ECH-associated protein 1; Nrf2, nuclear factor (erythroid-derived 2)-like 2; HSF-1, heat shock factor 1; GAPDH, glyceraldehyde 3phosphate dehydrogenase; HSA, human serum albumin.

ACCEPTED MANUSCRIPT 1. Introduction

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Nitroalkene fatty acids are unsaturated fatty acids that contain a nitro group adjacent to a double bond. They can be formed endogenously through the initial addition of nitrogen dioxide (NO2·) to an unsaturated fatty acid. Nitrogen dioxide originates from nitrite (NO2-) under acidic conditions, from nitric oxide (NO·) autooxidation, from peroxynitrite (ONOO-) decay or from peroxidase-mediated nitrite oxidation. Thus, the nitration of fatty acids is favored in the contexts of digestion and inflammation. The molecular pathways leading to nitroalkene fatty acid formation are the topic of another review in this series [1–3]. Nitroalkene fatty acids are able to exert pleiotropic signaling actions, usually through mechanisms that involve the post-translational modification of regulatory proteins [4]. Protective effects have been reported in a wide range of disease models such as atherosclerosis, restenosis, ischemia reperfusion, renal injury, diabetes, metabolic syndrome and endotoxemia [5–11]; and their endogenous formation has recently been associated with the cardioprotective benefits of the Mediterranean diet [12,13]. Nitroalkene fatty acids can also be administered exogenously and show promise as pharmacological agents. In this review we describe the reactions of nitroalkene fatty acids in biological contexts. We focus on the Michael addition-elimination reaction with thiols, a reaction that is in the basis of their signaling effects. 2. Nitroalkene fatty acids are electrophiles and react with thiols

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The structures of the best characterized nitroalkene fatty acids are shown in Fig. 1A-D. The electron withdrawing nitro group renders the β-carbon electron deficient and thus susceptible to attack by nucleophiles through Michael addition reactions [4] (Fig. 2). Electrophilicity is the main feature to account for the biological effects of nitroalkene fatty acids, as evidenced by the inhibitory effects of the nitroalkene reductase activity of prostaglandin reductase-1, an enzyme able to reduce fatty acid nitroalkenes to nitroalkanes [14]. Nitro-oleic (NO2-OA), nitro-linoleic (NO2-LA) and nitro-arachidonic (NO2-AA) acids contain one or more double bonds flanked by methylene groups and have only one electrophilic center. In contrast, nitro-conjugated linoleic acid (NO2-CLA) has two conjugated double bonds and has thus two non-equivalent electrophilic centers located in the β- and δ-carbons [15]. Importantly, conjugated linoleic acid is a preferential substrate for biological nitration and yields considerably more nitrated products than bis-allylic linoleic acid [16]. Thiols are organosulfur compounds with a sulfhydryl group bound to a carbon atom. In biological systems, thiols are found in cysteine and derived molecules of low and high molecular weight. The tripeptide glutathione (GSH) is present in the cytosolic compartment at concentrations of 2-17 mM and is mostly reduced. Protein thiols are even more abundant than GSH (10-50 mM) [17–20]. For most chemical and enzymatic reactions of thiols, reactivity involves the nucleophilic attack of the ionized thiolate (RS−) on an electrophile. Due to the negative charge, to the relatively low electronegativity of sulfur and to the high polarizability, thiolates are excellent nucleophiles. However, at a certain pH, only a fraction of the total thiol is ionized to thiolate. For example, the thiol in GSH has a pKa of 8.94 [21], so at pH 7.4, only 2.8% is available for reaction. Accordingly, the observed rate constant for a certain reaction increases as the pH increases because more thiolate is available. The pKa values of

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different thiols change depending on the particular molecular context. For instance, the proximity of a positive charge decreases the pKa value of a thiol. If we compare different compounds, as the pKa of the thiol decreases, more thiolate is available for reaction at a fixed pH. This has led to the misconception that reactive cysteines have low pKas. Although acidic thiols have increased thiolate availability at a certain pH, the intrinsic reactivity of the thiolate tends to diminish, because nucleophilicity depends on the electron density of the reacting atom, which actually decreases with acidity. Indeed, for several thiolate reactions, the intrinsic rate constants increase as the pKa increases. Nevertheless, in proteins, the environment where the thiol is located can modulate the nucleophilicity towards specific substrates. In some cases, the reactivity is increased towards some electrophiles but decreased towards others. Furthermore, this modulation can be achieved independently of the pKa [21,22].

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Figure 1. Structures of some nitroalkene fatty acids. A, Nitro-oleic acid; 9- and 10-nitrooctadec-9-enoic acid (9and 10-NO2-OA). B, Nitro-linoleic acid; 9-, 10-, 12- and 13-nitrooctadec-9,12-dienoic acid (9-, 10-, 12- and 13NO2-LA). C, Nitro-arachidonic acid, four mononitrated isomers; 9-, 12-, 14- and 15-nitroeicosa-5,8,11,14tetraenoic acid (9-, 12-, 14- and 15-NO2-AA). D, Nitroconjugated linoleic acid; 9- and 12-nitrooctadeca-9,11dienoic acid (9- and 12-NO2-CLA).

The kinetics of the reaction between three nitroalkene fatty acids (NO2-OA, NO2-LA and NO2-CLA) and several thiols have been studied (Table 1). The rate constant for the addition of GSH to NO2-OA is 64 M-1 s-1 (pH 7.4, 25 ºC). The product of thiol addition to a typical nitroalkene is a thioether-substituted nitroalkane (Fig. 2). This adduct can undergo an elimination reaction to regenerate thiol and nitroalkene (the reverse reaction in Fig. 2). The rate constants for the elimination reaction of the GSH adduct of NO2-OA is 6 x 10-3 s-1 [15,23]. From the ratios of the reverse and the forward rate constants, the equilibrium dissociation constant can be calculated as 90 µM [15]. As a point for comparison, for the three nitroalkene fatty acids evaluated, the rate constants

ACCEPTED MANUSCRIPT with GSH are 1-2 orders of magnitude higher than those of hydrogen peroxide with GSH (0.42 M-1 s-1, pH 7.06, 25 ºC, [24]).

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Figure 2. Reversible Michael addition elimination reaction between a thiolate and a nitroalkene.

β addition

NO2-CLA

Glutathionea a

konδ (M-1 s-1)

koffδ Keqδ pKa (s-1) (x10-4) (M) (x10-4)

34 ± 4

0.10 ± 0.02

2.8 ± 0.9

3.5 ± 0.5

3±1

0.9 ± 0.4

8.94c

0.196 ±0.002

6.0 ± 0.1

2.8 ± 0.4

6 ±3

2±1

8.29c

0.03± 0.02

2±1

1.4 ± 0.2

1 ±1

1±1

9.10c

0.28 ± 0.06

5±2

10 ± 2

10 ± 5

1.0 ± 0.7

7.95c

0.019 ± 0.005

1.3 ± 0.6

3.0 ±0.4

0.6 ± 0.5

0.2 ± 0.2

9.60c

~50

n. d.

0.09 ± 0.02

Absent

Absent

n.d.

Absent

Absent

n.d.

Absent

Absent

18.9 ± 0.5 51 ± 4 a

15 ± 2

Thionitrobenzoatea

~800

Glutathionea

64 ± 1 183 ± 6

Cysteineb

267 ± 33

Homocysteine

n. d.

(6 ± 1) x 10-3

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Glutathione

b

100 ± 2

n.d.

Absent

Absent

Cysteine methyl esterb

442 ± 9

n.d.

Absent

Absent

6.7d

Penicillamineb

71 ± 5

n.d.

Absent

Absent

7.9d

28 ± 2

n.d.

Absent

Absent

9.74c

13 ± 3

n.d.

Absent

Absent

10.7d

Glutathioneb

355 ± 5

n.d.

Absent

Absent

Cysteineb

522 ± 36

n.d.

Absent

Absent

N-Acetyl cysteineb

b

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Dihydrolipoic acid

a

Keqβ (M) (x10-3)

Homocysteinea

b

NO2-LA

koffβ (s-1)

32.6 ± 0.2

β-Mercaptoethanol NO2-OA

konβ (M-1 s-1)

Cysteine

Cysteinylglycinea

δ addition

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Nitroalkene Thiol fatty acid

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Table 1. Apparent rate and equilibrium constants at pH 7.4 of the reaction of nitroalkene fatty acids with low molecular weight thiols

b

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determined at 25 ° C, reported in [15]; determined at 37 °C, reported in [25]; reported in [24]; d reported in [26]; n.d. not determined

c

In contrast to the monophasic kinetics observed for NO2-OA and NO2-LA, in the case of NO2-CLA the kinetics are biphasic due to the presence of two electrophilic centers [15,25]. The β-adduct is formed faster (kinetic product) while the δ-adduct is more stable (thermodynamic product). The electron withdrawing potency of the nitro group is reflected in the rate constants for thiolate addition to nitroalkenes, which are higher than those of compounds containing other electron-withdrawing groups. For example, the rate constants for the addition of GSH to 4-hydroxynonenal and 15-deoxy-12,14prostaglandin J2 are 1.33 M-1 s-1 (pH 7.4, 23 ºC) [27] and 0.7 M-1 s-1 (pH 7.4, 37 °C) [25], respectively, while the corresponding value for NO2-OA is 64 M-1 s-1 (pH 7.4, 25 °C) [15] (Tables 1 and 2). A comparison of the rate constants for GSH addition to a series of Michael electrophiles shows the sequence of decreasing reactivity nitro > carbonyl or sulfone>>sulfoxide, nitrile or amide [28].

ACCEPTED MANUSCRIPT Table 2. Selected lipid-derived electrophiles: rate constants with glutathione at pH 7.4 and plasma concentration in health.

a

kon (M-1 s-1) 64a

koff (s-1) 6 x 10-3a

Keq (M) 9 x 10-5

34a 3.5 a 1.09d 0.7e

0.10a 3 x 10-4 a 9.60 x10-7d n.d.

2.8 x 10-3 0.9 x 10-4 8.8 x 10-7 n.d.

Plasma concentration (nM) 0.3-1b 1-3c 280-680d 47f

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Electrophile NO2-OA NO2-CLA β-addition δ-addition 4-Hydroxynonenal 15-deoxy-12,14-prostaglandin J2

determined at 25°C [29], b [30,31], c [16], d [32], e determined at 37 °C [33], f [34].

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3. The addition-elimination process of the reaction between nitroalkene fatty acids and thiols occurs through a stepwise mechanism with thiolate attack or release as rate-controlling steps.

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In general, the reversible Michael addition reactions between various electrophiles and nucleophiles involve the attack of the nucleophile on the electron deficient center and proton incorporation. These processes can occur through different mechanisms depending on the characteristics of the reaction partners and on the reaction conditions. Two types of mechanisms have been proposed: concerted, in which nucleophile attack and proton incorporation are simultaneous and no distinct intermediate is formed, or stepwise. The stepwise processes are mediated by anionic intermediates and the rate-controlling step can reside in the attack of the nucleophile on the electrophile to form the anionic intermediate or in the proton incorporation step. Conversely, the reverse elimination process can be concerted or stepwise, and the rate controlling step can either be the initial deprotonation or the release of the original nucleophile as leaving group [35–38]. The characteristics of the reactions of several low molecular weight thiols with both the β- and the δ-carbons of NO2-CLA are consistent with a stepwise mechanism in which thiolate attack is the rate-controlling step [15] (Fig. 3). This proposal is supported by the following evidence. First, the apparent rate constants for the forward addition processes increase with increasing pH, showing that the thiolates are the reacting species, while the rate constants of the reverse elimination processes are independent of pH, up to pH 9. At pH higher than 9, there is a contribution from an additional elimination mechanism. Second, the logarithm of the pH-independent rate constants for the forward addition processes (those corresponding to all thiol present as thiolate) increase as the pKa of the thiols increase; the Brønsted nucleophilic coefficients are ~ 0.6 for both the β- and the δ-additions. In fact, similar correlations can be calculated for the reaction of NO2-OA with thiols from the rate constants reported in [25]. Conversely, the rate constants for the reverse elimination processes increase as the acidity of the thiols increase, with Brønsted leaving group coefficients of ~ -0.7. These correlations are consistent with thiolate attack or thiolate departure being involved in the ratecontrolling steps of the forward or reverse processes, respectively. Third, when the reactions are performed in deuterium oxide instead of water, no kinetic isotope effects are detected for the β- nor the δ-addition processes, showing that proton incorporation is not involved in the rate-controlling step. Thus, although the protonation/deprotonation steps can be quite slow and limit the rate in some systems, in the reaction of NO2-CLA and thiols the rate-controlling step is thiolate attack/release [36,38].

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Figure 3. Mechanism of the reaction between a thiolate and NO2-CLA. Attack of the thiolate on the β-carbon (A) or on the δ-carbon (B) is the rate-controlling step and leads to the formation of a nitronate intermediate. This intermediate is protonated initially on one of the oxygens. The proton is then transferred to the α-carbon (A and B bottom product) or to the γ-carbon (B top product). β-addition and δaddition are the kinetically and thermodynamically preferred processes, respectively.

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The stepwise mechanism was confirmed through computational modeling [15]. The free energy barrier for the achievement of the transition state was higher by 1.3 kcal mol-1 for δ-addition than for β-addition, in line with the experimentally observed decrease in the rate constant by a factor of 9, and could be explained by a lag in S-C bond formation with respect to charge transfer, consistent with the principle of nonperfect synchronization [39]. The anionic intermediates have the charge located in the nitro groups and are thus nitronate anions [15]. Protonation occurs initially on the oxygens to form aci-nitro (nitronic acid) derivatives that then tautomerize to the more stable C-protonated adducts. The final products in the case of NO2-CLA are a β-thioether substituted unsaturated nitroalkane (top product in Fig. 3A) and two δ-adducts resulting from protonation in the γ- or in the α-carbons (Fig. 3B, top and bottom products respectively). The former maintains the nitroalkene group and can in principle undergo further Michael addition reactions, although double adducts have not been detected so far, suggesting that reactions are slowed by steric constraints. The δ-thioadduct is the most stable product, as expected from the retention of conjugation between the nitro group and the double bond. According to the equilibrium constants, the glutathionyl δ-adduct is 30 times more stable than the glutathionyl β-adduct at pH 7.4 (Table 1). 4. The reactivity of different thiols with a nitroalkene and the stability of the adducts vary with pH and with thiol pKa, and are affected by the protein environment From the mechanistic analysis performed in [15], several conclusions can be made regarding the rate constants for the forward and the reverse addition processes and the stability of the adducts as a function of pH or thiol pKa (Fig. 4). The pH dependence can be illustrated semiquantitatively for the reaction of a typical thiol with a pKa of 8.29, similar to that of free cysteine [24], with a nitroalkene

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(i. e., NO2-OA) (Fig. 4A). As reflected by the decrease in the equilibrium dissociation constant, the stability of the adducts increases as the pH increases to more alkaline values as a consequence of the increase in kon with pH (due to increased thiol ionization) and the non-dependence of koff up to pH 9. At non-physiological pHs higher than 9, the scenario may be different due to an increase in the elimination rate through the contribution of an additional elimination mechanism [15]. The dependence on thiol pKa is shown in Fig. 4B, which illustrates the reaction at pH 7.4 between various thiols with increasing pKa and a nitroalkene. The apparent kon at pH 7.4 has a bell shaped profile that reflects the compromise between the need for the thiolate to be ionized and the fact that nucleophilicity correlates with basicity. The koff increases as the pKa decreases, reflecting that acidic thiols are better leaving groups. The stability is higher for those adducts that are formed with thiols that have high pKa. Thus, in the absence of particular effects of the protein environment, it is expected that the adducts likely to predominate are those formed with cysteine residues that have relatively high pKa. However, with the paucity of available data regarding protein thiol pKa and rate or equilibrium constants for the reactions with nitroalkene fatty acids it is premature to extrapolate this observation to in vivo situations. For protein thiols, in addition to pKa considerations, it is likely that steric constraints, the presence or not of water, the proximity of charged or polar residues and specific interactions will affect the kinetic and thermodynamic aspects of the Michael addition and elimination processes. The possible outcome of these effects is hard to predict. First, it is likely that the presence of multiple residues that impede the correct approach of a nitroalkene fatty acid to a protein thiol will decrease the rate constant for adduct formation but, once formed, the adduct may have increased stability. Second, with regards to the effect of solvent polarity, it is important to consider that water may be excluded from the environment of a protein thiol. It has been observed for Michael addition-elimination reactions of amines with nitro-activated olefins that decreases in solvent polarity tend to moderately increase the kon rate constant for adduct formation, to strongly increase the koff rate constant and to overall increase the equilibrium dissociation constant, thus decreasing the stability of the adducts [40]. Third, the presence of properly positioned protein residues with positive charge or with the ability to form hydrogen bonds may preferentially stabilize the negatively charged transition state, giving rise to increased rate constants. Fourth, the presence of specific binding sites in the proteins (e.g., fatty acid binding sites that accommodate nitroalkenes) may increase the stability of the adducts formed.

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Figure 4. Apparent rate and equilibrium constants for adduct formation as a function of pH or pKa. A, apparent forward (kon) and reverse (koff) kinetic rate constants and equilibrium dissociation constant (Kdiss) for the reaction of a thiol with a pKa of 8.29 with a typical nitroalkene fatty acid at pHs 5-9. At pH higher than 9, the apparent koff can increase due to a further elimination mechanism. B, apparent rate and equilibrium constants at pH 7.4 for the reaction of thiols with pKa varying between 5 and 10. The dependence of the rate and equilibrium constants on pH and pKa is taken from [15].

5. Nitroalkene fatty acids modulate enzymatic activity

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Nitroalkylation of certain proteins has been evidenced both in vitro and in vivo and results in changes in protein structure, function and subcellular distribution [4,41]. In fact, nitroalkene fatty acids usually exert their cytoprotective and antiinflammatory effects through the reaction with thiol containing proteins. Mainly, they inhibit the activation of the nuclear factor κB (NF-κB) and downstream proinflammatory events by reacting with a cysteine involved in DNA binding [42]. Nitroalkene fatty acids also react with critical thiols in Kelch-like ECH-associated protein 1 (Keap1), stabilizing the Nuclear Factor (Erythroid-Derived-2)-like (Nrf2) complex and allowing for newly synthesized Nrf2 to translocate to the nucleus and transactivate the expression of cytoprotective and antiproliferative genes[43–45]. Besides, they activate the heat shock response by releasing heat shock factor-1 (HSF-1) through a mechanism that is not yet clear [46]. Since nitroalkene fatty acids have a relatively low abundance (1-3 nM basal NO2-CLA [1,16] and 0.3-1 nM basal NO2-OA [30,31], Table 2) and probably modify a relative low percentage of the target proteins, they are expected to exert their biological effects mostly involving a gain of function rather than a loss of function of the target proteins. For example, PPARγ is activated upon nitroalkene fatty acids binding. However, this is not always the case. Nitroalkene fatty acids are also shown to modify transcription factors or regulatory proteins leading to changes in gene expression. Thus, the consequences of the modification of a small subset of proteins is amplified by the generation of many transcript molecules, as is the case of Keap1, with the consequent nuclear translocation of Nrf2 [42], and of NF-κB [44]. Most of the proteins reported as nitroalkene fatty acids targets and the effect of the nitroalkylation reactions are summarized in Table 3.

ACCEPTED MANUSCRIPT Table 3. Protein targets of nitroalkene fatty acids and their effects on protein function Nitroalkene fatty acid

Modification site

Additional information

Effect

Ref.

Nuclear factor κB subunit p65 (NF-κB p65)

NO2-OA, NO2-LA

DNA binding domain Cys

In human: Cys38

Inhibition of NF-κBDNA binding abolishing proinflammatory responses

[42]

Kelch-like ECHassociating protein 1 (Keap 1)

NO2-OA, NO2-LA and NO2-AA

Cys

Inactivation, Nrf2 release

[43– 45,47]

Peroxisome proliferatoractivated receptor (PPARγ)

NO2-OA and NO2-LA

Cys in ligand binding domain

In mouse, NO2-OA: Cys38, 226, 257, 273, 288, 297 and 489 In human: Cys285

Agonist activation of PPARγ

[9]

Glyceraldehyde-3phosphate dehydrogenase (GAPDH)

NO2-OA

Catalytic Cys, other Cys and His

In rabbit: Cys149 (catalytic), 153 and 244; His108, 134, 303 and 327

Inhibition, increase in hydrophobicity and change in subcellular distribution

[41]

Pro-matrix metalloproteinases, (ProMMP7 and Pro-MMP9)

NO2-OA

Zinc coordination Cys in the active site

In human: Cys70 in Pro-MMP7 and Cys100 in ProMMP9

Zinc release, autocatalytic cleavage of the prodomain.MMP activation

[48]

Transient receptor potential (TRPV1, TRPA1)

NO2-OA

ND

Rat

Activation of TRP channels

[49,50]

Angiotensin II receptor (AT1R)

NO2-OA

ND

Reduction of coupling with G-protein, inhibition of downstream signaling

[51]

Adenine nucleotide translocase 1 (ANT1)

NO2-LA

Cys

In mouse: Cys57

Cardioprotection

[52]

Protein kinase G (PknG)

NO2-OA

In M. tuberculosis: Cys128 and 131

Inhibition of kinase activity

[53]

Xanthine oxidoreductase (XOR)

NO2-OA

Iron coordination Cys in a noncatalytic domain and His Pterindithiolene that coordinates molybdenum

Bovine XOR

Inhibition of electron transfer reactions at the molybdenum cofactor

[54]

Cys and noncovalent binding

Cys34

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Human serum albumin (HSA)

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Protein

[15]

Prostaglandin endoperoxide H synthase (PGHS)

NO2-AA

Disruption of heme binding to the protein

No covalent modification detected

Inhibition of PGHS-1 cyclooxygenase activity and both PGHS-1 and 2peroxidase activity

[55]

Protein kinase C (PKC)

NO2-AA

Probable covalent modification

Human

Inhibitory effect on PKC activation

[56]

Nitric oxide synthase 2 (NOS2 or iNOS)

NO2-AA

Inhibition of transcription

Mouse

Inhibition of nitric oxide production

[57]

NADPH oxidase 2 (NOX2)

NO2-AA

Inhibition of assembly

Mouse

Inhibition of superoxide production

[58]

Protein disulfide isomerase (PDI)

NO2-AA

Cys at active site aʼ

In human: Cys397 and 400

Inhibition of reductase and chaperone activities

[59]

ACCEPTED MANUSCRIPT 6. Nitroalkene fatty acids can have alternative fates in biological contexts The formation of adducts with low molecular weight and protein thiols (Fig. 5, pathways A and B), described in the previous sections, constitutes a predominant decay pathway and the basis for their signaling properties. In addition, nitroalkene fatty acids can undergo several processes in biological contexts.

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Reaction with nitrogen based nucleophiles. Lysine and histidine are also nucleophiles and thus potentially reactive with nitroalkene fatty acids. However, they are weaker nucleophiles than thiols and, in fact, no reaction was observed between NO2CLA and free histidine at concentrations up to 40 mM [15]. When histidine is in the context of a protein its reactivity can be enhanced as observed in glyceraldehyde 3phosphate dehydrogenase (GAPDH), for which histidine adducts with NO2-OA were detected [41] (Fig. 5, pathway B).

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Reaction with hydroxide anion. This reaction, in which hydroxide anion acts as a nucleophile, is proposed to happen via a Michael addition-like mechanism analogous to that with thiols. The reaction starts with the nucleophilic attack of hydroxide anion on the β-carbon of the nitroalkene to form an intermediate nitronate anion and yields finally the corresponding nitrohydroxy fatty acid (Fig. 5, pathway C). These derivatives have been detected in vivo [60–62]. It is worth noting that this reaction can contribute to the slow decay of nitroalkene fatty acids in aqueous solution and can participate in the formation of polymerized derivatives.

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Nitric oxide release. Nitroalkene fatty acids release nitric oxide (NO·) [57,61,63]. Two mechanisms have been proposed. The first one implies a modified Nef reaction that leads to the formation of NO· and a carbon-centered radical [63]. An alternative proposal implies the nitroalkene fatty acid isomerization to the corresponding nitrite ester followed by N-O bond homolysis and/or metal ion/ascorbateassisted reduction to form the corresponding functional enol group and NO· [61] (Fig. 5, pathway D). However, NO· release is inhibited in hydrophobic environments [63] thus rendering this decay pathway of little or no biological significance.

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Nitroalkene reduction. Reduction of the nitroalkene moiety to a nitroalkane implies its inability to undergo Michael addition reactions and thus is the main inactivation pathway for nitroalkene fatty acids. This reaction is catalyzed in vivo by the vertebrate enzyme prostaglandin reductase-1 (PtGR-1) in a NADPH-dependent reaction (Fig. 5, pathway E). Overexpression of PtGR-1 in cell culture inhibited Nrf2dependent heme oxygenase-1 expression by NO2-OA [14]. Fatty acid metabolism. Nitroalkene fatty acids are actively metabolized in vivo. Mitochondrial and peroxisomal β-oxidation is a well-known metabolic route for these molecules [64] (Fig. 5, pathway F). Recently, the profile of NO2-OA metabolites upon oral administration in humans has been described, including the identification of new metabolites, particularly dicarboxylic derivatives. The latter are formed through an initial hydroxylation step catalyzed by the CYP4A family of cytochrome P450dependent enzymes. The dicarboxylic acid products are further metabolized through βoxidation at both ends [65] (Fig. 5, pathway G).

ACCEPTED MANUSCRIPT Esterification. Nitroalkene fatty acids can be found esterified into triacylglycerols and glycerophospholipids [66,67] (Fig. 5, pathway H). The esters can be formed either through the nitration of unsaturated fatty acids already forming part of the complex lipid or through the incorporation of nitroalkene fatty acids into them. The nitroalkene esterification will impact its systemic distribution. Other modifications that can occur at the level of the carboxylic acid moiety are the formation of conjugates with taurine and sulfate observed in rat urine [65].

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Partition. Partitioning into membranes, micelles or lipoproteins (Fig. 5, pathway I) prevents or at least decreases nitroalkene fatty acid reactivity due to hydrophobic stabilization and steric hindrance. In fact, a decrease in the formation of NO2-OA adducts with GSH was observed in the presence of non-ionic detergent micelles [25]. Furthermore, NO· release from NO2-LA was inhibited by the presence of non-ionic detergent micelles and phosphatidylcholine/cholesterol liposomes [63].

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Protein non-covalent binding. Nitroalkene fatty acids can also bind to fatty acid binding proteins such as albumin. This is likely to impact on nitroalkene transport, storage and bioavailability [15] (Fig. 5, pathway J).

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Figure 5. A nitroalkene fatty acid, represented here by NO2-OA, can undergo different processes. A, reaction with glutathione (GSH) and other low molecular weight thiols through Michael addition reactions. Adducts with glutathione can be exported through the multidrug resistance protein 1 (MRP-1); B, reaction with protein residues, mainly cysteines and histidines, as exemplified by glyceraldehyde 3phosphate dehydrogenase (GAPDH); C, reaction with hydroxyl anion; D, nitric oxide (NO·) release; E, nitroalkene reduction to nitroalkane by prostaglandin reductase-1 (PtGR-1); F, mitochondrial or peroxisomal β-oxidation; G, ω-oxidation through an initial hydroxylation step catalyzed by the CYP4A family of cytochrome P450-dependent enzymes; H, esterification to complex lipids; I, partition into hydrophobic compartments; J, binding to fatty acid binding proteins such as albumin (HSA).

7. Nitroalkene fatty acids and other lipid-derived electrophiles in biological contexts Besides nitroalkene fatty acids, other lipid derived electrophiles are formed in vivo including 15-deoxy-12,14-prostaglandin J2 and 4-hydroxynonenal. Their effect will depend on the concentration they achieve in vivo and their reactivity. Unlike nitroalkene fatty acids, 4-hydroxynonenal is shown to exert deleterious effects. This electrophile is found at higher concentrations in plasma (Table 2, µM versus nM). Furthermore, it is

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able to undergo irreversible Schiff’s base formation and poorly reversible Michael addition as reflected by the almost negligible rate constant for the reverse reaction (koff) [32]. When considering biological contexts, glutathione transferases need to be taken into account. These enzymes can catalyze the Michael addition reaction between glutathione and electrophiles such as 4-hydroxynonenal [68] and 15-deoxy-12,14prostaglandin J2 [69], impacting their metabolism. However, no catalysis was observed with NO2-OA or NO2-LA as substrates when four human glutathione transferases were evaluated [70]. Because there are other nitroalkene fatty acids and many different glutathione transferases, this issue needs further investigation. The biological relevance of the reaction between nitroalkene fatty acids and low molecular weight thiols is underscored by the detection of cysteinyl or Nacetylcysteinyl adducts in urine [15,65]. It is likely that these adducts are formed intracellularly with GSH, exported through the multidrug resistance protein 1 (MRP-1) [70] and then further processed. 8. Final reflections

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Within the set of reactions in which nitroalkene fatty acids can participate in biological systems, the Michael addition-elimination reaction with thiols stands out for being in the basis of the signaling effects by leading to the covalent modification of cysteine residues in regulatory proteins. In addition, the formation of adducts with GSH mediates cellular export and urinary excretion. Thus, the reactions between nitroalkene fatty acids and both low and high molecular weight thiols have central importance in the modulation of nitroalkene fatty acid levels and signaling in vivo. The Michael addition reactions are reversible. This allows for adducts that are formed initially with relative high kinetic rates to undergo elimination reactions and release the free nitroalkenes, which are then available to react with other electrophiles forming other adducts. Thus, the nitroalkene pool is likely to be very dynamic and to be affected not only by the rate constants of the reactions but also by the thermodynamic stability of the adducts. Given the abundance of cellular low and high molecular weight thiols and the magnitudes of the rate and equilibrium constants, it can be calculated that more than 99 % of the nitroalkene pool is covalently bound to thiols, and that the time needed for a newly synthesized nitroalkene to reach equilibrium concentrations is less than 1 s. A recent study showed that the Michael addition-elimination reactions occur through stepwise mechanisms and that thiolate attack and release are the ratecontrolling steps in the forward and reverse reactions, respectively [15]. The mechanistic insights have allowed the prediction that, in the absence of particular effects of the protein environment, the adducts that will predominate will be those formed with cysteine residues that have high pKa, while other aspects that may determine the reactivity of certain proteins with nitroalkene fatty acids and the stability of the adducts are yet to be unveiled. The finding that nitroalkene fatty acids can be formed endogenously and can exert protective actions has contributed to build the concept that nitrooxidative processes can give origin to lipid derivatives with anti-inflammatory and cytoprotective effects, and raised hope for the development of novel drugs. The addition-elimination reaction between nitroalkene fatty acids and thiols provides a framework for understanding the molecular basis of the modulatory actions, and lies at the crossroads between thiol signaling and electrophilic lipid signaling.

ACCEPTED MANUSCRIPT Funding sources: This work was supported by Comisión Sectorial de Investigación Científica (CSIC, Universidad de la República, Uruguay), Agencia Nacional de Investigación e Innovación (ANII, Uruguay) and Programa de Desarrollo de las Ciencias Básicas (PEDECIBA, Uruguay).

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Acknowledgements: We thank Francisco Schopfer and Darío Vitturi for helpful discussions.

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Conflicts of interest: None

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Highlights Nitroalkene fatty acids react with thiols through Michael addition reactions

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The mechanism is stepwise and thiolate attack is the rate-controlling step The reactions are reversible and the adducts can undergo elimination

Reaction with thiols is in the basis of nitroalkene fatty acids signaling effects

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Besides reacting with thiols, they have alternative fates in biological contexts