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PerspectiVe Mechanisms of Protein Damage Induced by Cysteine Thiyl Radical Formation Christian Schöneich* Department of Pharmaceutical Chemistry, The UniVersity of Kansas, 2095 Constant AVenue, Lawrence, Kansas 66047 ReceiVed January 3, 2008
This perspective covers the reactions and underlying kinetics of peptide and protein thiyl radicals specifically with respect to reversible hydrogen atom transfer processes, which may ultimately lead to the oxidation and/or epimerization of amino acids adjacent to the thiyl radicals. Contents 1. Introduction 2. Thiyl Radicals as Precursors for Irreversible Protein Damage 3. Consequences for Biology and Biotechnology 4. Conclusion
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1. Introduction Oxidative post-translational modifications of proteins play an important role in modulating conformation, activity, and turnover in response to a variety of biologic stimuli (1). These modifications arise as a result of both controlled enzymatic and less-controlled nonenzymatic chemically induced processes. To date, more than 200 post-translational protein modifications are known (2), creating a vast array of possible protein isoforms when 30000 gene products have the opportunity to accumulate one or more of these modifications within their sequences. Hence, the quantitative and functional characterization of posttranslational protein modifications represents an active field in current biomedical research. However, this area of research is equally important to the biotechnology industry (3, 4), where even the presence of small levels of post-translational protein modifications raise concerns about potential antigenicity and stability during long-term storage. Diagnostic and therapeutic antibodies and antibody derivatives make up an increasing share of biopharmaceutical products, with currently ca. 200 antibody products under pharmaceutical development (3). Antibodies are specifically prone to chemical modification such as deamidation, pyroglutamate formation, isomerization, β-elimination, and oxidation, eventually leading to conformational changes, aggregation, and/or fragmentation (3, 5). Some of the underlying mechanisms and parameters controlling these reactions are currently unknown but must be characterized to ensure the rational development of stable formulations. An interesting example is the rather random hydrolysis between all amino acids in the IgG1 hinge sequence Cys220-Thr225 (6), where instead one would expect hydrolysis primarily between Asp221 and Lys222 (6). * To whom correspondence should be addressed. Tel: 785-864-4880. Fax: 785-864-5736. E-mail:
[email protected].
Generally, hydrolysis reactions of proteins are limited to a few amino acid hot spots [predominantly Asn and, to a lower extent, Asp and Gln (7)]. Instead, oxidation reactions can target all amino acids (1), and selectivities will be controlled by numerous parameters such as (i) the nature of the oxidizing species, (ii) the solvent-accessible surface areas of the respective amino acid targets, (iii) the presence of catalytically active transition metals and the geometry and nature of metal-binding sites, and (iv) neighboring group effects. For example, the highly reactive hydroxyl radical will react rather unselectively with all amino acids (1) via addition (aromatic amino acids), electron transfer (Met), and/or hydrogen abstraction (Cys and all CR-H and side chain C-H bonds). Importantly, protein damage is not restricted to the sites of initial attack, as secondary radicals (e.g., alkoxyl and peroxyl radicals) may react with additional amino acid targets, eventually propagating a chain oxidation mechanism (8). On the other hand, more selective oxidants such as hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and peroxynitrite (ONOO-) will restrict initial protein damage to a few amino acids (1). The current perspective will focus on a mechanism of amino acid oxidation that has received little attention in the literature but may be of paramount importance for protein degradation both in vivo and in vitro. Central to this mechanism is the formation of a Cys thiyl radical (CysS•), followed by the reaction of CysS• with surrounding amino acids.
2. Thiyl Radicals as Precursors for Irreversible Protein Damage The amino acid Cys has been regarded as a sink for oxidation processes in proteins. The two-electron oxidation of Cys yields sulfenic acid (CysSOH), which oxidizes further to sulfinic acid (CysSO2H) and sulfonic acid (cysteic acid; CysSO3H) or forms a disulfide bond through reaction with a second CysSH (9, 10). Further oxidation to thiolsulfinate has been detected upon exposure of a peptide sequence of the transcription factor Sp1 to hydrogen peroxide (11), and an intermediary oxidation to thioaldehyde has been detected during iron-dependent oxidation of a Cys residue in iron regulatory protein 2 (IRP2) (12). In contrast, the one-electron oxidation of Cys (and the one-electron
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reduction of cystine) yields Cys thiyl radicals. Well-characterized reactions of highly mobile thiyl radicals (RS•) in aqueous solution are the formation of disulfide radical anions (RSSR•-) through reaction with additional thiol/thiolate (reaction 1), the formation of thiyl peroxyl radicals (RSOO•) through the reversible reaction with O2 (reaction 2), and the reaction with ascorbate (Asc-) (reaction 3) (13).
RS• + RS- ) [RSSR]-• •
•
Scheme 1
(1)
RS +O2)RSOO
(2)
RS•+Asc-fRS- + Asc•
(3)
However, in proteins, reactions 1 and 3 may be significantly less efficient depending on the access of CysS• to additional protein Cys residues and/or endogenous glutathione or ascorbate. In addition, equilibrium 2 does not represent an efficient pathway for the removal of CysS• radicals in proteins, specifically at physiologic concentrations of O2 (ca. 3–30 µM) (14, 15), as k–2 ) 6.3 × 105 s-1 is relatively high (16). Hence, competitive reactions of CysS• may become important, specifically in proteins. In a series of calculations, Rauk and co-workers (17–20) determined homolytic bond dissociation enthalpies (BDE) for S-H and CR-H bonds of Cys and other amino acid residues. While the BDE of the Cys S-H bond was calculated to ca. 370 kJ/mol (18), the CR-H BDE of most amino acids (except Pro) in fully relaxed model peptides of the structure HC(O)-NH-CH(R)-C(O)NH2 was calculated to be e357 kJ/ mol (19). Generally, secondary structure increased the CR-H BDE [i.e., for Gly, BDE(CR-H) ) 348, 361, and 402 kJ/mol for the fully optimized structure, β-sheet, and R-helix, respectively] (17), but even for several amino acids (e.g., Gly, Ala, Ser, and Thr) within β-sheet conformation, BDE(CR-H) < BDE(S-H). Hence, these calculations would suggest a thermodynamic preference for H-atom abstraction by CysS• radicals within certain protein domains. Experiments confirm the propensity of thiyl radicals to abstract CR-H atoms from amino acids within simple peptide models such as diketopiperazines and N-acetyl amino acid amides, CH3-C(O)NH-CH(R)-C(O)NH2 (21). For example, rate constants as high as 3.2 × 105 and 6.4 × 104 M-1 s-1 were obtained for the reaction of cysteamine thiyl radicals with glycine anhydride and CH3-C(O)NH-CH2-C(O)NH2, respectively (21). In addition, thiyl radicals abstract H-atoms from amino acid sides chains, demonstrated for Met, Phe, Thr, and Ser, with rate constants between 9 × 103 and 1.6 × 105 M-1 s-1 (22). The potential consequences for the protein are illustrated in Schemes 1 and 3. In Scheme 1, CysS• exists in equilibrium 5 with an amino acid CR• radical and in equilibrium 2 with the thiylperoxyl radical. The rate constant for rearrangement reaction 4 is rather slow [ca. 2 × 103 s-1 at 37 °C (16)], while the amino acid radical reacts diffusion-controlled with O2 [reaction 6; k6 ca. 2 × 109 M-1 s-1 (23)], ultimately leading to fragmentation products (reaction 7) (1). In a first approximation (absence of glutathione and ascorbate), we calculate that in Scheme 1 g60% of CysS• will convert into alanine peroxyl radicals in air-saturated solution (reactions 5 and 6; R ) CH3), based on our measured value for the second-order rate constant k5 ) 1 × 104 M-1 s-1 (21), an assumed effective concentration of Ala and Cys of 1 M within a given protein, k-5 e 106 M-1 s-1 and [O2] ) 3 × 10-4 M. Such a situation (air saturation, absence of endogenous antioxidants) would apply well to formulation conditions of biotechnology products, indicating that fragmentation reactions of pharmaceutical proteins may well
Scheme 2
be initiated through the formation of protein thiyl radicals (vide infra). For oxygen concentrations in vivo [e30 µM (14, 15)], similar calculations in the absence of endogenous antioxidants would predict that g70% of CysS• could lead to the formation of alanyl peroxyl radicals. Here, the presence of up to 1 mM ascorbate or 10 mM glutathione could significantly reduce the amount of CysS• available for reaction 5, provided that the CysS• radical is fully accessible to the endogenous antioxidant. However, preliminary data on the intramolecular reaction of CysS• with Gly in the model heptapeptide N-Ac-Cys-Gly6 (reaction 8) indicate that k8 ca. 105 s-1 (Scheme 2).1 Therefore, even when 1 mM ascorbate is present, ca. 14% of CysS• would react via intramolecular H-atom transfer with peptide Gly residue(s) instead of intermolecular electron transfer with ascorbate. In Scheme 3, an additional set of reactions is presented, which could lead to protein damage even in the absence of oxygen such as epimerization at CR-H (analogous reactions would also be a problem for H-atom transfer from the Cβ-H position of Thr). Precedence for thiyl radical-dependent racemization of aliphatic amines is available in the organic synthetic literature (24). The literature contains a number of examples where reactions of protein thiyl radicals are or may be involved in irreversible protein damage and/or physiologic processes through H-atom transfer processes. These will be summarized briefly below, before a discussion of pathways of thiyl radical generation, which will show that thiyl radicals may well function as catalysts of protein damage. When Hao and Gross (25) investigated the behavior of S-nitroso peptides during electrospray MS/MS analysis, they noticed that the doubly charged sequence [GTFATLSELHC(NO)DK + 2H]2+ efficiently lost NO to yield the corresponding thiyl radical, [GTFATLSELH(CysS•)DK + 2H]2+. Subsequently, the thiyl radical abstracted an H-atom from Cβ-H of Thr at position i-6, resulting in a series of characteristic fragments (c - 1, z, a, x + 1) resolved by MS3 analysis. These 1 Nauser, T., Casi, G., Koppenol, W. H., and Schöneich, Ch. Unpublished results.
PerspectiVe
Chem. Res. Toxicol., Vol. 21, No. 6, 2008 1177 Scheme 3
CysS• + HCO2- f CysSH + •CO2-
Scheme 4
data confirm the sensitivity of the Thr Cβ-H bond toward thiyl radical attack, revealed by our solution NMR experiments (22), and also suggested by earlier pulse radiolysis experiments on the reaction of thiyl radicals with 2-propanol (26, 27). Previous radiation chemistry experiments had also indicated an intramolecular H-atom transfer to thiyl radicals from the γ-glutamyl residue of glutathione (Scheme 4; equilibrium 9) (28–30). Here, reaction 9 benefits from deprotonation of the N-terminal amino group. On the other hand, protonation of the N-terminal amino group usually deactivates the CR-H group (i.e., compare the rate constants for the reaction of cysteamine thiyl radicals with N-Ac-Gly-NH2 and Gly-NH2 in acidic solution, 6.4 × 104 and 0.7 × 104 M-1 s-1, respectively) (22) so that the intramolecular process observed in glutathione is not an ideal model for CR-H bonds of amino acids within peptides. Evidence for H-atom abstraction by thiyl radicals within a protein comes from experimental and theoretical studies on the enzyme pyruvate formate lyase (PFL) (31, 32). In the resting state, this enzyme features a glycyl radical at position Gly734, which, upon binding of pyruvate, abstracts an H-atom from Cys419 to initiate substrate turnover. However, dissociation of pyruvate (or, after turnover, formate) triggers the reverse reaction, H-atom transfer from Gly734 to the thiyl radical of Cys419. In the presence of oxygen, the glycyl radical at Gly734 adds oxygen to form a peroxyl radical (33, 34), ultimately resulting in protein cleavage (analogous to reaction 7 presented in Scheme 1). Further, albeit indirect, evidence for thiyl radical-dependent protein cleavage was provided by radiation chemical studies on lysozyme (35). The anaerobic reduction of lysozyme in aqueous solution by •CO2- radicals targets specifically one out of four disulfide bridges between Cys6 and Cys127, generating two equivalents of CysSH per lysozyme. This stoichiometry suggests the formation and reaction of thiyl radicals according to reactions 10–12, where reaction 12 propagates a chain reaction.
CO2- + CysSSCys f CO2 + [CysSSCys]•-
•
•-
+
•
[CysSSCys] + H ) CysS + CysSH
(10) (11)
(12)
However, SDS-PAGE analysis of the protein after reduction does reveal not only monomeric (14.4 kDa) and some dimeric (ca. 29 kDa) lysozyme but also a band with an apparent molecular mass of ca. 24 kDa (i.e., below that expected for the dimer). This band indicates fragmentation and either crosslinking between two lysozyme fragments or an intact and fragmented lysozyme. On the basis of the observed stoichiometry for the formation of CysSH, these data would be consistent with an alternative H-atom transfer of CysS• in competition to reaction 12, possibly H-atom abstraction from an amino acid CR-H bond. The resulting carbon-centered radical would be a precursor for protein fragmentation as well as cross-linking. It should be noted that the γ-irradiation of solid lysozyme at 77 K resulted in the generation of perthiyl radicals (36), likely through reactions 13–15 (P, P′ ) polypeptide chains), facilitated by the low protein mobility in the solid state.
P-CH2-S-S-CH2-P′ + e-/H+ f P-CH2-S-•S(H)CH2-P′(13) P-CH2-S-•S(H)-CH2-P′ f [P-CH2-S-S-H + CH2-P′](14)
•
[P-CH2-S-S-H + •CH2-P′] f P-CH2-S-S• + H3CP′(15) Perthiyl radicals are significantly (ca. 10-fold) less reactive toward H-atom abstraction as compared to thiyl radicals (37). Moreover, C-centered radicals abstract H-atoms ca. 10-fold faster from RSSH as compared with RSH (37). Hence, it is not expected that perthiyl radicals would be able to initiate protein fragmentation. On the other hand, if also in solution perthiyl radicals were formed through the reaction of lysozyme with • CO2-, the subsequent reduction of P-CH2-S-S• would yield P-CH2-S-S-H. The latter may be reductively cleaved to P-CH2-S•/HS- and/or P-CH2-S-/HS•, generating thiyl radicals available for H-atom abstraction from CR-H bonds.
3. Consequences for Biology and Biotechnology The chemical and biological consequences of the reactivity of protein thiyl radicals toward amino acid CR-H and Cβ-H bonds must be discussed. On one hand, if protein thiyl radicals are necessary intermediates in controlled biological processes, the rate of these processes must be sufficiently fast to outcompete the intramolecular H-atom transfer with adjacent amino acids. This can be achieved through (i) high substrate concentrations, (ii) a sufficiently restricted mobility of the protein thiyl radical, which would inhibit reactions with other amino acids, and/or (iii) a conformational rigidity of the target amino acid such that the forming C-centered radical cannot adopt a planar configuration, resulting in a situation where k5 85% of CysS• would react with protein amino acids rather than NO, provided the CysS• radical has access to amino acids with labile C-H bonds. These considerations would make it rather unlikely that the reaction of free protein thiyl radicals with NO represents an important pathway for biologically controlled S-nitrosothiol formation. Moreover, these considerations are consistent with time-resolved UV spectroscopic observations on the photolysis of S-nitrosoglutathione (GS-NO) (39). Here, the laser flash photolysis of GS-NO resulted in photobleaching of the 330 nm absorbance, rationalized by the homolytic cleavage of the S-N bond (reaction 17).
GS-NO f GS• + NO
(17)
However, over ca. 100 µs after the laser pulse, no radical recombination of GS• and NO, restoring GS-NO, was observed, even when the experiment was conducted in the additional presence of 1.8 mM NO. These data were rationalized by an efficient H-atom transfer (for example, reaction 9 and Scheme 3), which competed successfully against the reaction of GS• with NO (39). In the second case, we shall consider the reaction of a protein with superoxide radical anion (O2•-), the one-electron reduction product of molecular oxygen. The latter will usually not react efficiently with amino acids except with Cys. On the basis of electron spin resonance and product studies, the rate constant for the reaction of superoxide with glutathione (reaction 18) is on the order of k18 ) 220 M-1 s-1 (40). Glutathiyl radical adducts to DMPO were detected as a result of reaction 18 (40), although product studies also reported on an alternative mode of reaction, leading to sulfinyl radicals (GSO•) (41).
GSH + O2•- f GS• + HOO-
(18)
For biological systems, that means that superoxide will by no means have any chance to oxidize Cys residues in the presence of superoxide dismutase, an enzyme that catalyzes the disproportionation of superoxide to hydrogen peroxide and oxygen. However, if superoxide were generated in vitro in pharmaceutical formulations, it may well react with protein Cys residues, specifically if superoxide fluxes were small, lowering the probability for uncatalyzed superoxide dismutation. The
resulting cysteinyl radical, CysS•, will then have the opportunity to attack the surrounding amino acids according to the reactions displayed in Schemes 1 and 3. In other words, while superoxide will not directly react with any amino acid except Cys, the Cys residue may function as a catalyst for the irreversible breakdown of amino acid residues through superoxide. Similar considerations would hold for other “weak” oxidants such as, for example, redox-active transition metals, which could generate protein CysS• radicals. Such a catalytic role of thiols has been recognized for organic radical reactions, where H-transfer reactions (for example from alcohols) to carbon-centered radicals were catalyzed by organic thiols, a concept referred to as “polarity reversal catalysis” by Roberts and co-workers (42, 43). In pharmaceutical protein formulations, multiple pathways can lead to the formation of CysS•. For example, detergents such as Tween 80 can carry peroxide contaminations, which react with traces of redox-active transition metals to generate oxygen and carbon-centered radicals (44). Another source for CysS• radicals is the exposure of proteins to UV light. Specifically, Trp residues can function as endogenous photosensitizers for the reductive cleavage of disulfides, which generates free thiol and CysS•, where CysS• has been demonstrated to cross-link with Trp radicals (45, 46). Moreover, some as yet unidentified Cys-Lys cross-links have been detected (46). In more detail, these photosensitized reactions involve the photoionization of Trp (or Tyr) under formation of amino acid radical cations and solvated electrons. The latter efficiently reduce disulfide bonds analogous to reactions 10 and 11 (where the reductant is •CO2-). In addition, the disulfide bond can be cleaved homolytically through the direct absorption of light (47). Importantly, the photolysis of cystine leads predominantly to CysS• whereas the photolysis of β-substituted cystine, that is, penicillamine disulfide, predominantly yields perthiyl radicals (47, 48). Our own data2 on the photolysis of insulin demonstrate a rather efficient covalent H/D exchange of surrounding amino acids induced by protein CysS• radicals when reactions are carried out in D2O, consistent with the propensity of protein thiyl radicals to generate carbon-centered radicals, which could serve as precursors for some of these cross-links.
4. Conclusion The available experimental data suggest that thiyl radicals, no matter whether generated through the oxidation of Cys or the reduction of cystine, must be considered as highly reactive entities toward surrounding amino acids within peptides and proteins. Protein Cys residues are not only targets for oxidation but can function as catalysts for the oxidation of additional amino acids, eventually leading to protein damage remote from the initial oxidation event. Therefore, thiyl radical reactions must be considered when protein oxidation is studied either within the framework of the biology of oxidative stress or as part of (pre)formulation studies in the biotechnology industry with the goal to develop stable protein pharmaceuticals. Acknowledgment. This work was supported by the NIH (PO1AG12993).
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