Among the reactive species with oxidant action produced in organisms, hypochlorite (HOCl) is released in the extracellular environment as a key component of the inflammatory immune response in mammals. It has bactericidal activity but collateral damage to host tissues is also highly likely, and proteins are by far the major targets of HOCl-mediated modifications since are rich of potential reactive sites. Regarding the possible consequences of HOCl-induced chemical modification on protein behavior, at least two classes of proteins could be defined: those that do not aggregate and can even become effective chaperones, and those that become prone to precipitation.
We recently investigated the fate of Human Serum Albumin (HSA), the most abundant protein in blood plasma, undergoing HOCl-induced chemical damage at increasing doses of the oxidant, by applying a combination of techniques aimed at obtaining both chemical and structural information directly in solution. This led to discover a reproducible structural transition of the protein induced above a critical level of oxidative modification which could be relevant to explain the general suitability of the albumin structure to work as a scavenger in blood.
In the project we would like to characterize the consequences of HOCl-induced damage on another highly studied protein, Hen Egg White Lysozyme (HEWL). Preliminary experiments suggested that HEWL, differently from HSA, undergoes aggregation and precipitation when oxidized with HOCl.
Understanding the molecular basis of protein resistance to chemical damage, or, on the other hand, of the processes leading to the formation of insoluble precipitates as a consequence of these oxidative modifications, can be relevant for understanding physiological phenomena taking place in conditions of chronical inflammation and oxidative stress. In addition this knowledge can be useful to prevent inactivation of enzymes or protein carriers in applications.
Protein aggregation is a complex process, primarily determined by stress related factors revealing the hidden aggregation propensity of proteins that otherwise are fully soluble. We expect that a further understanding of the HOCl-induced aggregation of the protein lysozyme, with highlight on the critical phenomena that trigger the formation of precipitate, can represent a model case study which can shed some light on the complex picture of oxidant-induced protein aggregation. Even if the formation of insoluble precipitates in protein preparations which undergo some kind of stress is a quite widespread experience, [19] the elucidation of the critical aspects determining the phenomenon is usually not of immediate interest for experimenters who work with proteins.
Our preliminary approch is aimed at filling the gap between the knowledge of possible chemical modifications proteins undergo in oxidative stress conditions and the consequences that these have on the protein solution behavior as conformational changes and tendency to aggregate. In addition we hope to also obtain some possible interpretation at the molecular level of the chaperone-like acivity of some HOCl-modified proteins.
In the future, after gaining a solid knowledge of the experimental conditions, it could be of high interest to collaborate with other research groups to use mass-spectrometry or protein crystallography tools in order to identify with low abiguity the chemical modifications which determine a different protein behavior upon oxidative damage.
References:
[1] M.J. Davies, The oxidative environment and protein damage, Biochim. Biophys. Acta - Proteins Proteomics. 1703 (2005) 93-109.
[2] D.C. Thomas, The phagocyte respiratory burst: Historical perspectives and recent advances, Immunol. Lett. 192 (2017) 88-96.
[3] S.J. Klebanoff, Myeloperoxidase: friend and foe, J. Leukoc. Biol. 77 (2005) 598-625.
[4] C.C. Winterbourn, Comparative reactivities of various biological compounds with myeloperoxidase-hydrogen peroxide-chloride, and similarity of oxidant to hypochlorite, Biochim. Biophys. Acta - Gen. Subj. 840 (1985) 204-210.
[5] D.I. Pattison, M.J. Davies, Absolute rate constants for the reaction of hypochlorous acid with protein side chains and peptide bonds, Chem. Res. Toxicol. 14 (2001) 1453-1464.
[6] D.I. Pattison, C.L. Hawkins, M.J. Davies, What are the plasma targets of the oxidant hypochlorous acid? A kinetic modeling approach., Chem. Res. Toxicol. 22 (2009) 807-17.
[7] C. Hawkins, M. Davies, Hypochlorite-induced damage to proteins: formation of nitrogen-centred radicals from lysine residues and their role in protein fragmentation, Biochem. J. 625 (1998) 617-625.
[8] A. Müller, S. Langklotz, N. Lupilova, K. Kuhlmann, J.E. Bandow, L.I.O. Leichert, Activation of RidA chaperone function by N-chlorination., Nat. Commun. 5 (2014) 5804.
[9] A. Ulfig, A. V. Schulz, A. Müller, N. Lupilov, L.I. Leichert, N-chlorination mediates protective and immunomodulatory effects of oxidized human plasma proteins, BioRxiv. (2019) 584961.
[10] A. Del Giudice, C. Dicko, L. Galantini, N. V. Pavel, Structural Response of Human Serum Albumin to Oxidation: Biological Buffer to Local Formation of Hypochlorite, J. Phys. Chem. B. 120 (2016) 12261-12271.
[11] T. Peters, All About Albumin, Elsevier, 1995.
[12] M. Roche, P. Rondeau, N.R. Singh, E. Tarnus, E. Bourdon, The antioxidant properties of serum albumin, FEBS Lett. 582 (2008) 1783-1787.
[13] G. Colombo, M. Clerici, D. Giustarini, R. Rossi, A. Milzani, I. Dalle-Donne, Redox albuminomics: oxidized albumin in human diseases., Antioxid. Redox Signal. 17 (2012) 1515-27.
[14] D.J. Vocadlo, G.J. Davies, R. Laine, S.G. Withers, Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate, Nature. 412 (2001) 835-838.
[15] C.L. Hawkins, M.J. Davies, Inactivation of protease inhibitors and lysozyme by hypochlorous acid: Role of side-chain oxidation and protein unfolding in loss of biological function, Chem. Res. Toxicol. 18 (2005) 1600-1610.
[16] M.S. Petrônio, V.F. Ximenes, Effects of oxidation of lysozyme by hypohalous acids and haloamines on enzymatic activity and aggregation, Biochim. Biophys. Acta - Proteins Proteomics. 1824 (2012) 1090-1096.
[17] P. Arosio, T.C.T. Michaels, S. Linse, C. Månsson, C. Emanuelsson, J. Presto, J. Johansson, M. Vendruscolo, C.M. Dobson, T.P.J. Knowles, Kinetic analysis reveals the diversity of microscopic mechanisms through which molecular chaperones suppress amyloid formation, Nat. Commun. 7 (2016) 10948.
[18] M. Zaffagnini, C.H. Marchand, M. Malferrar, S. Murail, S. Bonacchi, D. Genovese, M. Montalti, G. Venturoli, F. G., M. Baaden, S.D. Lemaire, S. Fermani, P. Trost, Glutathionylation primes soluble GAPDH for late collapse into insoluble aggregates, BioRxiv. (2019) 545921.
[19] M.R.H. Krebs, G.L. Devlin, A.M. Donald, Protein particulates: Another generic form of protein aggregation?, Biophys. J. 92 (2007) 1336-1342.