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To adhere or not to adhere? This is the disulfide exchange question!

Submitted by redoxoma on
Foto by Chris Reading (https://pixabay.com/users/chrisreadingfoto-2723427/), under Pixabay License

Highlights by Marcela Franco Mineiro, PhD, from Instituto de Química da USP

Marcela got her PhD in 2019 under supervision of Flavia Carla Meotti at the Laboratory of Redox Processes in Inflammation, Department of Biochemistry, Institute of Chemistry, University of São Paulo, Brazil

The term ‘adhesion’ is simply defined as ‘steady or firm attachment’ [1]. In cell biology, however, the adhesion process is far more complex and involves countless well-orchestrated reactions and interactions between proteins at both extra and intracellular surface of the plasma membrane. Intra and intermolecular disulfide exchange in these proteins is one of the most relevant mechanism that drives cell adhesion/detachment. Therefore, oxidizing and reducing agents can directly affect cell adhesion. Nonetheless, it is hard to infer whether an oxidant would increase or decrease this phenomenon because adhesion involves disulfide linkage/break at specific cysteine residues within each protein. It is well known that catalytic cysteines from extracellular cell surface protein disulfide isomerases (PDIs, of which the prototype is PDIA1, refered to as PDI) are crucial in cell adhesion and disulfide exchange between PDI and integrin takes place during the adhesion process [2, 3]. Of relevance, our group has shown that cysteines at the catalytic site of PDI are rapidly oxidized by urate hydroperoxide (6 × 10³ M⁻¹s⁻¹) [4]; this is a peroxide generated from uric acid in the inflammatory oxidative burst [5]. In fact, the oxidation of PDI by urate hydroperoxide is much faster than that by glutathione disulfide (188 M⁻¹s⁻¹) [6] or hydrogen peroxide (17.3 M⁻¹s⁻¹) [7], but slower than the oxidation by peroxynitrite (6.9 × 10⁴ M⁻¹s⁻¹) [7]. We found that the oxidation of extracellular cell surface PDI by urate hydroperoxide impaired the adherence of vascular endothelial cells to fibronectin in the same way as the thiol alkylating p-CMBS, the PDI inhibitor Rutin and the integrin blocking peptide RGDS. Interestingly, adhesion was markedly inhibited in the first 30 min and, to the exception of the treatment with the irreversibly thiol alkylating p-CMBS, cells were able to adhere after 90 min of treatment [4]. These results show that the oxidation of thiols and inhibition of PDI or integrin disrupt cellular adhesion in a transient way. However, the continuous production of oxidants, as in vascular inflammation, might further recover cell adhesion. Since urate hydroperoxide can be formed extracellularly and efficiently targets cell surface PDI affecting cell adhesion, it might be a mechanism underlying the known vascular endothelial dysfunction described for uric acid. Analogous effects of other vascular oxidants in cell adhesion remain to be investigated.

References

  1. Merriam-Webster [on-line] 2020. | url: https://www.merriam-webster.com/dictionary/adhesion
  2. N. Rosenberg, R. Mor-Cohen, V. H. Sheptovitsky, O. Romanenco, O. Hess, J. Lahav. Integrin-mediated cell adhesion requires extracellular disulfide exchange regulated by protein disulfide isomerase Experimental Cell Research, 381(1): 77–85, 2019. | doi: 10.1016/j.yexcr.2019.04.017
  3. A. I. Soares Moretti, F. R. Martins Laurindo. Protein disulfide isomerases: Redox connections in and out of the endoplasmic reticulum Archives of Biochemistry and Biophysics, 617: 106–19, 2017. | doi: 10.1016/j.abb.2016.11.007
  4. M. F. Mineiro, E. de S. Patricio, Á. S. Peixoto, T. L. S. Araujo, R. P. da Silva, A. I. S. Moretti, F. S. Lima, F. R. M. Laurindo, F. C. Meotti. Urate hydroperoxide oxidizes endothelial cell surface protein disulfide isomerase-A1 and impairs adherence Biochimica et Biophysica Acta (BBA) - General Subjects, 1864(3): 129481, 2020. | doi: 10.1016/j.bbagen.2019.129481
  5. R. P. Silva, L. A. Carvalho, E. S. Patricio, J. P. Bonifacio, A. B. Chaves-Filho, S. Miyamoto, F. C. Meotti. Identification of urate hydroperoxide in neutrophils: A novel pro-oxidant generated in inflammatory conditions Free Radical Biology and Medicine, 126: 177–86, 2018. | doi: 10.1016/j.freeradbiomed.2018.08.011
  6. Á. S. Peixoto, R. R. Geyer, A. Iqbal, D. R. Truzzi, A. I. Soares Moretti, F. R. M. Laurindo, O. Augusto. Peroxynitrite preferentially oxidizes the dithiol redox motifs of protein-disulfide isomerase Journal of Biological Chemistry, 293(4): 1450–65, 2017. | doi: 10.1074/jbc.m117.807016
  7. A. Lappi, L. W. Ruddock. Reexamination of the Role of Interplay between Glutathione and Protein Disulfide Isomerase Journal of Molecular Biology, 409(2): 238–49, 2011. | doi: 10.1016/j.jmb.2011.03.024

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Inflammatory oxidative burst: a new actor in this scenario

Submitted by redoxoma on
neutrophil

Redoxoma Highlights by Flavia Carla Meotti

Activation of neutrophils either by invader microorganisms or by endogenous stimuli (sterile inflammation) triggers the assembling of NADPH oxidase at the membrane and the sequential production of free radicals and oxidants. This so-called inflammatory oxidative burst is initiated by the reduction of oxygen to superoxide, followed by the dismutation of superoxide to hydrogen peroxide. Hydrogen peroxide is a substrate to the heme-peroxidase, myeloperoxidase, oxidizing halides (mainly chloride) to hypohalous (hypochlorous) acid. Reaction between hydrogen peroxide and hypochlorous acid generates singlet oxygen. In parallel, the up-regulation of inducible nitric oxide synthase produces nitric oxide, which rapidly reacts with superoxide to form peroxynitrite. These are the main known players in the inflammatory oxidative burst. However, myeloperoxidase is a versatile enzyme and can effortlessly use hydrogen peroxide to oxidize other substrates in addition to halides. For instance, myeloperoxidase efficiently oxidizes uric acid to produce urate free radical, a short living reactive species that can rapidly combine with superoxide to generate an organic peroxide, urate hydroperoxide [1]. Although the formation of urate hydroperoxide was chemically feasible, the question was whether it would be biologically relevant since its precursor, urate free radical, could react with an unacountable number of biomolecules besides superoxide. This was one of the subjects addressed by our group at the Laboratory of Redox Process in Inflammation, CEPID-Redoxoma. By using mass spectrometry, we unambiguosly demonstrated the presence of urate hydroperoxide in stimulated peripheral blood neutrophils. Quantification of the amount of urate hydroperoxide that is formed by these inflammatory cells revealed that it is comparable to the amount of HOCl [2].

By analyzing the reactivity of urate hydroperoxide, we detected that it reacts preferentially with thiol groups in proteins and can rapidly oxidize the thiol peroxidases peroxiredoxin 1 (Prx1) and peroxiredoxin 2 (Prx2) [3]. These ubiquitous proteins are the fastest to react with hydrogen peroxide (~1 ⋅ 108 M-1s-1) [4] and have been proposed as hydrogen peroxide sensors, able to perform a signaling redox relay mechanism [5]. Urate hydroperoxide oxidizes purified Prx1 and Prx2 at the rate constants of 4.5 ⋅ 105 M-1s-1 and 2.3 ⋅ 106 M-1s-1, respectively, and oxidized Prx2 from intact erythrocytes at the same extent as hydrogen peroxide [3]. Together, these data suggest urate hydroperoxide as a new reported protagonist in the inflammatory oxidative burst. The oxidation of cytosolic Prx1 and Prx2 by urate hydroperoxide might affect cell function and be partially responsible for the harmfull effects attibuted to uric acid.

References

  1. F. C. Meotti, G. N. L. Jameson, R. Turner, D. T. Harwood, S. Stockwell, M. D. Rees, S. R. Thomas, A. J. Kettle. Urate as a Physiological Substrate for Myeloperoxidase Journal of Biological Chemistry, 286(15): 12901–11, 2011 | doi: 10.1074/jbc.m110.172460
  2. R. P. Silva, L. A. Carvalho, E. S. Patricio, J. P. Bonifacio, A. B. Chaves-Filho, S. Miyamoto, F. C. Meotti. Identification of urate hydroperoxide in neutrophils: A novel pro-oxidant generated in inflammatory conditions Free Radical Biology and Medicine, 126: 177–86, 2018 | doi: 10.1016/j.freeradbiomed.2018.08.011
  3. L. A. C. Carvalho, D. R. Truzzi, T. S. Fallani, S. V. Alves, J. C. Toledo, O. Augusto, L. E. S. Netto, F. C. Meotti. Urate hydroperoxide oxidizes human peroxiredoxin 1 and peroxiredoxin 2 Journal of Biological Chemistry, 292(21): 8705–15, 2017 | doi: 10.1074/jbc.m116.767657
  4. S. Portillo-Ledesma, L. M. Randall, D. Parsonage, J. Dalla Rizza, P. A. Karplus, L. B. Poole, A. Denicola, G. Ferrer-Sueta. Differential Kinetics of Two-Cysteine Peroxiredoxin Disulfide Formation Reveal a Novel Model for Peroxide Sensing Biochemistry, 57(24): 3416–24, 2018 | doi: 10.1021/acs.biochem.8b00188
  5. C. C. Winterbourn, M. B. Hampton. Signaling via a peroxiredoxin sensor Nature Chemical Biology, 11(1): 5–6, 2014 | doi: 10.1038/nchembio.1722

Flavia C. Meotti, Ph.D. Professor at Department of Biochemistry,
Institute of Chemistry, University of São Paulo, Brazil

 

Inflammation

 

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