Redox-dependent thiol modifications: implications for the release of extracellular vesicles

Abstract

Extracellular vesicles (EVs), including microvesicles and exosomes, are emerging as important regulators of homeostasis and pathophysiology. During pro-inflammatory and pro-oxidant conditions, EV release is induced. As EVs released under such conditions often exert pro-inflammatory and procoagulant effects, they may actively promote the pathogenesis of chronic diseases. There is evidence that thiol group-containing antioxidants can prevent EV induction by pro-inflammatory and oxidative stimuli, likely by protecting protein thiols of the EV-secreting cells from oxidation. As the redox state of protein thiols greatly impacts three-dimensional protein structure and, consequently, function, redox modifications of protein thiols may directly modulate EV release in response to changes in the cell's redox environment. In this review article, we discuss targets of redox-dependent thiol modifications that are known or expected to be involved in the regulation of EV release, namely redox-sensitive calcium channels, N-ethylmaleimide sensitive factor, protein disulfide isomerase, phospholipid flippases, actin filaments, calpains and cell surface-exposed thiols. Thiol protection is proposed as a strategy for preventing detrimental changes in EV signaling in response to inflammation and oxidative stress. Identification of the thiol-containing proteins that modulate EV release in pro-oxidant environments could provide a rationale for broad application of thiol group-containing antioxidants in chronic inflammatory diseases.

Keywords: Chronic inflammation; Exosomes; Microvesicles; N-acetyl-L-cysteine; Redox environment; Sulfhydryl groups.

Fig. 1

Schematic representation of thiol modifications by ROS, RNS and RCS. (1) Thiol oxidation by ROS leads to formation of an unstable cysteine sulfenic acid intermediate. This unstable intermediate can react with other thiol groups within the same or another molecule, which leads to formation of intramolecular or intermolecular disulfide bonds. (2) Thiol modification by RNS such as nitric oxide (NO) or peroxynitrite (ONOO⁻) leads to S-nitrosylation or S-nitration. (3) Thiol modification by RCS causes formation of relatively large and bulky carbonyl S-adducts as illustrated here for acrolein. This figure was created using Servier Medical Art

Fig. 2

Known modulators of EV release that are directly or indirectly regulated by redox-sensitive thiols. Active proteins are represented in green and inactive proteins in purple. Disulfide bonds (–S–S–) in this figure are used representatively for all oxidative thiol modifications. a Several calcium channels become activated upon thiol oxidation, resulting in calcium influx and increased cytoplasmic calcium concentration. b Upon the thiol-dependent calcium influx, SNAREs mediate calcium-dependent fusion of MVBs with the plasma membrane, resulting in exosome release. c Reduced, but not oxidize NSF catalyzes the separation of v-SNAREs and t-SNAREs, allowing their recovery for repeated membrane fusion events. d Flippases ensure localization of PE and PS in the inner membrane leaflet. Upon thiol oxidation or upon thiol-dependent calcium influx, the enzymatic activity of flippase is inhibited, resulting in accumulation of PE and PS in the outer membrane leaflet and consequently, in membrane blebbing. e Upon thiol-dependent calcium influx, scramblase becomes activated, allowing PE and PS to diffuse to the outer membrane leaflet, enhancing membrane blebbing. f The actin cytoskeleton depends on reduced thiols for retracting membrane blebs. Oxidation of actin thiols causes depolymerization of actin filaments and impairs their functionality. Moreover, actin filaments can be degraded by calpains, cysteine proteases which are activated by cytoplasmic calcium but inactivated by thiol oxidation. NSF, N-ethylmaleimide sensitive factor; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine SNARE, soluble NSF attachment protein receptor; t-SNARE, target membrane-associated SNARE; v-SNARE, vesicle-associated SNARE. This figure was created using Servier Medical Art