Increased Protein S-Glutathionylation in Leber’s Hereditary Optic Neuropathy (LHON)

Abstract

Leber’s hereditary optic neuropathy (LHON, MIM#535000) is the most common form of inherited optic neuropathies and mitochondrial DNA-related diseases. The pathogenicity of mutations in genes encoding components of mitochondrial Complex I is well established, but the underlying pathomechanisms of the disease are still unclear. Hypothesizing that oxidative stress related to Complex I deficiency may increase protein S-glutathionylation, we investigated the proteome-wide S-glutathionylation profiles in LHON (n = 11) and control (n = 7) fibroblasts, using the GluICAT platform that we recently developed. Glutathionylation was also studied in healthy fibroblasts (n = 6) after experimental Complex I inhibition. The significantly increased reactive oxygen species (ROS) production in the LHON group by Complex I was shown experimentally. Among the 540 proteins which were globally identified as glutathionylated, 79 showed a significantly increased glutathionylation (p < 0.05) in LHON and 94 in Complex I-inhibited fibroblasts. Approximately 42% (33/79) of the altered proteins were shared by the two groups, suggesting that Complex I deficiency was the main cause of increased glutathionylation. Among the 79 affected proteins in LHON fibroblasts, 23% (18/79) were involved in energetic metabolism, 31% (24/79) exhibited catalytic activity, 73% (58/79) showed various non-mitochondrial localizations, and 38% (30/79) affected the cell protein quality control. Integrated proteo-metabolomic analysis using our previous metabolomic study of LHON fibroblasts also revealed similar alterations of protein metabolism and, in particular, of aminoacyl-tRNA synthetases. S-glutathionylation is mainly known to be responsible for protein loss of function, and molecular dynamics simulations and 3D structure predictions confirmed such deleterious impacts on adenine nucleotide translocator 2 (ANT2), by weakening its affinity to ATP/ADP. Our study reveals a broad impact throughout the cell of Complex I-related LHON pathogenesis, involving a generalized protein stress response, and provides a therapeutic rationale for targeting S-glutathionylation by antioxidative strategies.

A plasma metabolomic signature of Leber hereditary optic neuropathy showing taurine and nicotinamide deficiencies

“…Taurine is present in high concentration in oxidative tissues, where it plays a role in stabilizing the inner mitochondrial membrane gradient and the equilibrium between NAD/NADH and GSH/GSSG (oxidized and reduced forms of glutathione) ( 41 ). Interestingly, we have recently reported a profound impact of the glutathione redox imbalance in LHON fibroblasts associated with the increased oxidative stress and at the origin of an increased level of glutathionyled proteins ( 19 ). Taurine is also a cytoprotective nutriment that ensures normal complex I and ER functions, both of which are altered in LHON ( 11 , 19 , 42 ).…”

“…Interestingly, we have recently reported a profound impact of the glutathione redox imbalance in LHON fibroblasts associated with the increased oxidative stress and at the origin of an increased level of glutathionyled proteins ( 19 ). Taurine is also a cytoprotective nutriment that ensures normal complex I and ER functions, both of which are altered in LHON ( 11 , 19 , 42 ). The taurine deficiency may be an attractive therapeutic target given its protective properties, especially since such protective effect has already been demonstrated in the retina ( 43 ).…”

“…Nevertheless, these data-driven and hypothesis-free approaches disclosed unexpected alterations of cell metabolism, going well beyond the mitochondrial respiration defects. For instance, we, along with others, saw evidence of a generalized stress protein response in patient fibroblasts by transcriptomics ( 17 ), metabolomics ( 11 ) and proteomics analyses ( 18 , 19 ). Since protein synthesis is one of the most energy-consuming molecular mechanisms ( 20 ), it is not surprising that altered mitochondrial energetic metabolism would have an impact on this mechanism.…”

Oxidative Stress in Optic Neuropathies

“…The plasma of LHON patients (m.11778G>A, m.14484T>C, or m.3460G>A) also showed higher levels of AOPP [136]. Furthermore, fibroblast proteins in LHON patients (m.11778G>A, m.14484T>C, or m.3460G>A) exhibit increased S-glutathionylation [150], which refers to the reversible binding of glutathione to cysteine residues in target proteins as a consequence of direct oxidation or due to thiol-disulfide exchange, leading to loss of function [203]. This protein modification was shown to affect proteins related to energy metabolism, catalytic activity, and cell protein quality control [150].…”

“…Furthermore, fibroblast proteins in LHON patients (m.11778G>A, m.14484T>C, or m.3460G>A) exhibit increased S-glutathionylation [150], which refers to the reversible binding of glutathione to cysteine residues in target proteins as a consequence of direct oxidation or due to thiol-disulfide exchange, leading to loss of function [203]. This protein modification was shown to affect proteins related to energy metabolism, catalytic activity, and cell protein quality control [150]. Importantly, inhibition of Complex I in fibroblasts from healthy subjects led to a similar pattern of S-glutathionylation, indicating that Complex I misfunction is primarily responsible for this oxidative stress-related molecular process [150].…”