Research Article: Origin and pathophysiology of protein carbonylation, nitration and chlorination in age-related brain diseases and aging

Date Published: May 17, 2018

Publisher: Impact Journals

Author(s): Efstathios S. Gonos, Marianna Kapetanou, Jolanta Sereikaite, Grzegorz Bartosz, Katarzyna Naparło, Michalina Grzesik, Izabela Sadowska-Bartosz.


Non-enzymatic protein modifications occur inevitably in all living systems. Products of such modifications accumulate during aging of cells and organisms and may contribute to their age-related functional deterioration. This review presents the formation of irreversible protein modifications such as carbonylation, nitration and chlorination, modifications by 4-hydroxynonenal, removal of modified proteins and accumulation of these protein modifications during aging of humans and model organisms, and their enhanced accumulation in age-related brain diseases.

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Aging, an inevitable part of the life process, is characterized by a progressive decline in physiological functions that ultimately leads to morbidity and mortality. Aging increases susceptibility to certain class of diseases. Age-related diseases constitute a considerable socioeconomic burden for contemporary societies. As human mean lifespan increases, growing incidence of these diseases has features of a pandemic. The number of people aged 65 or older is projected to grow from an estimated 524 million in 2010 to almost 1.5 billion in 2050, mostly in underdeveloped and developing countries [1]. These trends have obvious serious social and economic implications, such as healthcare costs [2].

Compared to other oxidative modifications, carbonyls are relatively difficult to induce and in contrast to, for example, methionine sulfoxide and cysteine disulfide bond formation, carbonylation is an irreversible oxidative process [11]. Protein carbonylation is an oxidative modification induced by ROS, RNS, RXS and reactive aldehydes. It consists in formation of reactive aldehyde or ketone residues on proteins, which can react with 2,4-dinitrophenylhydrazine (DNPH) forming hydrazones. There are two ways of protein carbonylation. “Primary protein carbonylation” is due to oxidation of some amino acid residues, initiated by ROS, RNS and RXS, often catalyzed by metals while “secondary protein carbonylation” is caused by addition of aldehydes. The aldehydes are formed mainly in the process of lipid peroxidation [malondialdehyde, MDA; 4-hydroxy-2,3-trans-nonenal, (4-HNE); 2-propenal (acrolein, ACR)], but may be also by-products of glycolysis and the glycation process (methylglyoxal, glyoxal).

Protein carbonyl content is the most general and broadly used biomarker of oxidative protein damage and, more generally, OS. However, protein carbonyls are important not only as a biomarker for protein oxidation in aging and disease. They have also been shown to impair protein structure and function and to participate in the etiology and progress of diseases and age-related changes in the body [39,40]. Carbonylation may alter the conformation of the polypeptide chain, which leads to partial or total inactivation of proteins. The consequent loss of function or structural integrity of carbonylated proteins can have a wide range of downstream functional consequences and may underlie the subsequent cellular dysfunctions and tissue damage [41]. Protein carbonylation was demonstrated to modify activities of enzymes and other protein functions like DNA binding of transcription factors [42]. Carbonylation can lead to functional impairment of proteins involved in insulin signaling, so the insulin signaling pathway gets disrupted by carbonylation [43].

It should be noted that protein Tyr nitration is observed in vivo in healthy tissues, indicating that there is a basal flux of RNS; nevertheless, physiological nitration levels are typically low. Possible biochemical consequences of protein Tyr nitration involve changes in activity (usually loss, but sometimes gain of function), induction of immune responses, interference with tyrosine-kinase-dependent pathways, alteration of protein assembly and polymerization, and effects of protein turnover: either facilitation of protein degradation or induction of formation of proteasome-resistant protein aggregates, depending on the dose [103,104].

Chlorinative stress undoubtedly contributes to the pathogenesis of neurodegenerative diseases [143]. In brain, chloride ions are present at the concentration of 10-2 – 10-1 M [144]. HOCl can be generated with the activation of microglia and myeloperoxidase secretion [145–148]. Moreover, infiltration of monocyte/macrophage and neuronal expression of myeloperoxidase also contribute to the formation of HOCl [149,150]. Supposedly, the brain has poor defence system against HOCl [79,151,152]. Thus, the toxicity of HOCl towards central nervous system tissue was shown [153–155]. Furthermore, MPO was reported to be expressed with increased levels in the cerebral tissue of patients affected by AD [156] and 3-chlorotyrosine as a biomarker of HOCl production was detected in proteins from AD hippocampus. The level of 3-chlorotyrosine in the samples from diseased brain was three-fold higher compared to control samples [150]. Halogenation has a clear effect on the self-assembly of the amyloid β peptide aggregates [157]. However, it can be concluded that a role of protein chlorination in neurodegenerative diseases is not analysed completely yet.

A special class of protein modification products, consisting of oxidized, dityrosine-containing, crosslinked proteins formed mainly by reactions of RXS with plasma proteins, predominantly albumin, are so-called advanced oxidation protein products (AOPP). In vivo, the generation of chlorinated oxidants is a feature of phagocytic cells containing MPO [158,159]. Witko-Sarsat et al. [160] first reported elevated plasma level of AOPPs in uremic patients. High levels of AOPPs were detected in patients on maintenance hemodialysis, followed by those on peritoneal dialysis. Patients with advanced chronic renal failure not yet on dialysis had almost three times higher AOPP levels than healthy subjects.

Post-mitotic neurons are notably vulnerable to lipid peroxidation since the brain has high levels of polyunsaturated fatty acids, high levels of redox transition metal ions, high oxygen consumption, relatively low levels of low-molecular weight antioxidants and antioxidant enzymes. Peroxidation of polyunsaturated fatty acids, especially linoleic acid, linolenic acid and arachidonic acid by non-enzymatic processes leads to the formation of aldehydes, among them 4-HNE is present at very low concentration in plasma, in the range of 0.28–0.68 μM under physiologic conditions, but its concentration in cells, where it is produced, may be higher (≤5 μM) [173]. 4-HNE concentration can be increased as much as by 100 times under OS conditions [174]. Esterbauer’s group demonstrated that 4-HNE formation from arachidonic acid is greater in the presence of NADPH-dependent microsomal enzymes [175]. 4-HNE possesses three reactive functions: a C2=C3 double bond, a C1=O carbonyl group and a hydroxyl group on C4. These functions make this electrophilic molecule highly reactive toward nucleophilic thiol and amino groups. 4-HNE can enter the reaction of Michael addition to thiol or amino groups, which involves the C3 of the C2=C3 double bond or can form Schiff bases between the C1 carbonyl group and primary amines. The kinetics of the Schiff base formation is slow and reversible, making Michael-adducts predominant adducts of 4-HNE to proteins. 4-HNE reacts mainly His, Cys and Lys residues in proteins [176,177] (Fig. 1F, Fig. 2). The formation of the 4-HNE-protein adducts is a bioactive marker of pathophysiological processes [178–180]. 4-HNE forms Michael adducts with enzyme peptidylprolyl cis/trans-isomerase A1 (Pin1), which catalyzes conversions of phosphoserine and phosphothreonine-proline from cis to trans conformation. These adducts were detected by matrix-assisted laser desorption ionization/time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry at the active site residues His157 and Cys113, with Cys113 being the primary site of 4-HNE modification [181–185]. Protein modifications by 4-HNE impairs glutamate and glucose transport, disrupts Ca2+ homeostasis, damages cholinergic neurons thus impairing visuospatial memory and induces apoptosis in PC12 cells (cell line derived from a pheochromocytoma of the rat adrenal medulla) and cultured rat hippocampal neurons [186–188]. Nam et al. (2014) compared N-methyl-D-aspartate receptor type 1 (NMDAR1) and 4-HNE in the hippocampus of D-galactose (D-gal)-induced and naturally aging models of mice [189]. These authors observed an age-dependent reduction of NMDAR1 and an increase in 4-HNE in the dentate gyrus, CA1 and CA3 regions of the hippocampus via immunohistochemistry and Western blot analyses. In the D-gal-induced chemical aging model they noted similar changes in NMDAR1 and 4-HNE although the degree of reduction/increase in NMDAR1/HNE was not as severe as that in the naturally aged mice.

The blood brain barrier (BBB) separates the brain and blood with a large surface area (between 12 and 18 m2 in the average human adult) [208,209]. The opposing membranes of endothelial cells are connected by tight junctions, which are formed through an intricate network of interacting proteins such as claudins, occludin, junctional adhesion molecules and cytoplasmic proteins [210]. Nitta et al. (2003) demonstrated that claudin-5 is a critical determinant of BBB permeability [211]. In the process of healthy aging an increased “leakage” of BBB may occur, not only due to alteration of thickness of basal lamina, endothelial cells, morphology of pericytes and astrocytes, but also as a result of the changes in expression of transporter proteins at the endothelial cell layer of BBB [212]. Bors et al. (2018) reported that the number of tight junctions decreases, the thickness of basal lamina increases as well as the size of astrocyte endfeet extends with advanced age. These authors also demonstrated that the function of P-glycoprotein 1 (P-gp, ABCB1 Abcb1a/Mdr1a), the most important efflux transporter located on the luminal surface of brain capillary endothelial cells is reduced in old Wistar rats [213]. Reduced BBB expression of P-gp was associated with increased brain parenchymal Aβ40 and Aβ42 levels in aged rats [214], in agreement with the idea that P-gp is an important efflux transporter to remove Aβ from the CNS [215]. Pan et al. (2018) showed that low density lipoprotein receptor-related protein 1 (LRP-1) expression declines with age, which may contribute to Aβ accumulation [209]. Van Assema et al. (2012) studied in vivo effects of gender and aging on human BBB P-gp function in a large sample size using PET and (R)-[11C]verapamil. These authors reported that decreased BBB P-gp is found with aging; nevertheless, effects of age on BBB P-gp function differ between men and women [216].

The level of posttranslationally modified proteins is a resultant of the rate of protein modification and rate of removal of modified proteins. Aging, as well as several age-related diseases are associated with a decreased ability to maintain proteostasis [221]. All cells have a number of quality control mechanisms in order to maintain the stability and functionality of their proteome. The proteostasis network includes both protein stabilization mechanisms (major heat shock proteins) and protein degradation systems (proteasome and lysosome) [222–224]. In addition, there are several modulators of proteotoxicity (like MOAG-4), that operate through distinct pathways [42]. All these systems work in concert to restore the structure of denatured proteins or to promote their degradation, thus preventing the accumulation of damaged components and ensuring the continuous renewal of the intracellular polypeptides. Many studies have shown that aging is accompanied by failure of proteostasis [225], while chronic exposure to denatured or aggregated proteins contributes to the development of age-related neurodegenerative diseases such as AD and PD [221,226].

Abundant evidence demonstrates accumulation of products of protein modifications by ROS, RNS and RXS during aging of humans and model organisms and enhanced accumulation of such products in age-related diseases. New methods of analysis, based mainly on the MS technique, became available allowing for more precise identification of protein modifications and perhaps introduction of specific disease markers. Elucidation of the role of such modifications in aging-related changes and in the progress of diseases is more difficult. Are they only markers or aging and diseases or play a primary role in their development? There are reasons to not exclude the second possibility as these modifications adversely affect protein functions and interactions. Prospective and intervention studies may be helpful in this respect and may point to the possible role of specific protein modifications as possible early disease markers.




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