Effects of Protein Glycation and Protective Mechanisms Against Glycative Stress

Abstract

Glycation is a post-translational modification of proteins that contributes to the vast array of biological information that can be conveyed via a singular proteome. Understanding the role of Advanced Glycation End-products (AGEs) in human health and pathophysiology can be difficult as the physiological effects of AGEs have been associated with multiple biological processes and disease state development including acute myocardial ischemia-reperfusion injury, heart failure, and atherosclerosis, as well as tumor cell migration. The critical role of the glyoxylase system in the detoxification of methylglyoxal and other AGEs has been well established. Recently, evidence has emerged that DJ-1 displays anti-glycative activity and may contribute to another mechanism of protection against protein glycation outside of the glyoxylase system. Identification of potential substrates of DJ-1 and determination of the pathways in which DJ-1 operates is needed to fully understand the role of this protein in modulating biological homeostasis and the development of disease.

Introduction

In regards to informational content, the proteome is more complex than the transcriptome and genome [1]. As such, the regulation of the quality and function of proteins is essential to cellular homeostasis. Under both physiological and pathophysiological conditions, the activation of cellular signaling pathways relies heavily on reversible post-translational modifications (PTMs) for controlling the function and stability of proteins [2]. PTMs are established and removed in a highly dynamic manner and exist in many different forms and flavors, such as glycosylation, phosphorylation, SUMOylation, and methylation, to name a few [3]. The purpose of this review article is to provide a brief overview of glycation, a process by which dicarbonyls react non-enzymatically with amino groups of proteins to form advanced glycation end-products (AGEs) [4]. We will also discuss emerging data demonstrating that the protein deglycase DJ-1 (PARK7) serves to oppose glycation in the heart.

Glycation

Glycation has been linked to a variety of biological processes from the mother-infant bond to aging as well as in the development of other widely distributed diseases including diabetic heart failure, chronic kidney disease, Parkinson’s disease, and Alzheimer’s disease [5]. The process of glycation is initiated by a chemical reaction in which reducing sugars/reactive dicarbonyls, such as glyoxal and methylglyoxal, bind to free amino groups in proteins, nucleotides, and lipids through Schiff base formation [6]. As described in Figure 1, Amadori rearrangement of the Schiff base leads to formation of AGEs. This reaction was first described by the French biochemist Louis Camille Maillard in 1912 in the context of the browning reaction observed by heating glucose and glycine [7]. How different sugars will react with different proteins depends on several factors including the size of the sugar molecule and the amino groups available on the protein. The initial alteration begins with the formation of fructosamine adducts through the reaction of glucose with N-terminal and lysyl side chain amino groups. Further reactions including rearrangement, dehydration, condensation, fragmentation, oxidation, and cyclization result in the formation of an array of AGEs, such as N-(carboxymethyl)-lysine (CML) [8]. Protein glycation most often leads to the loss of protein function or degradation of the protein. Essential in the detoxifying of AGEs are an enzymatic network of glyoxalases including glyoxalase 1 (GLO1), glyoxalase 2 (GLO2), and reduced glutathione (GSH) [9]. In the glyoxalase pathway, the presence of GSH is required for the catalytic isomerization of methylglyoxal to hemithioacetal. Next, Glo1 converts hemoithioacetal to the intermediate S-D-lactoylglutathione, which is then hydrolyzed by Glo2 to form nontoxic D-lactate and GSH. [9] It is estimated that around 99% of methylglyoxal is metabolized by the glyoxalase system [9]. When the build-up of AGEs outpaces the ability of the glycoxalases to detoxify them, glycation stress or dicarbonyl stress can occur. This excessive accumulation of dicarbonyl metabolites ultimately lead to this disruption of endogenous signaling cascades and contributes to the development of many pathophysiological states including diabetes, cardiovascular disease, neurodegenerative diseases, and cancer [10–12].

Figure 1. Process of protein glycation and potential downstream effects on physiology.

Through the nonenzymatic reaction between a nucleophilic free amino group of a protein and a reducing sugar, the process of glycation is initiated. Within hours, this reducing reaction, known as the Maillard reaction, leads to the formation of a highly unstable Schiff base. Over the course of days to weeks, this reversible reaction leads to the production of a more stable family of compounds known as Amadori products, such as hemoglobin A1c which is utilized to monitor blood glucose levels in people with diabetes. Additional rearrangements over weeks to months following the formation of an Amadori product leads to the production of Advanced Glycation End-Products or AGEs, such as carboxymethyl lysine (CML).

Glycation and Disease

Glycation products have been shown to possess both pro-inflammatory and pro-oxidant effects depending on the circumstance and thus have been found to be involved in a wide array of biological processes and human maladies. It is widely established that changes in rates of formation and degradation of altered proteins occur in a wide array of chronic disease states [13]. For example, research has shown that high excretion fractions of glycation products are linked to early decline in renal function in patients with type I diabetes [14]. Assessment of AGE accumulation in the skin via measurements of skin autofluorescence has served as a useful tool in predicting cardiac mortality in patients with diabetes [10,15,16].

One of the most studied glycative agents is glucose and applications of glycation biochemistry has been widely utilized in measurements of blood glucose concentrations. The well-known A1c measurement in diabetes care assesses levels of the glycation product HbA1c, which is an adduct of hemoglobin and glucose. Because this product is stable for several weeks, HbA1c reflects the mean blood glucose concentration and serves as a useful tool in the management of diabetes[17]. Research has also shown that the formation of β-sheet structures in proteins can be induced by glycation. Aggregation of these β-sheets occurs to form fibrillar structures resulting in several diseases including Alzheimer’s disease, Parkinson’s disease, prion disease and others [18]. In addition, increased oxidative stress due to protein glycation can trigger immune responses through cytokine signaling cascades and can lead to downstream tissue damage [19]. In cardiovascular disease development, the crosslinking of proteins due to the presence of AGEs can lead to stiffening of cardiac tissues resulting in high blood pressure and diastolic heart failure. Alterations to receptor binding sites and induction of ROS production can also affect heart health through chronic inflammation, development of atherosclerosis, and increased ischemia reperfusion injury [10].

AGE-RAGE Signaling

The accumulation of AGEs impacts cellular signaling and homeostasis through the activation of the receptor for AGEs (RAGE) [20], a multiligand receptor for the immunoglobulin superfamily [21]. When RAGE is activated, several signaling pathways are induced including the p21ras-MAP kinase pathway, the NADPH oxidase-ROS-NF-KB pathway and the Jak-Stat pathway, leading to inflammation and oxidative stress [22]. This further contributes to AGE generation and enhanced RAGE expression [23]. Thus, AGE-RAGE signaling maintains a vicious cycle that induces glycative stress, which contributes to cellular and tissue injury (Figure 2) [24]. Regarding cardiovascular disease, there are several lines of experimental data linking AGE-RAGE signaling to its development and progression. For instance, AGE-RAGE levels are increased in models of acute myocardial ischemia-reperfusion injury and heart failure [25–29]. Importantly, pharmacological antagonism of RAGE or genetic deletion of the receptor in these models has shown to be protective [25,26,30]. Further, RAGE deficient mice display less plaque accumulation in the arch, thoracic and abdominal aortas [31]. Additionally, therapeutic delivery of soluble RAGE (sRAGE) has been utilized successfully to inhibit the progression of atherosclerosis as well as kidney disease in diabetic mice [32]. In both in vitro and in vivo models of Alzheimer’s and Parkinson’s diseases, there has been some evidence that dietary supplements of polyphenols aid in the clearance of AGEs through autophagy [33]. This suggests that a nutraceutical approach is worth further exploration as a preentative or therapeutic option for these widespread diseases. As previously mentioned, Glo1 is an essential rate-limiting enzyme in the glutathione-dependent detoxification of the reactive carbonyl methylglyoxal (MG). In monocrotaline (MCT) rat models of pulmonary hypertension, overexpression of Glo1 by treatment with adeno-associated virus serotype 9 (AAV9) encoding a cardiac-specific Glo1 resulted in a reduction in total right ventricle protein glycation by about 50%, an increase in mitochondrial density, an elevation in fatty acid handling and FAO protein levels [34]. In addition, Glo1 transgenic mice are partially protected from myocardial infarction-induced cardiac dysfunction due to reduced cardiac inflammation [35]. Conversely, Glo1 deficient mice and those treated with a Glo1 inhibitor displayed significant increases in MG concentration [36–38]. In humans with carotid artery disease, significant reductions in Glo1 activity-to-protein ratio relative to control patients have been observed [39]. Additionally, Hanssen et al. have shown that rupture-prone plaques in humans display decreased Glo1 mRNA compared to stable, carotid atherosclerotic plaques [40] Together, this experimental data indicates that AGE-RAGE signaling contributes to the development of cardiovascular disease and that therapeutic antagonism of it might be a unique target for therapeutic intervention.

Figure 2. Proposed protective mechanisms against glycative stress.

High levels of reactive oxygen species, caused by general inflammation or by a myocardial infarction, results in the oxidation of lipids and glucose, which ultimately lead to the production of methylglyoxal (MG). The buildup of MG leads to protein glycation causing protein dysfunction and activation of the AGE-RAGE pathway in a negative feeback loop inducing additional inflammation. The glyoxalase system, consisting of Glo1, Glo2 and GSH, works to detoxify methyglyoxal to nontoxic D-Lactate, preventing the dangerous buildup of AGEs. DJ-1, which is activated by ROS and works to reduce glycative stress through its proteolytic activity, allowing proteins to maintain their cellular function. Both mechanisms appear to be important in the efforts to maintain cardiac function in response to stress conditions.

DJ-1

DJ-1 (PARK7, Parkinson’s Disease autosomal recessive, early onset 7) is a single-domain protein that is activated in response to various pathological stimuli [41–43]. It was originally associated with the development of Parkinson’s Disease. As such, much of the work centered on the characterization of DJ-1 has been focused on the brain, where it has been shown to have anti-apoptotic and anti-oxidative actions in several disease states [43–45]. However, DJ-1 is ubiquitously expressed throughout the body, including the heart, where it has been shown to have protective effects in models of acute myocardial ischemia-reperfusion injury and heart failure [28,46–48]. The mechanism(s) by which DJ-1 exerts its protective effects have not been fully characterized. DJ-1 localizes to the cytosol, nucleus, and mitochondria [48] where it regulates different cellular pathways to influence the cellular oxidative stress response, apoptosis, RNA-binding and transcriptional regulation [43–45]. DJ-1 is a small, dimeric, single-domain protein [49]. As such, it must have multiple functions that would explain its ability to regulate all the pathways, or it has a single function that can explain them all. Functionally, DJ-1 has been characterized as a protease, a molecular chaperone, and a transcriptional co-activator. The proteolytic activity of DJ-1 has been shown to be important to its cellular actions, as, studies have revealed that this activity is induced by oxidative stress [45,50–53]. Specifically, under oxidative stress conditions, DJ-1 undergoes a C-terminal cleavage of a 15aa peptide to an active form that possesses 26 times more proteolytic activity compared to its full-length counterpart and is more effective in providing cytoprotection against oxidative injury.

DJ-1 and Glycation

The physiological role for the proteolytic activity of DJ-1 is not completely understood. In recent years, there has been some compelling evidence that the proteolytic activity of DJ-1 acts in part to oppose glycation. Specifically, DJ-1 has been reported to be a glutathione-independent glyoxalase.[6] Additionally, two recent studies suggest that DJ-1 is a deglycase rather than a glyoxalase.[54,55] This characterization is important because a glyoxalase would metabolize the reactive carbonyls or AGE precursors, whereas a deglycase would remove the glycation moiety from a specific protein. Under this characterization, a deglycase would be considered a repair protein. The Richarme group argues that the kinetics of methylglyoxal degradation in the presence of recombinant DJ-1 (observed by both groups [6]) displays a lag, which results from the spontaneous formation of the substrate, glycated DJ-1 or glycated BSA. They also argue that the apparent glyoxalase activity of DJ-1 increased in a linear fashion with the square of DJ-1 concentration, in accordance with DJ-1 being both an enzyme (deglycase) and a substrate (glycated DJ-1). Together, they suggest that these results are consistent with DJ-1 substrates being glycated proteins instead of glyoxals. More importantly, they demonstrated that DJ-1 removes glycation moieties from lysine/arginine residues of proteins. Fernandes et al has confirmed this substrate binding capacity of DJ-1 to lysine and arginine sites [56] Neither group disputes the anti-glycative actions of DJ-1, just the way in which it performs the actions.

Recently, Shimizu et al [28] provided a physiological context to the anti-glycative actions of DJ-1 with evidence that it is an endogenous cytoprotective protein that protects against the development of ischemia-reperfusion–induced heart failure by reducing glycative stress. Together, this evidence provides some insights into the mechanism by which DJ-1 elicits its protective effects. Building on this evidence, Pantner et al [29] sought to provide insights into the cellular substrates that DJ-1 protects from glycation. In this study, the authors focused on the mitochondria. Breakdown of mitochondrial function can result in cell or organ injury through synthesis of damaging metabolites, generation of reactive oxygen species and dysregulation of apoptosis pathways. Within the eukaryotic cell, mitochondria are the site of energy production and cellular respiration, making them a critical component of the cellular redox system. In cardiomyocytes specifically, mitochondria occupy 35% of volume and provide 90% of ATP through oxidative phosphorylation [57]. Therefore, mitochondria play a critical role in the pathogenesis of many cardiovascular diseases including ischemia-reperfusion injury, heart failure and other myopathies of the heart [54]. Hence, maintaining cardiac mitochondrial health and function is critical to reducing myocardial ischemic/reperfusion injury. Mitochondria are susceptible to glycation, as there is evidence to suggest that glycation inhibits the activity of respiratory chain complexes [58,59]. Pantner et al [29] found that within 4 hours after reperfusion, the expression of both the full-length and cleaved forms of DJ-1 are both elevated in cardiac mitochondria [29]. Overexpression of the cleaved form of DJ1 in wild-type and DJ-1 KO mice through administration of and adeno-associated virus showed reduced glycation and enhanced activity of mitochondrial Complex I and III along with increased ATP synthesis. This suggests that cardiac DJ-1 maintains Complex I and Complex III efficiency and mitochondrial function during the recovery from ischemia-reperfusion injury through its anti-glycative actions. Further studies are warranted to determine additional cellular substrates that DJ-1 could potentially protect from glycation.

Summary and Future Directions

To maintain functionally acceptable levels of AGEs, cells must possess detoxification mechanisms which reduce the availablity AGE precurors or deglycation mechanisms which allow for the cleavage of glycation residues from proteins or the degradation and recycling of entire glycated proteins. The importance of glyoxalases in reducing the buildup of methylglyoxal and downstream AGEs is well established. In recent years within glycation research, DJ-1 has emerged as a novel route to modulating protein glycation and a potentially critical component of recovery mechanisms following ischemia of the heart. Deficiency of DJ-1 results in greater accumulation of reactive dicarbonyls in cardiac mitochondria following myocardial infarction, which can give rise to unfavorable levels of reactive oxygen species and lipid peroxidation, ultimately leading to an increase in inflammation and potential cardiac dysfunction. Given the extreme importance of functional mitochondria for maintenance of energy levels in cardiomyocytes, the enhanced glycation of Complex I and Complex III due to DJ-1 deficiency suggests an important role for this protein in heart energy homeostasis, likely through its deglycase capabilities (Figure 2). However, the proteins substrates on which DJ-1 exercises this deglycative action have not been fully elucidated. The low abundance of many AGEs combined with the sheer number of potential unique sugar moieties have made further elucidation of the role of glycation in development disease states challenging. Fortunately, more advanced proteomics techniques alongside improvements in biomarker development have primed the field of glycation research for major leaps forward towards diagnostic and therapeutic applications [60].

Agonists that limit accumulation of AGEs prevent complications in animal models, suggesting glycation as a useful target of research aimed at reducing diabetes-, heart failure-, and aging-related complications in humans [8]. Deciphering signaling pathways involved in the process of glycation can expand understanding of post-translational modifications, in general, as well as reveal molecular mechanisms which could lead to disruption in cellular homeostasis or disease states. Determining the specific recipients of protein glycation/deglycation, like the substrates of DJ-1, could reveal previously unknown protein functions and potential therapeutic targets for glycation-related diseases.