NDT Advance Access published April 9, 2015 Nephrol Dial Transplant (2015) 0: 1–11 doi: 10.1093/ndt/gfv048
Full Review Emerging role of post-translational modifications in chronic kidney disease and cardiovascular disease
1
Institute for Molecular Cardiovascular Research (IMCAR), University Hospital RWTH Aachen, Aachen, Germany and
2
Department of Internal Medicine 4, Saarland University Hospital, Homburg/Saar, Germany
Correspondence and offprint requests to: Joachim Jankowski, E-mail: [email protected] * These authors contributed equally to the manuscript.
A B S T R AC T Post-translational modifications (PTMs) of proteins and peptides have recently gained much attention, as they are involved in the pathogenesis of cardiovascular disease and eventually also play a role in the progression of chronic kidney disease (CKD). In this review, we provide an overview of post-translational protein modifications such as carbamylation, glycation and oxidation, starting with their definitions, mechanisms and clinical relevance in the setting of CKD and cardiovascular disease. The methods currently used for the identification and, in particular, quantification of PTMs are described and potential treatment options in the context of PTMs are reviewed. We foresee that advancements in mass spectrometry-based methods leading to the identification of novel disease markers and/or pathophysiologically relevant factors will certainly boost the clinical utility in sample analyses. Keywords: carbamylation, cardiovascular disease, chronic kidney disease, oxidation, post-translational modification
INTRODUCTION Chronic kidney disease (CKD) and cardiovascular disease (CVD) are strongly interlinked with each other, and they share common risk factors such as diabetes mellitus or hypertension [1]. In the context of CKD and CVD, the interest towards the post-translational modifications (PTMs) of proteins has grown
© The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
considerably. Analysis of PTMs might be useful for the identification of mechanisms which play a role in the genesis and/or progression of CKD and CVD because proteins are constantly being exposed to different plasma and tissue components under different pathophysiological conditions. PTMs are covalent changes of proteins or peptides that are altered either by proteolytic cleavage or by adding moieties to one or more amino acids. This enhances their complexity with respect to regulation of activity state, subcellular localization, turnover and interaction with other cellular molecules [2]. PTM proteins and peptides have gained attention as biomarkers and/ or mediators of CVD and CKD [3, 4]. Here, we describe the most relevant currently known PTMs reported in relation to CKD and CVD. We used the PubMed database search to retrieve relevant literature with phrases like ‘(“Post translational modifications” OR “PTM’s”) AND (“Chronic kidney disease” OR “Chronic renal failure” OR “Cardiovascular disease”)’ and obtained 42 publications up to July 2014. Out of these, 16 were found to be actually relevant to the topic. Thus, we explored the PubMed database further, this time for articles related to different PTMs with relation to CKD and CVD like carbamylation, oxidation, glycosylation, nitrosylation, phosphorylation, citrullination and cystenylation. Here, we provide an overview of the most commonly reported PTMs such as carbamylation, glycation and oxidation, and their clinical relevance in the setting of CKD and CVD. Phosphorylation is omitted because it is mostly involved in cell signalling pathways. Although, other PTMs have been reported, to our knowledge their role is not clearly understood and are in its infancy.
1
Downloaded from http://ndt.oxfordjournals.org/ at SUNY Health Science Center at Brooklyn on April 12, 2015
Prathibha R. Gajjala1, Danilo Fliser2, Thimoteus Speer2, Vera Jankowski1,* and Joachim Jankowski1,*
C A R B A M Y L AT I O N Definition Carbamylation is a non-enzymatic spontaneous reaction of a primary amine or a free sulfhydryl group of protein with isocyanate [OCN]−. Isocyanate is the active form of cyanate, resulting in an addition of the carbamoyl moiety [−CONH2] to the functional groups of proteins [5]. Carbamylated proteins subsequently undergo structural and functional changes [6].
FULL REVIEW
Relevance in CKD Since kidney function declines as CKD progresses, metabolic substances like urea accumulate dramatically in the
F I G U R E 1 : Schematic representation of synthesis of carbamylated proteins/aminoacids. The source for urea and thiocyanate are from the urea
cycle, diet, smoking and air. Urea undergoes deamination to form cyanate and the MPO in the presence of hydrogen peroxide as cosubstrate converts thiocyanate to isocyanate. Cyanate and isocyanate are interconvertible and electrophilically attacks the amino or sulphur groups on proteins or amino acids to form respective carbamylated products.
2
P. R. Gajjala et al.
Downloaded from http://ndt.oxfordjournals.org/ at SUNY Health Science Center at Brooklyn on April 12, 2015
Mechanism Under physiological conditions, the nitrogenous waste content of the organism is removed in the form of urea CO (NH2)2. Urea is a by-product of the urea cycle, which slowly dissociates into cyanate and ammonia. Urea is generally found to be present at a ratio of 1 to 100 for cyanate to urea at equilibrium, which in turn depends on pH and temperature [7]. Cyanate, an electrophile, attacks nucleophilic groups like amino groups in proteins, and this results in carbamylation [5, 6]. Thereby, the positively charged lysines are neutralized, modifying its interaction with the surrounding environment. In vitro carbamylation on proteins has a strong effect on the protein
conformation, whereby its biological activity is modified [8]. However, it might have limited changes in in vivo situation. Furthermore, cyanate is synthesized from myeloperoxidase (MPO), which is present in the granules of neutrophils, monocytes and certain tissue macrophages. At the inflammatory sites and at atherosclerotic plaques, MPO utilizes H2O2 and isothiocyanate (SCN−) as a co-substrate, and produces the main metabolite cyanate [9]. Isocyanic acid reacts irreversibly with both alpha amino group of peptides, proteins or amino acids or epsilon amino group of lysine but it reacts faster with lower pKa values [8]. In general, the pKa value of alpha amino group is less when compared with the side-chain amino group of lysine. However, the pKa values of lysine side chain in protein show marked variation, sometimes being less than the N-terminal amino group which depends on the microenvironment where it is buried [10]. The ε-amino-carbamyl-lysine is also known as homocitrulline which is commonly used as a biomarker for the carbamylation. Figure 1 demonstrates the formation of carbamylated proteins/amino acids.
Emerging role of PTMs in CKD and CVD
3
FULL REVIEW
Relevance for CVD Besides CKD, carbamylated proteins are highly involved in the genesis of CVD, since carbamylated proteins are mainly formed during inflammation as a result of the MPO-mediated cyanate formation. Recently, it was demonstrated that carbamylated HDL reduces the lecithin–cholesterol acyl transferase
(LCAT) activity which is essential for cholesterol esterification and HDL maturation. HDLs are also involved in lipid droplet formation in macrophages, resulting in the accumulation of cholesterol and thus the lipid droplet formation. ApoA-I, a component of HDL, is a major activator of LCAT; an elimination of its positive charge at the active site suppresses the LCAT activity leading to dysfunctional HDL [20]. HDLs are selective targets for carbamylation in atherosclerotic lesions, since apoA-I carbamyl-lysine’s presence is enhanced as the stage of lesions progresses. Carbamylation of LDL represents a well-studied example on how carbamylation of lysine residues may alter distinct biological properties of proteins. In 2005, Ok et al. demonstrated that cLDL induces endothelial apoptosis and proliferation of vascular smooth muscle cells; both processes are involved in the pathogenesis of atherosclerotic CVD [21]. This may be at least particularly relevant in patients with CKD in whom total plasma carbamylation was reported to be increased. Moreover, cLDL accumulated in the aortic wall of Apoe −/− mice that underwent partial nephrectomy, a model for chronic kidney failure [22]. These observations were corroborated with the studies of MPO-derived cyanate besides urea derived cyanate as a prevailing source for protein carbamylation in patients with inflammatory disease states and smokers [9]. They were able to show that such carbamylated modified LDL associates with macrophage scavenger receptor A1, leading to cholesterol accumulation and foam cell formation. Moreover, protein-bound homocitrulline was associated with poorer cardiovascular outcomes in clinical conditions [9]. Besides the effects on macrophages, cLDL was shown to alter distinct vasoprotective endothelial properties. Scavenger receptors expressed on the surface of endothelial cells such as lectin-like oxidized LDL receptor-1 (LOX-1), CD36, scavenger receptor A1 and scavenger receptor of endothelial cells-1(SREC-1) were shown to mediate a rapid endothelial internalization as well as transcytosis of cLDL [23]. Besides that, cLDL itself stimulated endothelial surface expression of LOX-1 [23]. In addition, cLDL induces an endothelial pro-inflammatory phenotype by increasing the expression of the intercellular adhesion molecules (ICAM-1) and vascular adhesion molecules (VCAM-1) mediating the adhesion of monocytes to the endothelium in an LOX-1-dependent manner [24]. The pivotal role of LOX-1 for mediating the adverse vascular effects of cLDL was further enhanced by the observation that LOX-1 stimulates endothelial apoptosis in response to cLDL by endonuclease G mediated DNA fragmentation in a mitogen-activated protein kinase (MAPK)-dependent manner [25]. Moreover, we recently demonstrated that cLDL directly induces endothelial dysfunction [26]. Thereby, the interaction of cLDL with LOX-1 leads to a p38-MAPK-dependent production of ROS by endothelial NADPH oxidation. LOX-1 induced uncoupling of the endothelial nitric oxide synthase (eNOS) by S-glutathionylation, which resulted in a reduced nitric oxide (NO) bioavailability and an impaired vasodilation in vivo. Additionally, we demonstrated that the number of carbamylated lysine residues was substantially increased in LDL from dialysis patients but not in LDL from healthy subjects. However, these findings demonstrate that the carbamylation of LDL alters the vascular properties of lipoprotein and may convey to a proatherogenic particle. However, there is a strong debate on the
Downloaded from http://ndt.oxfordjournals.org/ at SUNY Health Science Center at Brooklyn on April 12, 2015
blood and tissues of patients. The concentration of urea and thiocyanate in the serum of healthy subjects was found to be
Full Review Emerging role of post-translational modifications in chronic kidney disease and cardiovascular disease
1
Institute for Molecular Cardiovascular Research (IMCAR), University Hospital RWTH Aachen, Aachen, Germany and
2
Department of Internal Medicine 4, Saarland University Hospital, Homburg/Saar, Germany
Correspondence and offprint requests to: Joachim Jankowski, E-mail: [email protected] * These authors contributed equally to the manuscript.
A B S T R AC T Post-translational modifications (PTMs) of proteins and peptides have recently gained much attention, as they are involved in the pathogenesis of cardiovascular disease and eventually also play a role in the progression of chronic kidney disease (CKD). In this review, we provide an overview of post-translational protein modifications such as carbamylation, glycation and oxidation, starting with their definitions, mechanisms and clinical relevance in the setting of CKD and cardiovascular disease. The methods currently used for the identification and, in particular, quantification of PTMs are described and potential treatment options in the context of PTMs are reviewed. We foresee that advancements in mass spectrometry-based methods leading to the identification of novel disease markers and/or pathophysiologically relevant factors will certainly boost the clinical utility in sample analyses. Keywords: carbamylation, cardiovascular disease, chronic kidney disease, oxidation, post-translational modification
INTRODUCTION Chronic kidney disease (CKD) and cardiovascular disease (CVD) are strongly interlinked with each other, and they share common risk factors such as diabetes mellitus or hypertension [1]. In the context of CKD and CVD, the interest towards the post-translational modifications (PTMs) of proteins has grown
© The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
considerably. Analysis of PTMs might be useful for the identification of mechanisms which play a role in the genesis and/or progression of CKD and CVD because proteins are constantly being exposed to different plasma and tissue components under different pathophysiological conditions. PTMs are covalent changes of proteins or peptides that are altered either by proteolytic cleavage or by adding moieties to one or more amino acids. This enhances their complexity with respect to regulation of activity state, subcellular localization, turnover and interaction with other cellular molecules [2]. PTM proteins and peptides have gained attention as biomarkers and/ or mediators of CVD and CKD [3, 4]. Here, we describe the most relevant currently known PTMs reported in relation to CKD and CVD. We used the PubMed database search to retrieve relevant literature with phrases like ‘(“Post translational modifications” OR “PTM’s”) AND (“Chronic kidney disease” OR “Chronic renal failure” OR “Cardiovascular disease”)’ and obtained 42 publications up to July 2014. Out of these, 16 were found to be actually relevant to the topic. Thus, we explored the PubMed database further, this time for articles related to different PTMs with relation to CKD and CVD like carbamylation, oxidation, glycosylation, nitrosylation, phosphorylation, citrullination and cystenylation. Here, we provide an overview of the most commonly reported PTMs such as carbamylation, glycation and oxidation, and their clinical relevance in the setting of CKD and CVD. Phosphorylation is omitted because it is mostly involved in cell signalling pathways. Although, other PTMs have been reported, to our knowledge their role is not clearly understood and are in its infancy.
1
Downloaded from http://ndt.oxfordjournals.org/ at SUNY Health Science Center at Brooklyn on April 12, 2015
Prathibha R. Gajjala1, Danilo Fliser2, Thimoteus Speer2, Vera Jankowski1,* and Joachim Jankowski1,*
C A R B A M Y L AT I O N Definition Carbamylation is a non-enzymatic spontaneous reaction of a primary amine or a free sulfhydryl group of protein with isocyanate [OCN]−. Isocyanate is the active form of cyanate, resulting in an addition of the carbamoyl moiety [−CONH2] to the functional groups of proteins [5]. Carbamylated proteins subsequently undergo structural and functional changes [6].
FULL REVIEW
Relevance in CKD Since kidney function declines as CKD progresses, metabolic substances like urea accumulate dramatically in the
F I G U R E 1 : Schematic representation of synthesis of carbamylated proteins/aminoacids. The source for urea and thiocyanate are from the urea
cycle, diet, smoking and air. Urea undergoes deamination to form cyanate and the MPO in the presence of hydrogen peroxide as cosubstrate converts thiocyanate to isocyanate. Cyanate and isocyanate are interconvertible and electrophilically attacks the amino or sulphur groups on proteins or amino acids to form respective carbamylated products.
2
P. R. Gajjala et al.
Downloaded from http://ndt.oxfordjournals.org/ at SUNY Health Science Center at Brooklyn on April 12, 2015
Mechanism Under physiological conditions, the nitrogenous waste content of the organism is removed in the form of urea CO (NH2)2. Urea is a by-product of the urea cycle, which slowly dissociates into cyanate and ammonia. Urea is generally found to be present at a ratio of 1 to 100 for cyanate to urea at equilibrium, which in turn depends on pH and temperature [7]. Cyanate, an electrophile, attacks nucleophilic groups like amino groups in proteins, and this results in carbamylation [5, 6]. Thereby, the positively charged lysines are neutralized, modifying its interaction with the surrounding environment. In vitro carbamylation on proteins has a strong effect on the protein
conformation, whereby its biological activity is modified [8]. However, it might have limited changes in in vivo situation. Furthermore, cyanate is synthesized from myeloperoxidase (MPO), which is present in the granules of neutrophils, monocytes and certain tissue macrophages. At the inflammatory sites and at atherosclerotic plaques, MPO utilizes H2O2 and isothiocyanate (SCN−) as a co-substrate, and produces the main metabolite cyanate [9]. Isocyanic acid reacts irreversibly with both alpha amino group of peptides, proteins or amino acids or epsilon amino group of lysine but it reacts faster with lower pKa values [8]. In general, the pKa value of alpha amino group is less when compared with the side-chain amino group of lysine. However, the pKa values of lysine side chain in protein show marked variation, sometimes being less than the N-terminal amino group which depends on the microenvironment where it is buried [10]. The ε-amino-carbamyl-lysine is also known as homocitrulline which is commonly used as a biomarker for the carbamylation. Figure 1 demonstrates the formation of carbamylated proteins/amino acids.
Emerging role of PTMs in CKD and CVD
3
FULL REVIEW
Relevance for CVD Besides CKD, carbamylated proteins are highly involved in the genesis of CVD, since carbamylated proteins are mainly formed during inflammation as a result of the MPO-mediated cyanate formation. Recently, it was demonstrated that carbamylated HDL reduces the lecithin–cholesterol acyl transferase
(LCAT) activity which is essential for cholesterol esterification and HDL maturation. HDLs are also involved in lipid droplet formation in macrophages, resulting in the accumulation of cholesterol and thus the lipid droplet formation. ApoA-I, a component of HDL, is a major activator of LCAT; an elimination of its positive charge at the active site suppresses the LCAT activity leading to dysfunctional HDL [20]. HDLs are selective targets for carbamylation in atherosclerotic lesions, since apoA-I carbamyl-lysine’s presence is enhanced as the stage of lesions progresses. Carbamylation of LDL represents a well-studied example on how carbamylation of lysine residues may alter distinct biological properties of proteins. In 2005, Ok et al. demonstrated that cLDL induces endothelial apoptosis and proliferation of vascular smooth muscle cells; both processes are involved in the pathogenesis of atherosclerotic CVD [21]. This may be at least particularly relevant in patients with CKD in whom total plasma carbamylation was reported to be increased. Moreover, cLDL accumulated in the aortic wall of Apoe −/− mice that underwent partial nephrectomy, a model for chronic kidney failure [22]. These observations were corroborated with the studies of MPO-derived cyanate besides urea derived cyanate as a prevailing source for protein carbamylation in patients with inflammatory disease states and smokers [9]. They were able to show that such carbamylated modified LDL associates with macrophage scavenger receptor A1, leading to cholesterol accumulation and foam cell formation. Moreover, protein-bound homocitrulline was associated with poorer cardiovascular outcomes in clinical conditions [9]. Besides the effects on macrophages, cLDL was shown to alter distinct vasoprotective endothelial properties. Scavenger receptors expressed on the surface of endothelial cells such as lectin-like oxidized LDL receptor-1 (LOX-1), CD36, scavenger receptor A1 and scavenger receptor of endothelial cells-1(SREC-1) were shown to mediate a rapid endothelial internalization as well as transcytosis of cLDL [23]. Besides that, cLDL itself stimulated endothelial surface expression of LOX-1 [23]. In addition, cLDL induces an endothelial pro-inflammatory phenotype by increasing the expression of the intercellular adhesion molecules (ICAM-1) and vascular adhesion molecules (VCAM-1) mediating the adhesion of monocytes to the endothelium in an LOX-1-dependent manner [24]. The pivotal role of LOX-1 for mediating the adverse vascular effects of cLDL was further enhanced by the observation that LOX-1 stimulates endothelial apoptosis in response to cLDL by endonuclease G mediated DNA fragmentation in a mitogen-activated protein kinase (MAPK)-dependent manner [25]. Moreover, we recently demonstrated that cLDL directly induces endothelial dysfunction [26]. Thereby, the interaction of cLDL with LOX-1 leads to a p38-MAPK-dependent production of ROS by endothelial NADPH oxidation. LOX-1 induced uncoupling of the endothelial nitric oxide synthase (eNOS) by S-glutathionylation, which resulted in a reduced nitric oxide (NO) bioavailability and an impaired vasodilation in vivo. Additionally, we demonstrated that the number of carbamylated lysine residues was substantially increased in LDL from dialysis patients but not in LDL from healthy subjects. However, these findings demonstrate that the carbamylation of LDL alters the vascular properties of lipoprotein and may convey to a proatherogenic particle. However, there is a strong debate on the
Downloaded from http://ndt.oxfordjournals.org/ at SUNY Health Science Center at Brooklyn on April 12, 2015
blood and tissues of patients. The concentration of urea and thiocyanate in the serum of healthy subjects was found to be
