Non-enzymatic Modification Of Proteins Research Articles

Thermodynamic Overview of Bioconjugation Reactions Pertinent to Lysine and Cysteine Peptide and Protein Residues

Bioconjugation reactions are critical to the modification of peptides and proteins, permitting the introduction of biophysical probes onto proteins as well as drugs for use in antibody-targeted medicines. A diverse set of chemical reagents can be employed in these circumstances to covalently label protein side chains, such as the amine moiety in the side chain of lysine and the thiol functionality in cysteine residues, two of the more frequently employed sites for modification. To provide researchers with a thermodynamic survey of the reaction of these residues with frequently employed chemical modification reagents as well as reactive cellular intermediates also known to modify proteins non-enzymatically, a theoretical investigation of the overall thermodynamics of models of these reactions was undertaken at the T1 and G3(MP2) thermochemical recipe levels (gas phase), the M06-2X/6-311+G(2df,2p)/B3LYP/6-31G(d) (gas and water phase), and the M06-2X/cc-PVTZ(-f)++ density functional levels of theory (water phase). Discussions of the relationship between the reagent structure and the overall thermodynamics of amine or thiol modification are presented. Of additional interest are the observations that routine cellular intermediates such as certain thioesters, acyl phosphates, and acetyl-L-carnitine can contribute to non-enzymatic protein modifications. These reactions and representative click chemistry reactions were also investigated. The computational survey presented herein (>320 reaction computations were undertaken) should serve as a valuable resource for researchers undertaking protein bioconjugation. A concluding section addresses the ability of computation to provide predictions as to the potential for protein modification by new chemical entities, with a cautionary note on protein modification side reactions that may occur when employing synthetic substrates to measure enzyme kinetic activities.

Plasma and cerebrospinal fluid nonenzymatic protein damage is sustained in Alzheimer's disease

BackgroundOxidative stress is considered to play an important role in the pathogenesis of Alzheimer's disease (AD). It has been observed that oxidative damage to specific protein targets affecting particular functional networks is one of the mechanisms by which oxidative stress contributes to neuronal failure and consequently loss of cognition and AD progression. Studies are lacking in which oxidative damage is measured at both systemic and central fluid levels and in the same cohort of patients. We aimed to determine the levels of both plasma and cerebrospinal fluid (CSF) nonenzymatic protein damage in patients in the continuum of AD and to evaluate the relation of this damage with clinical progression from mild cognitive impairment (MCI) to AD. MethodsDifferent markers of nonenzymatic post-translational protein modification, mostly from oxidative processes, were detected and quantified in plasma and CSF by isotope dilution gas chromatography‒mass spectrometry using selected ion monitoring (SIM-GC/MS) for 289 subjects: 103 AD, 92 MCI, and 94 control subjects. Characteristics of the study population such as age, sex, Mini-mental state examination, CSF AD biomarkers, and APOE ϵ4, were also considered. ResultsForty-seven (52.8%) MCI patients progressed to AD during follow-up (58 ± 12.5 months). After controlling for age, sex, and APOE ϵ4 allele, plasma and CSF concentrations of protein damage markers were not associated with either diagnosis of AD or MCI. The CSF levels of nonenzymatic protein damage markers were associated with none of the CSF AD biomarkers. In addition, neither in CSF nor in plasma were the levels of protein damage associated with the MCI to AD progression. ConclusionThe lack of association between both CSF and plasma concentrations of nonenzymatic protein damage markers and AD diagnosis and progression suggests that oxidative damage in AD is a pathogenic mechanism specifically expressed at the cell-tissue level, not in extracellular fluids.

Protein Carbamylation and the Risk of ESKD in Patients with CKD.

Protein carbamylation, a nonenzymatic post-translational protein modification partially driven by elevated blood urea levels, associates with mortality and adverse outcomes in patients with ESKD on dialysis. However, little is known about carbamylation's relationship to clinical outcomes in the much larger population of patients with earlier stages of CKD. In this prospective observational cohort study of 3111 individuals with CKD stages 2-4, higher levels of carbamylated albumin (a marker of protein carbamylation burden) were associated with a greater risk of developing ESKD and other significant adverse clinical outcomes. These findings indicate that protein carbamylation is an independent risk factor for CKD progression. They suggest that further study of therapeutic interventions to prevent or reduce carbamylation is warranted. Protein carbamylation, a post-translational protein modification partially driven by elevated blood urea levels, associates with adverse outcomes in ESKD. However, little is known about protein carbamylation's relationship to clinical outcomes in the much larger population of patients with earlier stages of CKD. To test associations between protein carbamylation and the primary outcome of progression to ESKD, we measured baseline serum carbamylated albumin (C-Alb) in 3111 patients with CKD stages 2-4 enrolled in the prospective observational Chronic Renal Insufficiency Cohort study. The mean age of study participants was 59 years (SD 10.8); 1358 (43.7%) were female, and 1334 (42.9%) were White. The mean eGFR at the time of C-Alb assessment was 41.8 (16.4) ml/minute per 1.73 m 2 , and the median C-Alb value was 7.8 mmol/mol (interquartile range, 5.8-10.7). During an average of 7.9 (4.1) years of follow-up, 981 (31.5%) individuals developed ESKD. In multivariable adjusted Cox models, higher C-Alb (continuous or quartiles) independently associated with an increased risk of ESKD. For example, compared with quartile 1 (C-Alb ≤5.80 mmol/mol), those in quartile 4 (C-Alb >10.71 mmol/mol) had a greater risk for ESKD (adjusted hazard ratio, 2.29; 95% confidence interval, 1.75 to 2.99), and the ESKD incidence rate per 1000 patient-years increased from 15.7 to 88.5 from quartile 1 to quartile 4. The results remained significant across numerous subgroup analyses, when treating death as a competing event, and using different assessments of eGFR. Having a higher level of protein carbamylation as measured by circulating C-Alb is an independent risk factor for ESKD in individuals with CKD stages 2-4. This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_04_24_JSN_URE_EP22_042423.mp3.

Gas Plasma Protein Oxidation Increases Immunogenicity and Human Antigen-Presenting Cell Maturation and Activation.

Protein vaccines rely on eliciting immune responses. Inflammation is a prerequisite for immune responses to control infection and cancer but is also associated with disease onset. Reactive oxygen species (ROSs) are central during inflammation and are capable of inducing non-enzymatic oxidative protein modifications (oxMods) associated with chronic disease, which alter the functionality or immunogenicity of proteins that are relevant in cancer immunotherapy. Specifically, antigen-presenting cells (APCs) take up and degrade extracellular native and oxidized proteins to induce adaptive immune responses. However, it is less clear how oxMods alter the protein's immunogenicity, especially in inflammation-related short-lived reactive species. Gas plasma technology simultaneously generates a multitude of ROSs to modify protein antigens in a targeted and controlled manner to study the immunogenicity of oxMods. As model proteins relevant to chronic inflammation and cancer, we used gas plasma-treated insulin and CXCL8. We added those native or oxidized proteins to human THP-1 monocytes or primary monocyte-derived cells (moDCs). Both oxidized proteins caused concentration-independent maturation phenotype alterations in moDCs and THP-1 cells concerning surface marker expression and chemokine and cytokine secretion profiles. Interestingly, concentration-matched H2O2-treated proteins did not recapitulate the effects of gas plasma, suggesting sufficiently short diffusion distances for the short-lived reactive species to modify proteins. Our data provide evidence of dendric cell maturation and activation upon exposure to gas plasma- but not H2O2-modified model proteins. The biological consequences of these findings need to be elucidated in future inflammation and cancer disease models.

AGE/Non-AGE Glycation: An Important Event in Rheumatoid Arthritis Pathophysiology.

Rheumatoid arthritis (RA) is a chronic inflammatory, autoimmune disease that gradually affects the synovial membrane and joints. Many intrinsic and/or extrinsic factors are crucial in making RA pathology challenging throughout the disease. Substantial enzymatic or non-enzymatic modification of proteins driving inflammation has gained a lot of interest in recent years. Endogenously modified glycated protein influences disease development linked with AGEs/non-AGEs and is reported as a disease marker. In this review, we summarized current knowledge of the differential abundance of glycated proteins by compiling and analyzing a variety of AGE and non-AGE ligands that bind with RAGE to activate multi-faceted inflammatory and oxidative stress pathways that are pathobiologically associated with RA-fibroblast-like synoviocytes (RA-FLS). It is critical to comprehend the connection between oxidative stress and inflammation generation, mediated by glycated protein, which may bind to the receptor RAGE, activate downstream pathways, and impart immunogenicity in RA. It is worth noting that AGEs and non-AGEs ligands play a variety of functions, and their functionality is likely to be more reliant on pathogenic states and severity that may serve as biomarkers for RA. Screening and monitoring of these differentially glycated proteins, as well as their stability in circulation, in combination with established pre-clinical characteristics, may aid or predict the onset of RA.

Proteostasis in the Male and Female Germline: A New Outlook on the Maintenance of Reproductive Health.

For fully differentiated, long lived cells the maintenance of protein homeostasis (proteostasis) becomes a crucial determinant of cellular function and viability. Neurons are the most well-known example of this phenomenon where the majority of these cells must survive the entire course of life. However, male and female germ cells are also uniquely dependent on the maintenance of proteostasis to achieve successful fertilization. Oocytes, also long-lived cells, are subjected to prolonged periods of arrest and are largely reliant on the translation of stored mRNAs, accumulated during the growth period, to support meiotic maturation and subsequent embryogenesis. Conversely, sperm cells, while relatively ephemeral, are completely reliant on proteostasis due to the absence of both transcription and translation. Despite these remarkable, cell-specific features there has been little focus on understanding protein homeostasis in reproductive cells and how/whether proteostasis is “reset” during embryogenesis. Here, we seek to capture the momentum of this growing field by highlighting novel findings regarding germline proteostasis and how this knowledge can be used to promote reproductive health. In this review we capture proteostasis in the context of both somatic cell and germline aging and discuss the influence of oxidative stress on protein function. In particular, we highlight the contributions of proteostasis changes to oocyte aging and encourage a focus in this area that may complement the extensive analyses of DNA damage and aneuploidy that have long occupied the oocyte aging field. Moreover, we discuss the influence of common non-enzymatic protein modifications on the stability of proteins in the male germline, how these changes affect sperm function, and how they may be prevented to preserve fertility. Through this review we aim to bring to light a new trajectory for our field and highlight the potential to harness the germ cell’s natural proteostasis mechanisms to improve reproductive health. This manuscript will be of interest to those in the fields of proteostasis, aging, male and female gamete reproductive biology, embryogenesis, and life course health.

Recent advances in histone glycation: emerging role in diabetes and cancer.

Ever increasing information on genome and proteome has offered fascinating details and new opportunities to understand the molecular biology. It is now known that histone proteins surrounding the DNA play a crucial role in the chromatin structure and function. Histones undergo a plethora of posttranslational enzymatic modifications that influence nucleosome dynamics and affect DNA activity. Earlier research offered insights into the enzymatic modifications of histones; however, attention has been diverted to histone modifications induced by by-products of metabolism without enzymatic engagement in the last decade. Nonenzymatic modifications of histones are believed to be crucial for epigenetic landscape, cellular fate and for role in human diseases. Glycation of histone proteins constitutes the major nonenzymatic modifications of nuclear proteins that have implications in diabetes and cancer. It has emerged that glycation damages nuclear proteins, modifies amino acids of histones at crucial locations, generates adducts affecting histone chromatin interaction, develops neo-epitopes inducing specific immune response and impacts cell function. Presence of circulating antibodies against glycated histone proteins in diabetes and cancer has shown immunological implications with diagnostic relevance. These crucial details make histone glycation an attractive focus for investigators. This review article, therefore, makes an attempt to exclusively summarize the recent research in histone glycation, its impact on structural integrity of chromatin and elaborates on its role in diabetes and cancer. The work offers insights for future scientists who investigate the link between metabolism, biomolecular structures, glycobiology, histone-DNA interactions in relation to diseases in humans.

The melibiose-derived glycation product mimics a unique epitope present in human and animal tissues

Non-enzymatic modification of proteins by carbohydrates, known as glycation, leads to generation of advanced glycation end-products (AGEs). In our study we used in vitro generated AGEs to model glycation in vivo. We discovered in vivo analogs of unusual melibiose-adducts designated MAGEs (mel-derived AGEs) synthesized in vitro under anhydrous conditions with bovine serum albumin and myoglobin. Using nuclear magnetic resonance spectroscopy we have identified MAGEs as a set of isomers, with open-chain and cyclic structures, of the fructosamine moiety. We generated a mouse anti-MAGE monoclonal antibody and show for the first time that the native and previously undescribed analogous glycation product exists in living organisms and is naturally present in tissues of both invertebrates and vertebrates, including humans. We also report MAGE cross-reactive auto-antibodies in patients with diabetes. We anticipate our approach for modeling glycation in vivo will be a foundational methodology in cell biology. Further studies relevant to the discovery of MAGE may contribute to clarifying disease mechanisms and to the development of novel therapeutic options for diabetic complications, neuropathology, and cancer.

Minimal invasive fluorescence methods to quantify advanced glycation end products (AGEs) in skin and plasma of humans.

Advanced glycation end products (AGEs) are non-enzymatic modifications of proteins and lipids, which are spontaneously produced in the body in relation with several human diseases. Their relevance on protein functions alteration, either structural or enzymatic is under study, but their value as biomarkers or predictors of disease progression and clinical outcomes is unquestionable. The heterogeneity and amplitude of these modifications make their analysis difficult, although, different methods have been developed for specific AGEs based on colorimetric reactions, immunoassays or chromatography. However, for a massive application on human population, methods based on the autofluorescence of some AGEs stand out. Several qualities of these methods such as label-free measurement, rapidity, cost-effectiveness, and minimal invasiveness make them very useful for periodic measurements in critically ill patients and for the analysis of large populations. Here we explain the rationale of these methods, and we present a step-by-step protocol and the equipment requirements to carry out the estimation of AGE content in skin and plasma. AGE plasma content and skin accumulation are temporally related, so AGE plasmatic levels are a possible predictor of skin AGE content. On the other hand, AGE skin accumulation is a surrogate or an indicator of past AGE levels in plasma and in the rest of the body. AGE levels or their variations have shown to be related with prognosis of several diseases, so they can be used as predictor biomarkers for clinicians.

A possible role of glycation in the regulation of amyloid β precursor protein processing leading to amyloid β accumulation

Alzheimer’s disease (AD) is one of the most common forms of neurodegenerative diseases amongst the aged population. The disease is multifactorial, and diabetes has been considered as one of the major risk factors for the development of AD. Chronic hyperglycemic condition in diabetes promotes non-enzymatic protein modification by glucose termed as glycation, which affects protein structure and function. Previous studies have shown that many of the enzymes, including proteases, are affected by glycation. Conversely, glycated proteins are known to become resistant to protease action. In these hypotheses, we have extended these two concepts to the regulation of amyloid-β protein precursor (AβPP) by secretases leading to amyloid-β (Aβ) accumulation. The first hypothesis deals with the glycation of α-secretases leading to its reduced activity, while in the second hypothesis, AβPP glycation may prevent α-secretases action, rendering its processing by β secretase. As diabetes is a risk factor for the development of AD, either or both these pathways may operate, leading to the manifestation of AD.

Serum albumin cysteine trioxidation is a potential oxidative stress biomarker of type 2 diabetes mellitus

Metabolic disorders in T2DM generate multiple sources of free radicals and oxidative stress that accelerate nonenzymatic degenerative protein modifications (DPMs) such as protein oxidation, disrupt redox signaling and physiological function, and remain a major risk factor for clinical diabetic vascular complications. In order to identify potential oxidative biomarkers in the blood plasma of patients with T2DM, we used LC-MS/MS-based proteomics to profile plasma samples from patients with T2DM and healthy controls. The results showed that human serum albumin (HSA) is damaged by irreversible cysteine trioxidation, which can be a potential oxidative stress biomarker for the early diagnosis of T2DM. The quantitative detection of site-specific thiol trioxidation is technically challenging; thus, we developed a sensitive and selective LC-MS/MS workflow that has been used to discover and quantify three unique thiol-trioxidized HSA peptides, ALVLIAFAQYLQQC(SO3H)PFEDHVK (m/z 1241.13), YIC(SO3H)ENQDSISSK (m/z 717.80) and RPC(SO3H)FSALEVDETYVPK (m/z 951.45), in 16 individual samples of healthy controls (n = 8) and individuals with diabetes (n = 8). Targeted quantitative analysis using multiple reaction monitoring mass spectrometry revealed impairment of the peptides with m/z 1241.13, m/z 717.80 and m/z 951.45, with significance (P < 0.02, P < 0.002 and P < 0.03), in individuals with diabetes. The results demonstrated that a set of three HSA thiol-trioxidized peptides, which are irreversibly oxidatively damaged in HSA in the plasma of patients with T2DM, can be important indicators and potential biomarkers of oxidative stress in T2DM.

Hypoxia-Induced Degenerative Protein Modifications Associated with Aging and Age-Associated Disorders.

Aging is an inevitable time-dependent decline of various physiological functions that finally leads to death. Progressive protein damage and aggregation have been proposed as the root cause of imbalance in regulatory processes and risk factors for aging and neurodegenerative diseases. Oxygen is a modulator of aging. The oxygen-deprived conditions (hypoxia) leads to oxidative stress, cellular damage and protein modifications. Despite unambiguous evidence of the critical role of spontaneous non-enzymatic Degenerative Protein Modifications (DPMs) such as oxidation, glycation, carbonylation, carbamylation, and deamidation, that impart deleterious structural and functional protein alterations during aging and age-associated disorders, the mechanism that mediates these modifications is poorly understood. This review summarizes up-to-date information and recent developments that correlate DPMs, aging, hypoxia, and age-associated neurodegenerative diseases. Despite numerous advances in the study of the molecular hallmark of aging, hypoxia, and degenerative protein modifications during aging and age-associated pathologies, a major challenge remains there to dissect the relative contribution of different DPMs in aging (either natural or hypoxia-induced) and age-associated neurodegeneration.

Cyanate Induces Oxidative Stress Injury and Abnormal Lipid Metabolism in Liver through Nrf2/HO-1.

Chronic kidney disease (CKD) is problem that has become one of the major issues affecting public health. Extensive clinical data suggests that the prevalence of hyperlipidemia in CKD patients is significantly higher than in the general population. Lipid metabolism disorders can damage the renal parenchyma and promote the occurrence of cardiovascular disease (CVD). Cyanate is a uremic toxin that has attracted widespread attention in recent years. Usually, 0.8% of the molar concentration of urea is converted into cyanate, while myeloperoxidase (MPO) catalyzes the oxidation of thiocyanate to produce cyanate at the site of inflammation during smoking, inflammation, or exposure to environmental pollution. One of the important physiological functions of cyanate is protein carbonylation, a non-enzymatic post-translational protein modification. Carbamylation reactions on proteins are capable of irreversibly changing protein structure and function, resulting in pathologic molecular and cellular responses. In addition, recent studies have shown that cyanate can directly damage vascular tissue by producing large amounts of reactive oxygen species (ROS). Oxidative stress leads to the disorder of liver lipid metabolism, which is also an important mechanism leading to cirrhosis and liver fibrosis. However, the influence of cyanate on liver has remained unclear. In this research, we explored the effects of cyanate on the oxidative stress injury and abnormal lipid metabolism in mice and HL-7702 cells. In results, cyanate induced hyperlipidemia and oxidative stress by influencing the content of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), superoxide dismutase (SOD), catalase (CAT) in liver. Cyanate inhibited NF-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and the phosphorylation of adenosine 5′monophosphate-activated protein kinase (AMPK), activated the mTOR pathway. Oxidative stress on the cells reduced significantly by treating with TBHQ, an antioxidant, which is also an activator of Nrf2. The activity of Nrf2 was rehabilitated and phosphorylation of mTOR decreased. In conclusion, cyanate could induce oxidative stress damage and lipid deposition by inhibiting Nrf2/HO-1 pathway, which was rescued by inhibitor of Nrf2.

Insights into the effects of N-glycosylation on the characteristics of the VC1 domain of the human receptor for advanced glycation end products (RAGE) secreted by Pichia pastoris.

Advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs), resulting from non-enzymatic modifications of proteins, are potentially harmful to human health. They directly act on proteins, affecting structure and function, or through receptor-mediated mechanisms. RAGE, a type I transmembrane glycoprotein, was identified as a receptor for AGEs. RAGE is involved in chronic inflammation, oxidative stress-based diseases and ageing. The majority of RAGE ligands bind to the VC1 domain. This domain was successfully expressed and secreted by Pichia pastoris. Out of two N-glycosylation sites, one (Asn25) was fully occupied while the other (Asn81) was under-glycosylated, generating two VC1 variants, named p36 and p34. Analysis of N-glycans and of their influence on VC1 properties were here investigated. The highly sensitive procainamide labeling method coupled to ES-MS was used for N-glycan profiling. N-glycans released from VC1 ranged from Man9GlcNAc2- to Man15GlcNAc2- with major Man10GlcNAc2- and Man11GlcNAc2- species for p36 and p34, respectively. Circular dichroism spectra indicated that VC1 maintains the same conformation also after removal of N-glycans. Thermal denaturation curves showed that the carbohydrate moiety has a small stabilizing effect on VC1 protein conformation. The removal of the glycan moiety did not affect the binding of VC1 to sugar-derived AGE- or malondialdehyde-derived ALE-human serum albumin. Given the crucial role of RAGE in human pathologies, the features of VC1 from P. pastoris will prove useful in designing strategies for the enrichment of AGEs/ALEs from plasma, urine or tissues, and in characterizing the nature of the interaction.

New Evidence for the Diversity of Mechanisms and Protonated Schiff Bases Formed in the Non-Enzymatic Covalent Protein Modification (NECPM) of HbA by the Hydrate and Aldehydic Forms of Acetaldehyde and Glyceraldehyde.

Acetaldehyde is a physiological species existing in blood. Glyceraldehyde is a commonly-used surrogate for glucose in studies of nonenzymatic glycation. Both species exist in dynamic equilibrium between two forms, an aldehyde and a hydrate. Nonenzymatic covalent protein modification (NECPM) is a process whereby a protein is covalently modified by a non-glucose species. The purpose here was to elucidate the NECPM mechanism(s) for acetaldehyde and glyceraldehyde with human hemoglobin (HbA). For the first time, both aldehydic and hydrate forms of acetaldehyde and glyceraldehyde were considered. Computations and model reactions followed by 1H NMR were employed. Results demonstrated that the aldehyde and hydrate forms of acetaldehyde bind and covalently-modify Val1 of HbA via different chemical mechanisms, yet generated an identical protonated Schiff base (PSB). The aldehyde and hydrate of glyceraldehyde also covalently-modified Val1 via mechanisms distinct from one another, yet generated an identical PSB. It is noteworthy that the PSB from acetaldehyde and glyceraldehyde were different structures. The PSB from acetaldehyde is proposed to proceed to covalent adducts that have been implicated in alcohol toxicity. Conversely, the PSB generated from glyceraldehyde can form an Amadori which has been implicated in diabetic complications. Thus, the PSB structure generated from acetaldehyde versus glyceraldehyde may be central to pathophysiological outcomes because it determines the structure of the stable covalent adduct formed.

The Role of Shelterin Complex and Post-Translational Non-Enzymatic Modification in Telomere Maintenance in Chronic Myeloid Leukemia

The introduction of tyrosine kinase inhibitors (TKI) revolutionized therapy of chronic myeloid leukemia (CML) and gives long-lasting remission in majority of patients. However, a significant proportion of patients develops resistance and are at risk of progression. Genomic instability is one of the key features associated with CML progression and as in majority of human malignancies, reduction in telomere length characterize transition from chronic phase (CML-CP) to the blastic phase (CML-BP). Telomere maintenance depends on telomerase nucleoprotein complex consisting of reverse transcriptase TERT, RNA template TERC and structural proteins, such as dyskerin (DKC1), but also on telomere-associated proteins including but not limited to shelterin complex(TRF1, TRF2, RAP1, TIN2, TPP1, POT1) and other telomeric-associated proteins (such as TEP1 and Tankyrase). The role of shelterin complex and its post-translational non-enzymatic modifications in BCR/ABL1-mediated genomic instability in CML progression and resistance to TKIs is not fully elucidated, thus we decided to examine the possible role of different components of shelterin complex and its glycation and carbonylation in promoting genomic instability and CML progression. We employed CML CD34+ primary cells isolated from peripheral blood leukocytes or bone marrow of CML patients at different stages of disease: CML-CP, CML-BP and CML-TKIres (TKI-resistant cells). Blood samples were taken after informed consent. The length of telomeres was determined by Southern blotting and PNA-FISH technique. Enzymatic activity of telomerase, as well as protein glycation was measured immunoenzymatically, while expression of subunits of telomerase and shelterin complexes was examined by RT-qPCR and by Western Blot.We confirmed that reduction in telomere length was positively correlated with CML progression. However, in samples from CD34+ CML-TKIres patients in comparison to CML-CP patients, an increase in telomere length was observed (Fig 1). In addition, the negative correlation between mean telomere length and expression of BCR-ABL1 in CML samples was observed. This suggests involvement of alternative telomere lengthening mechanisms, since dynamic changes in telomere length were neither associated with enzymatic activity of telomerase nor with gene copy number/expression of TERT/TERC. Therefore we postulate that telomere dynamics in CML may have a bi-phasic scenario. We found that the expression of RAP1, DKC1 and Tankyrase1 was increased in CML-BP as compared to CML-CP. Moreover, RAP1 is involved in increased metabolism, which is associated with elevated glycolytic and oxidative rates leading to post-translational non-enzymatic modification of proteins and damage of cellular proteostasis which may indirectly influence telomere length in CML. We found that telomere shortening was associated with accumulation of post-translational non-enzymatic modification ofselected proteins of shelterin complex, namely glycation and carbonylation.In conclusion, we postulate that aberrant expression of members of the shelterin complex such as RAP1 may be responsible for aberrant telomere maintenance mechanisms in CML cells and may play role in genomic instability associated with CML progression as well as the clonal selection and resistance to TKIs. In addition, the changes in the level of protein glycation may influence telomere length in CML. DisclosuresNo relevant conflicts of interest to declare.

Analysis of Protein Oxidative Modifications in Follicular Fluid from Fertile Women: Natural Versus Stimulated Cycles.

Oxidative stress is associated with obstetric complications during ovarian hyperstimulation in women undergoing in vitro fertilization. The follicular fluid contains high levels of proteins, which are the main targets of free radicals. The aim of this work was to determine specific biomarkers of non-enzymatic oxidative modifications of proteins from follicular fluid in vivo, and the effect of ovarian stimulation with gonadotropins on these biomarkers. For this purpose, 27 fertile women underwent both a natural and a stimulated cycle. The biomarkers, glutamic semialdehyde (GSA), aminoadipic semialdehyde (AASA), Nε-(carboxymethyl)lysine (CML), and Nε-(carboxyethyl)lysine (CEL), were measured by gas-liquid chromatography coupled to mass spectrometry. Results showed that follicular fluid contained products of protein modifications by direct metal-catalyzed oxidation (GSA and AASA), glycoxidation (CML and CEL), and lipoxidation (CML). GSA was the most abundant biomarker (91.5%). The levels of CML amounted to 6% of the total lesions and were higher than AASA (1.3%) and CEL (1.2%). In the natural cycle, CEL was significantly lower (p < 0.05) than in the stimulated cycle, suggesting that natural cycles are more protected against protein glycoxidation. These findings are the basis for further research to elucidate the possible relevance of this follicular biomarker of advanced glycation end product in fertility programs.

Abstract 16744: Advanced Glycation End Products and the Pathophysiology of Bioprosthetic Heart Valve Structural Degeneration

Introduction: Bioprosthetic heart valves (BHV) are widely used to treat severe heart valve disease. BHV function deteriorates after 10-15 years due to structural degeneration (SVD) often involving calcification. SVD occurs regardless of biomaterial, requiring prosthesis replacement. Advanced glycation end products (AGEs) are post-translational, non-enzymatic protein modifications associated with diabetic complications and cardiovascular disease. Serum proteins, such as albumin, are also AGE modified. Hypothesis: AGEs, exacerbated by serum protein infiltration, play a role in SVD. Methods: Clinical BHV explants (n = 45) of glutaraldehyde-crosslinked bovine pericardium (BP, n = 35) or porcine aortic valve (PAV, n = 10) and equivalent unimplanted BHV were probed by immunostaining per semi-quantitative scoring (range 0-4) for generalized AGE, N-carboxymethyllysine (CML, an AGE-receptor ligand), and human serum albumin (HSA), the most abundant serum protein. Demographics comprise ages 34 to 86 (mean: 64.95), 28 males and 17 females, 8 diabetics, and 15 previous and concomitant coronary artery bypass patients (both conditions associated with SVD). BHV calcification was quantified by atomic absorption spectroscopy. BHV glycation was modeled by exposing BP to 50mM glyoxal +/- 5% albumin for 24hr, followed by either immunostaining for CML and albumin or 14 C glyoxal scintillation. Albumin incorporation was verified by mass uptake. Results: All explants showed positive immunostaining for AGE (mean ± SE = 3.52 ± 0.10); most showed HSA (2.30 ± 0.18) and less intense CML (1.55 ± 0.16) staining; unimplanted BHV staining was negative. Score comparisons showed no significant differences for BP vs PAV, coronary bypass or diabetic patients vs all patients. BHV Ca levels (122.01 ± 18.01ug/mg, binned as &lt;30 vs &gt;100ug/mL) did not correlate with AGE immunostaining. In vitro, glyoxal-generated CML and infiltrated albumin were evident throughout BP per immunostaining. 14 C glyoxal uptake by BP was 12.01 ± 0.50nmol/mg after 28 days and was increased 57.2 ± 7.5% by albumin co-incubation. Albumin co-incubation increased BP mass by 4.10 ± 0.19%. Conclusions: AGEs are associated with SVD regardless of BHV type and SVD risk factors. Albumin exacerbates AGE accumulation.

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

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.

Analysis of Advanced Glycation Endproducts in Rat Tail Collagen and Correlation to Tendon Stiffening.

Methylglyoxal is a major 1,2-dicarbonyl compound in vivo and leads to nonenzymatic protein modifications, known as advanced glycation endproducts. Especially long-lived proteins like collagen are prone to changes of the mechanical or biological function, respectively, by accumulation of Maillard-derived modifications. Specifically, the resulting nonenzymatic cross-link structures in parallel to the natural maturation process of collagen fibrils lead to complications with age or during disease. A novel lysine-lysine amide cross-link derived from methylglyoxal, 2,15-diamino-8-methyl-9-oxo-7,10-diaza-1,16-hexadecanedioic acid, named MOLA, was synthesized and identified in vitro and in vivo. Tail tendons of young, adult, and old rats (3, 12, and 22 months) were enzymatically digested prior to analysis of acid-labile glycation products via liquid chromatography-tandem mass spectrometry (LC-MS/MS). As a result, nine monovalent amino acid modifications, mostly originating from methylglyoxal (36 μmol/mol leucine-equivalents in total), and four glycation cross-links (0.72 μmol/mol glucosepane, 0.24 μmol/mol DODIC (3-deoxyglucosone-derived imidazoline cross-link), 0.04 μmol/mol MODIC (methylglyoxal-derived imidazoline cross-link), 0.34 μmol/mol MOLA) were quantitated in senescent tendon collagen. The results correlated with increased tail tendon breaking time from 10 to 190 min and indicate that methylglyoxal is a major player in the aging process of connective tissue.