Irreversible Post-translational Modification Research Articles

Post-translational modifications in prion diseases.

More than 650 reversible and irreversible post-translational modifications (PTMs) of proteins have been listed so far. Canonical PTMs of proteins consist of the covalent addition of functional or chemical groups on target backbone amino-acids or the cleavage of the protein itself, giving rise to modified proteins with specific properties in terms of stability, solubility, cell distribution, activity, or interactions with other biomolecules. PTMs of protein contribute to cell homeostatic processes, enabling basal cell functions, allowing the cell to respond and adapt to variations of its environment, and globally maintaining the constancy of the milieu interieur (the body's inner environment) to sustain human health. Abnormal protein PTMs are, however, associated with several disease states, such as cancers, metabolic disorders, or neurodegenerative diseases. Abnormal PTMs alter the functional properties of the protein or even cause a loss of protein function. One example of dramatic PTMs concerns the cellular prion protein (PrPC), a GPI-anchored signaling molecule at the plasma membrane, whose irreversible post-translational conformational conversion (PTCC) into pathogenic prions (PrPSc) provokes neurodegeneration. PrPC PTCC into PrPSc is an additional type of PTM that affects the tridimensional structure and physiological function of PrPC and generates a protein conformer with neurotoxic properties. PrPC PTCC into PrPSc in neurons is the first step of a deleterious sequence of events at the root of a group of neurodegenerative disorders affecting both humans (Creutzfeldt-Jakob diseases for the most representative diseases) and animals (scrapie in sheep, bovine spongiform encephalopathy in cow, and chronic wasting disease in elk and deer). There are currently no therapies to block PrPC PTCC into PrPSc and stop neurodegeneration in prion diseases. Here, we review known PrPC PTMs that influence PrPC conversion into PrPSc. We summarized how PrPC PTCC into PrPSc impacts the PrPC interactome at the plasma membrane and the downstream intracellular controlled protein effectors, whose abnormal activation or trafficking caused by altered PTMs promotes neurodegeneration. We discussed these effectors as candidate drug targets for prion diseases and possibly other neurodegenerative diseases.

Determination of the derivatization reactivity between α/β-dicarbonyl compounds and standard citrullinated peptides based on matrix-assisted laser desorption ionization-time-of-flight mass spectrometry

Protein citrullination is an irreversible post-translational modification process regulated by peptidylarginine deiminases (PADs) in the presence of Ca2+. This process is closely related to the occurrence and development of autoimmune diseases, cancers, neurological disorders, cardiovascular and cerebrovascular diseases, and other major diseases. The analysis of protein citrullination by biomass spectrometry confronts great challenges owing to its low abundance, lack of affinity tags, small mass-to-charge ratio change, and susceptibility to isotopic and deamidation interferences. The methods commonly used to study the protein citrullination mainly involve the chemical derivatization of the urea group of the guanine side chain of the peptide to increase the mass-to-charge ratio difference of the citrullinated peptide. Affinity-enriched labels are then introduced to effectively improve the sensitivity and accuracy of protein citrullination by mass spectrometry. 2,3-Butanedione or phenylglyoxal compounds are often used as derivatization reagents to increase the mass-to-charge ratio difference of the citrullinated peptide, and the resulting derivatives have been observed to contain α-dicarbonyl structures. To date, however, no relevant studies on the reactivity of dicarbonyl compounds with citrullinated peptides have been reported. In this study, we determined whether six α-dicarbonyl and two β-dicarbonyl compounds undergo derivatization reactions with standard citrullinated peptides using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Among the α-dicarbonyl compounds, 2,3-butanedione and glyoxal reacted efficiently with several standard citrullinated peptides, but yielded a series of by-products. Phenylglyoxal, methylglyoxal, 1,2-cyclohexanedione, and 1,10-phenanthroline-5,6-dione also derivated efficiently with standard citrullinated peptides, generating a single derivative. Thus, a new derivatization method that could yield a single derivative was identified. Among the β-dicarbonyl compounds, 1,3-cyclohexanedione and 2,4-pentanedione successfully reacted with the standard citrullinated peptides, and generated a single derivative. However, their reaction efficiency was very low, indicating that the β-dicarbonyl compounds are unsuitable for the chemical derivatization of citrullinated peptides. The above results indicate that the α-dicarbonyl structure is necessary for realizing the efficient and specific chemical derivatization of citrullinated peptides. Moreover, the side chains of the α-dicarbonyl structure determine the structure of the derivatives, derivatization efficiency, and generation (or otherwise) of by-products. Therefore, the specific enrichment and precise identification of citrullinated peptides can be achieved by synthesizing α-dicarbonyl structured compounds containing affinity tags. The proposed method enables the identification of citrullinated proteins and their modified sites by MS, thereby providing a better understanding of the distribution of citrullinated proteins in different tissues. The findings will be beneficial for studies on the mechanism of action of citrullinated proteins in a variety of diseases.

N-Terminomic Identification of Intracellular MMP-2 Substrates in Cardiac Tissue.

Proteases are enzymes that induce irreversible post-translational modifications by hydrolyzing amide bonds in proteins. One of these proteases is matrix metalloproteinase-2 (MMP-2), which has been shown to modulate extracellular matrix remodeling and intracellular proteolysis during myocardial injury. However, the substrates of MMP-2 in heart tissue are limited, and lesser known are the cleavage sites. Here, we used degradomics to investigate the substrates of intracellular MMP-2 in rat ventricular extracts. First, we designed a novel, constitutively active MMP-2 fusion protein (MMP-2-Fc) that we expressed and purified from mammalian cells. Using this protease, we proteolyzed ventricular extracts and used subtiligase-mediated N-terminomic labeling which identified 95 putative MMP-2-Fc proteolytic cleavage sites using mass spectrometry. The intracellular MMP-2 cleavage sites identified in heart tissue extracts were enriched for proteins primarily involved in metabolism, as well as the breakdown of fatty acids and amino acids. We further characterized the cleavage of three of these MMP-2-Fc substrates based on the gene ontology analysis. We first characterized the cleavage of sarco/endoplasmic reticulum calcium ATPase (SERCA2a), a known MMP-2 substrate in myocardial injury. We then characterized the cleavage of malate dehydrogenase (MDHM) and phosphoglycerate kinase 1 (PGK1), representing new cardiac tissue substrates. Our findings provide insights into the intracellular substrates of MMP-2 in cardiac cells, suggesting that MMP-2 activation plays a role in cardiac metabolism.

Pathological Impact of Redox Post-Translational Modifications.

Oxidative stress is involved in the development of several pathologies. The different reactive oxygen species (ROS) produced during oxidative stress are at the origin of redox post-translational modifications (PTMs) on proteins and impact nucleic acids and lipids. This review provides an overview of recent data on cysteine and methionine oxidation and protein carbonylation following oxidative stress in a pathological context. Oxidation, like nitration, is a selective process and not all proteins are impacted. It depends on multiple factors, including amino acid environment, accessibility, and physical and chemical properties, as well as protein structures. Thiols can undergo reversible oxidations and others that are irreversible. On the contrary, carbonylation represents irreversible PTM. To date, hundreds of proteins were shown to be modified by ROS and reactive nitrogen species (RNS). We reviewed recent advances in the impact of redox-induced PTMs on protein functions and activity, as well as its involvement in disease development or treatment. These data show a complex situation of the involvement of redox PTM on the function of targeted proteins. Many proteins can have their activity decreased by the oxidation of cysteine thiols or methionine S-methyl thioethers, while for other proteins, this oxidation will be activating. This complexity of redox PTM regulation suggests that a global antioxidant therapeutic approach, as often proposed, is unlikely to be effective. However, the specificity of the effect obtained by targeting a cysteine or methionine residue to be able to inactivate or activate a particular protein represents a major interest if it is possible to consider this targeting from a therapeutic point of view with our current pharmacological tools. Antioxid. Redox Signal. 41, 152-180.

Advancing In-Depth N-Terminomics Detection with a Cleavable 2-Pyridinecarboxyaldehyde Probe.

Proteolysis, an irreversible post-translational modification catalyzed by proteases, plays a crucial role in various biological processes. Exploring abnormally hydrolyzed proteins in pathological tissues is a valuable approach for elucidating the mechanisms underlying disease development. Herein, we have developed a cleavable 2-pyridinecarboxyaldehyde probe (2PCA-Probe) that enables efficient and in-depth N-terminomics detection, addressing limitations of previous methods. Furthermore, we unexpectedly discovered a new marker capable of identifying N-terminal chemical labeling with the 2PCA-Probe and elucidated the reaction mechanism. Using this probe, we identified 4686 N-terminal peptides in colorectal cancer and adjacent tissues, significantly expanding the depth of the N-terminome and revealing the potential role of abnormal protein hydrolysis in colorectal cancer development.

Abstract C103: Non-enzymatic glycoxidation reports on society, environment and biology to inform on enduring cancer disparities

Abstract Disparities in overall cancer outcomes have been reduced across racial and ethnic divides, but still persist for some tumor types and have increased for others. This has stimulated significant debate regarding whether the enduring nature of cancer disparities arises solely out of inherent failings in addressing health inequities, or does an individual’s inherent biology also play an accompanying role. As a newly termed “bio-social determinant of health”, non-enzymatic glycoxidation profiles may represent an unexploited opportunity to consider how health inequity constructs combine to impact cancer outcomes across transdisciplinary studies. Resulting from the combined processes of glycation and oxidation, non-enzymatic glycoxidation is a complex series of reactions, leading to the irreversible posttranslational modification of peptides, lipids and nucleic acids by Advanced Glycation End Products (AGEs). Evidence is presented to support that health inequity constructs can serve to provide a superfluous source of the reactive substrates that sustains non-enzymatic glycoxidation resulting in a chronic cycle of AGE formation, bioavailability, and pathogenicity. AGEs are significant because: 1. Communities who bear the highest cancer burden are more likely to be exposed to health inequity risk factors that serve to increase AGE bioavailability 2. When health equity is compromised complex relationships are created between social and biological determinates of health that serve to overwhelm the fragile rheostat that maintains AGE homeostasis.; and 3. Increases in AGE bioavailability are associated with increased tumor growth and progression through their impact on the tumor microenvironment. Glycoxidation patterns visualized by AGE accumulation and pathogenic function may serve as a “bio-social determinant of health” as they may reflect the dynamic and varied nature of multiple health inequity constructs and their combined effects on cancer incidence and mortality. In turn this may allow a greater understanding of the complex relationships that exist between societal, ancestral, environmental, and biological constructs, and lifestyle associated risk factors associated with enduring cancer disparities. This would provide significant insight on novel cancer prevention and control strategies aimed at reducing AGE exposure due to health inequity. Citation Format: David P. Turner. Non-enzymatic glycoxidation reports on society, environment and biology to inform on enduring cancer disparities [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C103.

A novel two-way rebalancing strategy for identifying carbonylation sites

BackgroundAs an irreversible post-translational modification, protein carbonylation is closely related to many diseases and aging. Protein carbonylation prediction for related patients is significant, which can help clinicians make appropriate therapeutic schemes. Because carbonylation sites can be used to indicate change or loss of protein function, integrating these protein carbonylation site data has been a promising method in prediction. Based on these protein carbonylation site data, some protein carbonylation prediction methods have been proposed. However, most data is highly class imbalanced, and the number of un-carbonylation sites greatly exceeds that of carbonylation sites. Unfortunately, existing methods have not addressed this issue adequately.ResultsIn this work, we propose a novel two-way rebalancing strategy based on the attention technique and generative adversarial network (Carsite_AGan) for identifying protein carbonylation sites. Specifically, Carsite_AGan proposes a novel undersampling method based on attention technology that allows sites with high importance value to be selected from un-carbonylation sites. The attention technique can obtain the value of each sample’s importance. In the meanwhile, Carsite_AGan designs a generative adversarial network-based oversampling method to generate high-feasibility carbonylation sites. The generative adversarial network can generate high-feasibility samples through its generator and discriminator. Finally, we use a classifier like a nonlinear support vector machine to identify protein carbonylation sites.ConclusionsExperimental results demonstrate that our approach significantly outperforms other resampling methods. Using our approach to resampling carbonylation data can significantly improve the effect of identifying protein carbonylation sites.

CaaX-motif-adjacent residues influence G protein gamma (Gγ) prenylation under suboptimal conditions

Prenylation is an irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Dysregulation of prenylation contributes to multiple disorders, including cancers and vascular and neurodegenerative diseases. Prenyltransferases tether isoprenoid lipids to proteins via a thioether linkage during prenylation. Pharmacological inhibition of the lipid synthesis pathway by statins is a therapeutic approach to control hyperlipidemia. Building on our previous finding that statins inhibit membrane association of G protein γ (Gγ) in a subtype-dependent manner, we investigated the molecular reasoning for this differential inhibition. We examined the prenylation of carboxy-terminus (Ct) mutated Gγ in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged Gγ subunits and their mutants using live-cell confocal imaging. Reversible optogenetic unmasking-masking of Ct residues was used to probe their contribution to prenylation and membrane interactions of the prenylated proteins. Our findings suggest that specific Ct residues regulate membrane interactions of the Gγ polypeptide, statin sensitivity, and extent of prenylation. Our results also show a few hydrophobic and charged residues at the Ct are crucial determinants of a protein's prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different Gγ subtypes, our findings indicate a plausible mechanism allowing for statins to differentially perturb heterotrimeric G protein signaling in cells depending on their Gγ-subtype composition. Our results may also provide molecular reasoning for repurposing statins as Ras oncogene inhibitors and the failure of using prenyltransferase inhibitors in cancer treatment.

Probing Tyrosine Nitration with a Small TbIII -Metallopeptide.

Tyrosine nitration, a post-translational modification (PTM) that takes place under nitrosative stress conditions, occurs through a non-enzymatic peroxynitrite-mediated reaction. Although protein nitration has long been considered an irreversible PTM involved in nitrosative stress-associated diseases, it has also been suggested to be a regulatory mechanism of signal transduction. Therefore, the development of tools that help to understand this protein modification is of great interest. Herein, we explore a TbIII -chelating metallopeptide to monitor tyrosine nitration. The luminescence of this probe decreases significantly between its non-nitrated and nitrated states, and this reduction in the luminescence intensity is directly related to the degree of tyrosine nitration after treatment with peroxynitrite. Remarkably, the luminescence intensity changes after nitration are not affected in the presence of complex biological media, which makes it a promising tool for understanding this protein modification.

Matrix metalloproteinases in arthritis: towards precision medicine

Proteolysis of structural molecules of the extracellular matrix (ECM) is an irreversible post-translational modification in all arthropathies. Common joint disorders, including osteoarthritis and rheumatoid arthritis, have been associated with increased levels of matrix remodelling enzymes, including matrix metalloproteinases (MMPs). MMPs, in concert with other host proteinases and glycanases, destroy proteoglycans, collagens and other ECM molecules. MMPs may also control joint remodelling indirectly by signalling through cell-surface receptors or by proteolysis of cytokines and receptor molecules. After synthesis as pro-forms, MMPs can be activated by various types of post-translational modifications, including proteolysis. Once activated, MMPs are controlled by general and specific tissue inhibitors of metalloproteinases (TIMPs). In rheumatoid arthritis, proteolysis of the ECM results in so-called remnant epitopes that enhance and perpetuate autoimmune processes in susceptible hosts. In osteoarthritis, the considerable production of MMP-13 by chondrocytes, often concurrent with mechanical overload, is a key event. Hence, information about the regulation, timing, localization and activities of MMPs in specific disease phases and arthritic entities will help to develop better diagnostics. Insights into beneficial and detrimental effects of MMPs on joint tissue inflammation are also necessary to plan and execute (pre)clinical studies for better therapy and precision medicine with MMP inhibitors. With the advances in proteomics and single-cell transcriptomics, two critical points need attention: neglected neutrophil MMP biology, and the analysis of net proteolytic activities as the result of balances between MMPs and their inhibitors.

Enzymatic proteolysis during the conversion of milk into cheese

The transformation of milk into cheese occurs under the influence of many physicochemical, biochemical and microbiological processes, among which proteolysis plays a very important role. Proteolysis belongs to the most complex type of irreversible post-translational modification of proteins. Enzymatic proteolysis catalysts at different stages of cheese production are native milk enzymes, exo- and endopeptidases of starter and non-starter microorganisms, and milk-clotting enzymes. The article presents a brief overview of modern ideas about the properties, mechanism of action and specificity of the main representatives of enzymes that hydrolyze milk proteins at the stages of preparing milk for coagulation, during rennet coagulation and subsequent maturation of cheeses. These include the plasmin system of milk, enzymes of psychrotrophic bacteria and lactic acid microorganisms that enter milk both accidentally (non-starter microflora) and planned in the form of starter cultures from specially selected strains. Milk-clotting enzymes, having fulfilled their main function — milk coagulation — partially pass into cheese and, along with enzymes of starter microorganisms and plasmin, participate in proteolytic processes during cheese ripening. It is generally accepted that proteolysis in ripening cheeses is the most significant biochemical process that affects the formation of taste, aroma and texture along with lipolysis and glycolysis. The combination of proteolysis products (peptides, amino acids, amines, etc.) is individual for different types of cheese and varies depending on the technological parameters of production, including the duration of maturation. Proteolysis in cheeses has been studied by many scientists in various aspects. This review supplements the known information with new information, without claiming to be comprehensive.

Novel method to quantify peptidylarginine deiminase activity shows distinct citrullination patterns in rheumatoid and juvenile idiopathic arthritis.

Peptidylarginine deiminases (PADs) mediate citrullination, an irreversible posttranslational modification that converts arginine to citrulline residues in proteins. Rheumatoid arthritis (RA) is characterized by unique autoantibodies that recognize citrullinated peptides, which are highly specific for this disease. However, the mechanism preceding the anti-citrulline response remains largely unclear. PAD enzymes are known to fuel the autoimmune response by generating autoreactive epitopes, and sustain local synovial inflammation through neutrophil extracellular trap formation. Therefore, detecting endogenous PAD activity is important to understand the pathogenesis of arthritis. In this study, we improved a fluorescent in vitro assay to enable endogenous PAD activity characterization in complex samples. We combine the use of an in-house synthetic, arginine-rich substrate and a negatively charged dye molecule to visualize enzyme activity. This pioneering PAD assay allowed profiling of active citrullination in leukocytes and in local and systemic samples of an arthritis cohort. Our results reveal that RA and juvenile idiopathic arthritis (JIA) synovial fluids display similar levels of PAD activity. In contrast, citrullination was limited in joints of patients suffering from gout or Lyme's disease. Interestingly, in blood, a higher level of extracellular citrullination was only found in anti-CCP-positive RA patients. Our finding suggests that enhanced synovial PAD activity drives the loss in tolerance towards citrullinated proteins and that systemic citrullination may indicate the risk for developing citrulline-specific autoimmunity.

Ultrasensitive HPLC-MS Quantification of S-(2-Succino) Cysteine Based on Ethanol/Acetyl Chloride Derivatization in Fumarate Accumulation Cells.

Succination is a nonenzymatic and irreversible post-translational modification (PTM) with important biological significance, yielding S-(2-succino) cysteine (2SC) residue. This PTM is low in abundance and often requires a large amount of protein samples for 2SC quantification. In this work, an efficient quantification method based on ethanol/acetyl chloride chemical derivatization was developed. The three carboxyl groups of 2SC were all esterified to increase hydrophobicity, greatly improving its ionization efficiency. The sensitivity was increased by 112 times; the limit of detection was reduced to 0.885 fmol, and the protein usage was reduced by at least 10 times. The established method was used to detect the overall concentration of 2SC in fumarate accumulation cells quantitatively.

Proteome-wide analysis of hydrogen peroxide-induced protein carbonylation in Arabidopsis thaliana.

Protein carbonylation is a non-enzymatic and irreversible post-translational modification that occurs naturally in living organisms under the direct or indirect effect of reactive oxygen species (ROS). In animals, signaling pathways involving numerous carbonylated proteins have been identified, highlighting the dual role of these molecules in ROS signal transduction. In plants, studies on phytohormone signaling (auxin, methyl jasmonate, abscisic acid) have shown that reactive carbonyl species (RCS: acrolein, malondialdehyde, 4-hydroxynonenal, etc.), derived from the action of ROS on lipids, play important roles in secondary root formation and stomatal closure. However, the carbonylated proteins involved in these signaling pathways remain to be identified. In this study, we analyzed proteins responsive to carbonylation by exogenous hydrogen peroxide (H2O2) by profiling the carbonyl proteome extracted from Arabidopsis thaliana leaves after H2O2 treatment. Carbonylated proteins were enriched at the peptide level and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We identified 35 and 39 uniquely carbonylated proteins in the untreated and the H2O2-treated plant samples, respectively. In comparison to the control treatment, gene ontology enrichment analysis revealed that most of the carbonylated proteins identified in the H2O2-treated plant samples are related to sulfate adenylyl transferases and amidophosphoribosyl transferases involved in the immune system response, defense response, and external stimulus-response. These results indicated that exogenous H2O2 caused a change in the pattern of protein carbonylation in A. thaliana leaves. Protein carbonylation may thus influence the plant transcriptome and metabolism in response to H2O2 and ROS-triggering external stimuli.

Insights into peptidylarginine deiminase expression and citrullination pathways.

Peptidylarginine deiminases (PADs) are calcium-dependent enzymes that mediate citrullination, an irreversible post-translational modification (PTM). PAD enzymes have received increasing attention in (patho-)physiology since multi-omics analysis accelerated their expression profiling. Here, we provide a comprehensive overview of PAD expression at the RNA and protein levels, and a list of annotated substrates per PAD isozyme. We discuss novel roles of citrullination in cellular growth, epigenetic regulation, tissue remodeling, inflammation, and cancer in mouse models and humans. Additionally, we cluster similar effects of protein deimination to offer a different perspective and improve our understanding of citrullination in health and disease. Citrullination should no longer be considered as a rare PTM, but as an important regulatory mechanism in physiology and pathology.

A Pilot Study on Peptidylarginine Deiminases and Protein Deimination in Animal Cancers across Vertebrate Species.

PADs are a group of calcium-dependent enzymes that play key roles in inflammatory pathologies and have diverse roles in cancers. PADs cause irreversible post-translational modification of arginine to citrulline, leading to changes in protein function in different cellular compartments. PAD isozyme diversity differs throughout phylogeny in chordates, with five PAD isozymes in mammals, three in birds, and one in fish. While the roles for PADs in various human cancers are mounting (both in regards to cancer progression and epigenetic regulation), investigations into animal cancers are scarce. The current pilot-study therefore aimed at assessing PAD isozymes in a range of animal cancers across the phylogeny tree. In addition, the tissue samples were assessed for total protein deimination and histone H3 deimination (CitH3), which is strongly associated with human cancers and also indicative of gene regulatory changes and neutrophil extracellular trap formation (NETosis). Cancers were selected from a range of vertebrate species: horse, cow, reindeer, sheep, pig, dog, cat, rabbit, mink, hamster, parrot, and duck. The cancers chosen included lymphoma, kidney, lung, testicular, neuroendocrine, anaplastic, papilloma, and granulosa cell tumour. Immunohistochemical analysis revealed that CitH3 was strongly detected in all of the cancers assessed, while pan-deimination detection was overall low. Both PAD2 and PAD3 were the most predominantly expressed PADs across all of the cancers assessed, while PAD1, PAD4, and PAD6 were overall expressed at lower, albeit varying, levels. The findings from this pilot study provide novel insights into PAD-mediated roles in different cancers across a range of vertebrate species and may aid in the understanding of cancer heterogeneity and cancer evolution.

Proteolysis: a key post-translational modification regulating proteoglycans.

Proteoglycans are composite molecules comprising a protein backbone, i.e., the core protein, with covalently attached glycosaminoglycan chains of distinct chemical types. Most proteoglycans are secreted or attached to the cell membrane. Their specialized structures, binding properties, and biophysical attributes underlie diverse biological roles, which include modulation of tissue mechanics, cell adhesion, and the sequestration and regulated release of morphogens, growth factors, and cytokines. As an irreversible post-translational modification, proteolysis has a profound impact on proteoglycan function, abundance, and localization. Proteolysis is required for molecular maturation of some proteoglycans, clearance of extracellular matrix proteoglycans during tissue remodeling, generation of bioactive fragments from proteoglycans, and ectodomain shedding of cell-surface proteoglycans. Genetic evidence shows that proteoglycan core protein proteolysis is essential for diverse morphogenetic events during embryonic development. In contrast, dysregulated proteoglycan proteolysis contributes to osteoarthritis, cardiovascular disorders, cancer, and inflammation. Proteolytic fragments of perlecan, versican, aggrecan, brevican, collagen XVIII, and other proteoglycans are associated with independent biological activities as so-called matrikines. Yet, proteoglycan proteolysis has been investigated to only a limited extent to date. Here, we review the actions of proteases on proteoglycans and illustrate their functional impact with several examples. We discuss the applications and limitations of strategies used to define cleavage sites in proteoglycans and explain how proteoglycanome-wide proteolytic mapping, which is desirable to fully understand the impact of proteolysis on proteoglycans, can be facilitated by integrating classical proteoglycan isolation methods with mass spectrometry-based proteomics.

The pattern of apolipoprotein A-I lysine carbamylation reflects its lipidation state and the chemical environment within human atherosclerotic aorta

Protein lysine carbamylation is an irreversible post-translational modification resulting in generation of homocitrulline (N-ε-carbamyllysine), which no longer possesses a charged ε-amino moiety. Two distinct pathways can promote protein carbamylation. One results from urea decomposition, forming an equilibrium mixture of cyanate (CNO−) and the reactive electrophile isocyanate. The second pathway involves myeloperoxidase (MPO)-catalyzed oxidation of thiocyanate (SCN−), yielding CNO− and isocyanate. Apolipoprotein A-I (apoA-I), the major protein constituent of high-density lipoprotein (HDL), is a known target for MPO-catalyzed modification in vivo, converting the cardioprotective lipoprotein into a proatherogenic and proapoptotic one. We hypothesized that monitoring site-specific carbamylation patterns of apoA-I recovered from human atherosclerotic aorta could provide insights into the chemical environment within the artery wall. To test this, we first mapped carbamyllysine obtained from in vitro carbamylation of apoA-I by both the urea-driven (nonenzymatic) and inflammatory-driven (enzymatic) pathways in lipid-poor and lipidated apoA-I (reconstituted HDL). Our results suggest that lysine residues within proximity of the known MPO-binding sites on HDL are preferentially targeted by the enzymatic (MPO) carbamylation pathway, whereas the nonenzymatic pathway leads to nearly uniform distribution of carbamylated lysine residues along the apoA-I polypeptide chain. Quantitative proteomic analyses of apoA-I from human aortic atheroma identified 16 of the 21 lysine residues as carbamylated and suggested that the majority of apoA-I carbamylation in vivo occurs on “lipid-poor” apoA-I forms via the nonenzymatic CNO− pathway. Monitoring patterns of apoA-I carbamylation recovered from arterial tissues can provide insights into both apoA-I structure and the chemical environment within human atheroma.

Abstract 11607: Protein Citrullination Landscape of Human Coronary Atherosclerosis

Introduction: Recently, an irreversible enzymatic post-translational modification, citrullination, has been shown to be present in the cardiovascular system (e.g. aorta). Citrullination, in which arginine is converted to citrulline, can alter protein structure and function, and produce autoantigens Hypothesis: Emerging evidence suggest a potential role for autoimmunity in the pathogenesis of atherosclerosis. However, it remains unknown whether of post-translational citrullination of arterial proteins contributes to local inflammatory processes that initiate or accelerate the atherosclerotic process. The aim of this study was to investigate changes in protein citrullination in human left anterior descending (LAD) coronary artery specimens with and without early atherosclerosis obtained from autopsied young adults with no clinical manifestations of coronary disease. Methods: Segments of the mid-LAD from 74 autopsied individuals (age range:15-55 yrs., 75% male, 67% White) were graded by a pathologist for percent of surface area involved with fatty streaks (FS) or fibrous plaque (FP). The frozen samples were homogenized and digested with Lys-C for subsequent proteomic mass-spectrometry using a data-independent acquisition (DIA) proteomic strategy with a hyper-citrullinated spectral library approach and our published CitFinder software. Results: We found 133 differentially expressed proteins (FDR<0.05) between control, FS and FP groups. Among them, 19 citrullinated proteins with 26 modified amino acid residues were identified. Citrullinated peptides from fibrinogen alpha chain (R84) and transgelin (R146) were upregulated in FP samples in comparison to FS and normal samples. Similarly, the citrullination site (R269) on alpha-enolase was significantly increased in the FP samples compared to normal. Interestingly, citrullination (R110) on myosin light polypeptide 6 was downregulated in FP samples. Conclusions: These data represent the most comprehensive interrogation of citrullinated proteins in human arterial samples to-date and suggest a possible association between this modification of several key proteins involved in tight junction, vascular smooth muscle contraction and inflammation and early atherosclerosis.

PreCar_Deep：A deep learning framework for prediction of protein carbonylation sites based on Borderline-SMOTE strategy

Carbonylation is an irreversible post-translational modification of proteins and regulates various cellular physiological processes. Due to the limitations of experimental methods, it is necessary to predict carbonylation sites by computational methods. In this paper, a new prediction model of carbonylation, Precar_Deep, is proposed. First, six feature extraction methods are used to obtain the original feature space from the protein sequences. Then, the Group LASSO method is used to remove redundant information and the oversampling Borderline-SMOTE method is employed to balance the data to obtain a new feature space. Finally, the processed data is input into the deep learning framework constructed in this paper to predict the carbonylation sites, and the performance of the model is evaluated by using 10-fold cross-validation and independent test datasets. The AUC values of the four datasets are all more than 90%. The experimental results show that PreCar_Deep is superior to other existing models and is helpful to identify protein carbonylation sites. The source code and all datasets are available at https://github.com/QUST-SHULI/PreCar_Deep/.