Covalent Modification Of Proteins Research Articles

Drug allergy to CFTR modulator therapy associated with lumacaftor-specific CD4+ T lymphocytes

Drug allergy to CFTR modulator therapy associated with lumacaftor-specific CD4+ T lymphocytes

A Review on: “Recent Updates on Poly-Adp-Ribosylation (Parylation) in Physiology and Pathology”

One of the most common cellular responses to DNA breakage is the covalent post-translational modification of the nuclear proteins with poly ADP ribose from the NAD+ as a precursor, which is mostly catalyzed by the poly-ADP-ribose polymerase1(PARP1).

Novel biomarkers for the evaluation of aging-induced proteinopathies.

Proteinopathies are characterized by aging related accumulation of misfolded protein aggregates. Irreversible covalent modifications of aging proteins may significantly affect the native three dimentional conformation of proteins, alter their function and lead to accumulation of misfolded protein as dysfunctional aggregates. Protein misfolding and accumulation of aberrant proteins are known to be associated with aging-induced proteinopathies such as amyloid ß and tau proteins in Alzheimer's disease, α-synuclein in Parkinson's disease and islet amyloid polypeptides in Type 2 diabetes mellitus. Protein oxidation processes such as S-nitrosylation, dityrosine formation and some of the newly elucidated processes such as carbamylation and citrullination recently drew the attention of researchers in the field of Gerontology. Studying over these processes and illuminating their relations between proteinopathies may help to diagnose early and even to treat age related disorders. Therefore, we have chosen to concentrate on aging-induced proteinopathic nature of these novel protein modifications in this review.

Selective Covalent Targeting of Mutated EGFR(T790M) with Chlorofluoroacetamide-Pyrimidines.

Covalent modification of disease-associated proteins with small molecules is a powerful approach for achieving an increased and sustained pharmacological effect. To reduce the potential risk of nonselective covalent modification, molecular design of covalent inhibitors is critically important. We report herein the development of a targeted covalent inhibitor for mutated epidermal growth factor receptor (EGFR) (L858R/T790M) using α-chlorofluoroacetamide (CFA) as the reactive group. The chemically tuned weak reactivity of CFA was suitable for the design of third-generation EGFR inhibitors that possess the pyrimidine scaffold. The structure-activity relationship study revealed that CFA inhibitor 18 (NSP-037) possessed higher inhibition selectivity to the mutated EGFR over wild-type EGFR when compared to clinically approved osimertinib. Mass-based chemical proteomics analyses further revealed that 18 displayed high covalent modification selectivity for the mutated EGFR in living cells. These findings highlight the utility of CFA as a warhead of targeted covalent inhibitors and the potential application of the CFA-pyrimidines for treatment of non-small-cell lung cancer.

Characterization of covalent protein modification by triclosan in vivo and in vitro via three-dimensional liquid chromatography-mass spectrometry: New insight into its adverse effects

Triclosan (TCS), an antimicrobial agent widely used in personal care products and ubiquitously exists in environment, has drawn increasing concern due to its potential to exert multiple adverse effects, ranging from endocrine disruption to carcinogenesis. However, the mechanism of these adverse effects is still not fully elucidated. More and more studies have shown that chemical reactive metabolites (RMs) covalently binding to proteins is a possible reason for these adverse effects, but there is still a lack of appropriate methods to predict or evaluate these adverse effects due to the extremely low abundance of the modified proteins in complex biological samples. In this study, we attempted to address this problem and investigate the possible mechanism of TCS adverse effects by a shotgun proteomics approach based on three-dimensional-liquid chromatography-mass spectrometry (3D-LC-MS). First, the in vitro incubation with model amino acids and protein in microsomes showed that TCS could react with cysteine residue of proteins through 3 types of RMs. Then, a 3D-LC-MS approach was developed to sensitively determine the low abundant modified proteins, which resulted in the identification of 45 TCS-modified proteins, including albumin, haptoglobin and NR1I2, in rats. STRING analysis indicated that these modified proteins mainly were involved in reproductive and development system, endocrine and immune system, and carcinogenesis, which were in accord with the main reported TCS-induced adverse effects and suggested that the covalent modification of TCS RMs for proteins might affect their activities and functions, thus inducing serious adverse effects. This study provided a new insight into the mechanism of TCS adverse effects and may serve as a valuable method to predict or evaluate adverse effects of ubiquitous chemicals.

Association of Enzymatically and Nonenzymatically Functionalized Caseins Analyzed by Size-Exclusion Chromatography and Light-Scattering Techniques.

The influence of covalent protein modifications resulting from the Maillard reaction (glycation) of casein and lactose on the noncovalent association behavior of the protein was studied. Nonenzymatic cross-linking with methylglyoxal (MGO) and glutaraldehyde (GTA) as well as enzymatic cross-linking with microbial transglutaminase (mTG) was investigated in comparison. Molar mass, particle size, and conformational characteristics of nonmicellar casein associates as well as the extent of intraparticle protein cross-linking were examined utilizing size-exclusion chromatography (SEC) combined with UV detection and static and dynamic light scattering. Cross-linking resulted in the stabilization of a certain fraction of casein associates, with particle sizes of approximately 30 nm in radius of gyration (Rg), and promoted an incorporation of further casein molecules into those particles, yielding molar masses (Mw) of 1.0-1.2 × 106 g/mol. When caseins were additionally conjugated with lactose during the early Maillard reaction, a further growth of the associates up to approximately 50 nm in Rg with a Mw of 2.1 × 106 g/mol was observed. Furthermore, glycation reactions induced a transition from slightly elongated, random-coil structures toward more anisotropic conformations. Associates consisting of caseins cross-linked with GTA appeared to preserve the original particle conformation.

Rhodopsin Oligomerization in Synthetic Lipid Bilayers and Native Cellular Membranes as Studied by DEER of a Spin-labeled Retinal Analog

Oligomerization of integral membrane proteins is often the final step of protein folding. This final step is likely to be affected by a number of factors including biophysical interactions with lipids and other proteins and so is the oligomers’ stability and their further organization into clusters/superlattices. Thus, the protein oligomers observed in different membrane mimetics including synthetic liposomes could be different from those formed in native membranes. Here we employed DEER spectroscopy to measure a distribution of long range (ca., 2-7 nm) constraints between specific protein sidechains labeled with paramagnetic tags. Spin-labeled derivative of retinal (4-[2,2,5,5-tetramethyl-1- pyrrolidinyloxy-3-carboxyl]-retinal) was synthesized and added to apo-rhodospsin for a tight non-covalent binding similar to the native retinal. Such an approach eliminated the need for covalent modification of proteins with conventional nitroxides. Further, the retinal binding is a useful probe for oligomerization of functional rhodopsins as the protein is known to lose the retinal upon denaturing. Thus, non-functional rhodopsins would be EPR-silent. Experimental DEER signals from such spin-labeled rhodopsin revealed significant differences in protein oligomerization in synthetic vs. native membranes. Specifically, while oligomers with well-defined distances were found to form in DMPC-DMPA, some shorter distances were determined to be present in the native cellular membranes. The latter distances were attributed to smaller oligomers (dimers, trimers, etc.) that could be intermediates to larger oligomers or incomplete oligomers which further oligomerization was terminated by the membrane defects and/or other protein components. The work at NCSU was supported by U.S. DOE Contract DE-FG02-02ER15354.

Sample preparation approaches for qualitative and quantitative analysis of lipid-derived electrophile modified proteomes by mass spectrometry.

Lipid-derived electrophile (LDE) modifications, which are covalent modifications of proteins by endogenous LDEs, are essential types of protein posttranslational modifications. LDE modifications alter the protein structure and regulate their biological processes in cells. LDE modifications of proteins are also closely associated with several diseases and function as potential biomarkers for clinical diagnosis. The crucial step in studying the LDE modifications is to enrich the LDE modified proteins/peptides from complex biological samples with high efficiency and high selectivity and quantify modified proteins/peptides with high accuracy. In this review, we summarize the recent progress in MS-based proteomic technologies to globally identify and quantify LDE modified proteomes, mainly focusing on discussing the qualitative and quantitative technologies.

Click chemistry-based imaging to study the tissue distribution of the curcumin-protein complex in mice.

Previous studies have shown that curcumin, a bioactive dietary compound with a thiol-reactive α,β-unsaturated carbonyl moiety, can covalently modify protein thiols. However, most of the previous studies were performed in cultured cells or cell-free enzyme systems, and so it remains unknown whether curcumin could covalently modify proteins after oral administration in vivo. Using click chemistry-based fluorescence imaging, here we show that oral administration of dialkyne-curcumin (Di-Cur), a "click" probe mimicking curcumin, results in covalent modifications of cellular proteins in colon and liver tissues, but not in other tissues, in mice. This result suggests that oral administration of curcumin leads to the formation of the curcumin-protein complex in a tissue-specific manner, which could contribute to the biological effects and/or pharmacokinetics of curcumin. Further studies to elucidate the identities of curcumin-binding proteins could greatly help us to better understand the molecular mechanisms of curcumin, and develop novel strategies for disease prevention.

Gene silencing is one of the most efficient and promising functional genomics tools

Gene silencing is defined as epigenetic of gene expression leading to inactivation of previously active individual genes or larger chromosome regions. Silencing of a target gene can be achieved at two levels-transcriptional and post-transcriptional stages. The transcriptional gene silencing occurs through the repression of the process of transcription and the post transcriptional gene silencing occurs by the degradation of mRNA. There is also another type called Meiotic. Mechanisms responsible for repression of genes involve changes in level of DNA methylation and alterations in covalent modifications of histone proteins which leads to chromatin compaction. These are the effects of small RNA regulators such as small interfering RNAs (siRNAs), microRNAs (miRNA), Piwi-associated RNAs (piRNAs) etc. Virus can also conduct gene silencing which is known as Virus Induced Gene Silencing by cloning and inserting plant endogenous gene sequence in recombinant viral vectors. Even Gene Silencing helps in buildup resistance against biotic and abiotic stress and helps in knock down any kind of undesirable genes.

Learning processes in elementary nervous systems§.

Invertebrate animal models show simple behaviors supported by neural circuits easily accessible for experimentation and yet complex enough to provide necessary information on the cellular and molecular mechanisms that govern the vertebrate nervous system's function. The mechanisms underlying simple forms of learning have been extensively studied in the marine gastropod Aplysia californica, in which elementary non-associative learning of the behavioral habituation and sensitization type has been studied using the gill withdrawal reflex. A strong stimulus applied to the neck or tail improves the reflex response through heterosynaptic facilitation. The neurotransmitter serotonin is involved in both behavioral sensitization and dishabituation by acting through the second messenger cyclic adenosine monophosphate, protein kinase A, the phosphorylation of a K+ channel, causing its closure. This broadens the action potential profile, increases the influx of Ca2+ through voltage-gated Ca2+ channels, and enhances the neurotransmitter glutamate's release. Short-term memory is based on covalent modifications of pre-existing proteins, while long-term memory requires gene transcription, protein translation and growth of new synapses. Another simple invertebrate model is the leech Hirudo medicinalis. In nearly-intact preparations, the repetitive application of light electrical stimuli at the level of the caudal portion of the body wall can induce the habituation of swimming induction. At the same time, the stroke on the dorsal skin generates behavioral sensitization or dishabituation. Knowledge of the molecular mechanisms of activity-dependent forms of synaptic plasticity provides a basis for understanding the mechanisms underlying learning, memory, other forms of brain plasticity, and pathological conditions and suggests potential therapeutic interventions.

Protein Modification of Lysine with 2-(2-Styrylcyclopropyl)ethanal.

A new lysine-reactive cyclopropropyl aldehyde for the covalent modification of proteins was developed. The reagent exploits a divinylcyclopropane-cycloheptadiene rearrangement to render the initial condensation irreversible. A labeling study on eGFP demonstrated excellent chemoselectivity for the modification of amine-nucleophiles with the possibility of subsequent modifications.

Binding to medium and long chain fatty acyls is a common property of HEAT and ARM repeat modules

Covalent post-translational modification (PTM) of proteins with acyl groups of various carbon chain-lengths regulates diverse biological processes ranging from chromatin dynamics to subcellular localization. While the YEATS domain has been found to be a prominent reader of acetylation and other short acyl modifications, whether additional acyl-lysine reader domains exist, particularly for longer carbon chains, is unclear. Here, we employed a quantitative proteomic approach using various modified peptide baits to identify reader proteins of various acyl modifications. We discovered that proteins harboring HEAT and ARM repeats bind to lysine myristoylated peptides. Recombinant HEAT and ARM repeats bind to myristoylated peptides independent of the peptide sequence or the position of the myristoyl group. Indeed, HEAT and ARM repeats bind directly to medium- and long-chain free fatty acids (MCFA and LCFA). Lipidomic experiments suggest that MCFAs and LCFAs interact with HEAT and ARM repeat proteins in mammalian cells. Finally, treatment of cells with exogenous MCFAs and inhibitors of MCFA-CoA synthases increase the transactivation activity of the ARM repeat protein β-catenin. Taken together, our results suggest an unappreciated role for fatty acids in the regulation of proteins harboring HEAT or ARM repeats.

Same same but different: Wie Cyanobakterien ein zentrales Enzym regulieren

In most bacteria the activity of glutamine synthetase (GS) is regulated by covalent protein modifications. Remarkably, cyanobacteria, which are of increasing interest in biotechnology, have evolved fundamentally different regulatory mechanisms for the same enzyme. This includes GS inhibition by the interaction with small proteins, the inactivating factors (IF). In addition, they evolved unique mechanisms to monitor GS activity and to respond with IF biosynthesis accordingly.

Functions of SETD7 during development, homeostasis and cancer

A controlled organ homeostasis is essential to sustain the integrity of any living organism. This homeostasis relies on stem cells, which maintain themselves and give rise to the various types of differentiated cells. It is well established that the differentiation of pluripotent embryonic stem cells (ESCs) depends on major changes in transcriptional programs. Covalent modifications of DNA, RNA and proteins are instrumental in setting up the genetic programs associated with cell fates. Another important mechanism for regulation of gene expression is protein localisation and stability. In addition to ubiquitination, SUMOylation and phosphorylation, methylation of proteins is an important determinant of their properties.

Skin sensitizing effects of sulfur mustard and other alkylating agents in accordance to OECD guidelines

Vesicants cause a multitude of cutaneous reactions like erythema, blisters and ulcerations. After exposure to sulfur mustard (SM) and related compounds, patients present dermal symptoms typically known for chemicals categorized as skin sensitizer (e.g. hypersensitivity and flare-up phenomena). However, although some case reports led to the assumption that SM and other alkylating compounds represent sensitizers, a comprehensive investigation of SM-triggered immunological responses has not been conducted so far. Based on a well-structured system of in chemico and in vitro test methods, the Organization for Economic Co-operation and Development (OECD) established procedures to categorize agents on their skin sensitizing abilities. In this study, the skin sensitizing potential of SM and three related alkylating agents (AAs) was assessed following the OECD test guidelines. Besides SM, investigated AAs were chlorambucil (CHL), nitrogen mustard (HN3) and 2-chloroethyl ethyl sulfide (CEES). The methods are described in detail in the EURL ECVAM DataBase service on ALternative Methods to animal experimentation (DB-ALM). In accordance to OECD recommendations, skin sensitization is a pathophysiological process starting with a molecular initiating step and ending with the in vivo outcome of an allergic contact dermatitis. This concept is called adverse outcome pathway (AOP). An AOP links an adverse outcome to various key events which can be assayed by established in chemico and in vitro test methods. Positive outcome in two out of three key events indicates that the chemical can be categorized as a skin sensitizer. In this study, key event 1 “haptenation” (covalent modification of epidermal proteins), key event 2 “activation of epidermal keratinocytes” and key event 3 “activation of dendritic cells” were investigated. Covalent modification of epidermal proteins measured by using the DPRA-assay provided distinct positive results for all tested substances. Same outcome was seen in the KeratinoSens assay, investigating the activation of epidermal keratinocytes. The h-CLAT assay performed to determine the activation of dendritic cells provided positive results for SM and CEES but not for CHL and HN3. Altogether, following OECD requirements, our results suggest the classification of all investigated substances as skin sensitizers. Finally, a tentative AOP for SM-induced skin sensitization is suggested.

A Metabolomics-Inspired Strategy for the Identification of Protein Covalent Modifications.

Identification of protein covalent modifications (adducts) is a challenging task mainly due to the lack of data processing approaches for adductomics studies. Despite the huge technological advances in mass spectrometry (MS) instrumentation and bioinformatics tools for proteomics studies, these methodologies have very limited success on the identification of low abundant protein adducts. Herein we report a novel strategy inspired on the metabolomics workflows for the identification of covalently-modified peptides that consists on LC-MS data preprocessing followed by statistical analysis. The usefulness of this strategy was evaluated using experimental LC-MS data of histones isolated from HepG2 and THLE2 cells exposed to the chemical carcinogen glycidamide. LC-MS data was preprocessed using the open-source software MZmine and potential adducts were selected based on the m/z increments corresponding to glycidamide incorporation. Then, statistical analysis was applied to reveal the potential adducts as those ions are differently present in cells exposed and not exposed to glycidamide. The results were compared with the ones obtained upon the standard proteomics methodology, which relies on producing comprehensive MS/MS data by data dependent acquisition and analysis with proteomics data search engines. Our novel strategy was able to differentiate HepG2 and THLE2 and to identify adducts that were not detected by the standard methodology of adductomics. Thus, this metabolomics driven approach in adductomics will not only open new opportunities for the identification of protein epigenetic modifications, but also adducts formed by endogenous and exogenous exposure to chemical agents.

Apolipoprotein E binds to and reduces serum levels of DNA-mimicking, pyrrolated proteins

Lysine N-pyrrolation, converting lysine residues to Nϵ-pyrrole-l-lysine, is a recently discovered post-translational modification. This naturally occurring reaction confers electrochemical properties onto proteins that potentially produce an electrical mimic to DNA and result in specificity toward DNA-binding molecules such as anti-DNA autoantibodies. The discovery of this unique covalent protein modification provides a rationale for establishing the molecular mechanism and broad functional significance of the formation and regulation of Nϵ-pyrrole-l-lysine-containing proteins. In this study, we used microbeads coupled to pyrrolated or nonpyrrolated protein to screen for binding activities of human serum-resident nonimmunoglobin proteins to the pyrrolated proteins. This screen identified apolipoprotein E (apoE) as a protein that innately binds the DNA-mimicking proteins in serum. Using an array of biochemical assays, we observed that the pyrrolated proteins bind to the N-terminal domain of apoE and that oligomeric apoE binds these proteins better than does monomeric apoE. Employing surface plasmon resonance and confocal microscopy, we further observed that apoE deficiency leads to significant accumulation of pyrrolated serum albumin and is associated with an enhanced immune response. These results, along with the observation that apoE facilitates the binding of pyrrolated proteins to cells, suggest that apoE may contribute to the clearance of pyrrolated serum proteins. Our findings uncover apoE as a binding target of pyrrolated proteins, providing a key link connecting covalent protein modification, lipoprotein metabolism, and innate immunity.

Role of intestinal microbiota-mediated genipin dialdehyde intermediate formation in geniposide-induced hepatotoxicity in rats

Geniposide is a natural hepatotoxic iridoid glycoside. Its hydrolysate of intestinal microbiota, genipin, is thought to be responsible for the hepatotoxicity. However, the underlying mechanism that genipin contributes to the hepatotoxicity of geniposide is not well understood. In this study, we found that genipin spontaneously converted into a reactive dialdehyde intermediate and covalently bond to the primary amine group of free amino acids in both of the phosphate buffers and geniposide-treated rats. Furthermore, genipin dialdehyde can form the covalent linkage to the primary amino group (ε) of lysine side chains of the hepatic proteins in geniposide-treated rats. Pretreatment with β-glucosidase or antibiotics significantly modulated the genipin dialdehyde formation and protein modification, revealing the essential role of microbial glycosidases. The levels of protein adduct were that mapped onto the hepatotoxicity of geniposide. In summary, this study demonstrates that the intestinal microbiota mediated covalent modification of the hepatic protein by genipin dialdehyde may play a crucial role in the liver injury of geniposide. The study is also helpful for understanding the contribution of intestinal microbiota to the metabolic activation of xenobiotics.

PARP1 and Poly(ADP-ribosyl)ation Signaling during Autophagy in Response to Nutrient Deprivation

Autophagy is considered to be the primary degradative pathway that takes place in all eukaryotic cells. Morphologically, the autophagy pathway refers to a process by which cytoplasmic portions are delivered to double-membrane organelles, called autophagosomes, to fuse with lysosomes for bulk degradation. Autophagy, as a prosurvival mechanism, can be stimulated by different types of cellular stress such as nutrient deprivation, hypoxia, ROS, pH, DNA damage, or ER stress, promoting adaptation of the cell to the changing and hostile environment. The functional relevance of autophagy in many diseases such as cancer or neurodegenerative diseases remains controversial, preserving organelle function and detoxification and promoting cell growth, although in other contexts, autophagy could suppress cell expansion. Poly(ADP-ribosyl)ation (PARylation) is a covalent and reversible posttranslational modification (PTM) of proteins mediated by Poly(ADP-ribose) polymerases (PARPs) with well-described functions in DNA repair, replication, genome integrity, cell cycle, and metabolism. Herein, we review the current state of PARP1 activation and PARylation in starvation-induced autophagy.