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Urea solution is one of the most commonly employed protein denaturants for protease digestion in proteomic studies. However, it has long been recognized that urea solution can cause carbamylation at the N termini of proteins/peptides and at the side chain amino groups of lysine and arginine residues. Protein/peptide carbamylation blocks protease digestion and affects protein identification and quantification in mass spectrometry analysis by blocking peptide amino groups from isotopic/isobaric labeling and changing peptide charge states, retention times, and masses. In addition, protein carbamylation during sample preparation makes it difficult to study in vivo protein carbamylation. In this study, we compared the peptide carbamylation in urea solutions of different buffers and found that ammonium-containing buffers were the most effective buffers to inhibit protein carbamylation in urea solution. The possible mechanism of carbamylation inhibition by ammonium-containing buffers is discussed, and a revised procedure for the protease digestion of proteins in urea and ammonium-containing buffers was developed to facilitate its application in proteomic research. 相似文献
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KR Shreder EC Lin J Wu J Cajica CM Amantea Y Hu E Okerberg HE Brown LM Pham de M Chung AS Fraser E McGee JS Rosenblum JW Kozarich 《Bioorganic & medicinal chemistry letters》2012,22(17):5748-5751
KIAA1363 is a serine hydrolase whose activity has been shown to be positively associated with tumor cell invasiveness. Thus, inhibitors of KIAA1363 represent a novel targeted therapy approach towards cancer. AX11890 ((1-bromo-2-naphthyl) N,N-dimethylcarbamate) was identified as a KIAA1363 inhibitor with an IC(50) value of 1.2μM and was shown using ESI-MS to carbamylate the catalytic residue Ser(191). SAR studies explored both substitution of the 1-bromo group and derivatization of the 6-position. Activity-based protein profiling demonstrated AX13057 inhibited tumor-localized KIAA1363 in SK-OV-3 xenograft-bearing mice. 相似文献
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Verma V Testero SA Amini K Wei W Liu J Balachandran N Monoharan T Stynes S Kotra LP Golemi-Kotra D 《The Journal of biological chemistry》2011,286(43):37292-37303
Carbapenem-hydrolyzing class D β-lactamases (CHDLs) represent an emerging antibiotic resistance mechanism encountered among the most opportunistic Gram-negative bacterial pathogens. We report here the substrate kinetics and mechanistic characterization of a prominent CHDL, the OXA-58 enzyme, from Acinetobacter baumannii. OXA-58 uses a carbamylated lysine to activate the nucleophilic serine used for β-lactam hydrolysis. The deacylating water molecule approaches the acyl-enzyme species, anchored at this serine (Ser-83), from the α-face. Our data show that OXA-58 retains the catalytic machinery found in class D β-lactamases, of which OXA-10 is representative. Comparison of the homology model of OXA-58 and the recently solved crystal structures of OXA-24 and OXA-48 with the OXA-10 crystal structure suggests that these CHDLs have evolved the ability to hydrolyze imipenem, an important carbapenem in clinical use, by subtle structural changes in the active site. These changes may contribute to tighter binding of imipenem to the active site and removal of steric hindrances from the path of the deacylating water molecule. 相似文献
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The rate and duration of inhibition of recombinant human acetylcholinesterase (AChE) and human butyrylcholinesterase (BuChE) by nine N-methyl,N-alkyl derivatives of (R)-3-prop-2-ynylamino-indan, designed as potential treatment of Alzheimer's disease, was obtained from measurement of the carbamylation k(i) and decarbamylation k(3) rate constants. This also provided information about the rate of formation of the leaving group, 6-OH-(R)-3-prop-2-ynylamino-indan, designed as an MAO-B inhibitor with neuroprotective activity. The N-dimethyl derivative had the highest k(i) of the alkyl derivatives. Substitution of one N-methyl by N-ethyl resulted in a 14-fold decrease in k(i) and 28-fold decrease in k(3). A progressive increase in k(i) occurred as the length of the alkyl chain progressed from propyl to n-hexyl and cyclo-hexyl, with relatively little or no increase in k(3). Higher k(i) values than that of the dimethyl analogue were obtained with the N-aryl substitutes, N-phenyl and N-methoxy-phenyl. Six of the compounds had much higher k(i) values for BuChE than AChE, but the N-cyclo-hexyl and N-methoxy-phenyl compounds were inactive. However, an inverse relation was found between k(i) and the degree of brain AChE inhibition ex vivo after parenteral administration of the compounds in rats. This could have resulted from more rapid hydrolysis of the compounds with high k(i) values by esterases in blood and liver. Only the N-ethyl and N-propyl derivatives showed AChE and BuChE inhibitory activity in vivo of a suitably slow onset and long duration, together with MAO-B inhibition. 相似文献
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Summary. The stability of felinine, an amino acid present in feline urine, was investigated. Synthetic felinine was unstable in the
urine of a selection of mammals. Felinine was found to stable in feline urine in which urea had been degraded. Synthetic felinine
was found to react specifically with urea and did not react with urea analogues such as biuret or thiourea or other nucleophilic
compounds such as ammonia which is more nucleophilic or acetamide and water which are less nucleophilic than urea. The reaction
of urea and felinine was independent of pH over the range of 3–10. Urea did not react with N-acetyl-felinine suggesting a felinine N-terminal interaction with urea. Mass spectral analysis of the reaction products showed
the presence of carbamylated felinine and fragmentation ions derived from carbamyl-felinine. The physiological relevance of
felinine carbamylation is yet to be determined. 相似文献
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Macrophage migration inhibitory factor (MIF) is an important player in the regulation of the inflammatory response. Elevated plasma MIF is found in sepsis, arthritis, cystic fibrosis and atherosclerosis. Immunomodulatory activities of MIF include the ability to promote survival and recruitment of inflammatory cells and to amplify pro-inflammatory cytokine production. MIF has an unusual nucleophilic N-terminal proline with catalytic tautomerase activity. It remains unclear whether tautomerase activity is required for MIF function, but small molecules that inhibit tautomerase activity also inhibit the pro-inflammatory activities of MIF. A prominent feature of the acute inflammatory response is neutrophil activation and production of reactive oxygen species, including myeloperoxidase (MPO)-derived hypochlorous acid and hypothiocyanous acid. We hypothesized that MPO-derived oxidants would oxidize the N-terminal proline of MIF and alter its biological activity. MIF was exposed to hypochlorous acid and hypothiocyanous acid and the oxidative modifications on MIF were examined by LC-MS/MS. Imine formation and carbamylation was observed on the N-terminal proline in response to MPO-dependent generation of hypochlorous and hypothiocyanous acid, respectively. These modifications led to a complete loss of tautomerase activity. However, modified MIF still increased CXCL-8/IL-8 production by peripheral blood mononuclear cells (PBMCs) and blocked neutrophil apoptosis, indicating that tautomerase activity is not essential for these biological functions. Pre-treatment of MIF with hypochlorous acid protected the protein from covalent modification by the MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP). Therefore, oxidant generation at inflammatory sites may protect MIF from inactivation by more disruptive electrophiles, including drugs designed to target the tautomerase activity of MIF. 相似文献
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Protein regulation by reversible phosphorylation is fundamental in nature, and large-scale phosphoproteomic analyses are becoming routine in proteomics laboratories. These analyses utilise phosphopeptide separation and enrichment techniques linked to LC-MS/MS. Herein, we report that IMAC and TiO(2) also enrich for non-phosphorylated modified peptides such as acetylated, deamidated and carbamylated peptides. Urea and digestion conditions commonly used in phosphoproteomic workflows are the likely sources of the induced modifications (deamidation and carbamylation) and can easily modify phosphopeptides. Including these variable modifications in database searches increased the total number of identified phosphopeptides by 15%. We also show that strong cation exchange fractionation provides poor resolution of phosphopeptides and actually enriches these alternatively modified peptides. By switching to reverse-phase chromatography, we show a significant improvement in the number of identified phosphopeptides. We recommend that the users of phosphopeptide enrichment strategies avoid using urea as a denaturant and that careful consideration is given to chromatographic conditions and the types of variable modifications used in database searches. Thus, the capacity of IMAC and TiO(2) to enrich phosphopeptides bearing modifications other than phosphorylation is a previously unappreciated property of these chromatographies with practical implications for the field of phosphoproteomics. 相似文献
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V. Guru KrishnaKumar Lokesh Baweja Krittika Ralhan Sharad Gupta 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(12):2590-2604
Background
Carbamylation is a non-enzymatic post-translational modification (PTM), which involves the covalent modification of N-terminus of protein or ε-amino group of Lys. The role of carbamylation in several age-related disorders is well documented, however, the relationship between carbamylation and neurodegenerative disorders including Alzheimer's disease remains uncharted.Methods
In the present study, using aggregation-prone tau-core hexapeptide fragments 306VQIVYK311 (PHF6) and 275VQIINK280 (PHF6*) as models, we have elucidated the effect of carbamylation on aggregation kinetics and the changes occurring in the 3-dimensional architecture of fibrils using biophysical assays and molecular dynamics simulations.Results
We found that carbamylation aids in amyloid formation and can convert the unstructured off-pathway aggregates into robust amyloids, which were toxic to cells. Electron microscopy images and molecular dynamics simulations of PHF6 fibrils showed that carbamylated peptides can form excess hydrogen bonds and modulate the pitch length and twist of peptides fibrils. We have also compared N-terminal carbamylation to acetylation and further extended our finding to full length tau that exhibits aggregation upon carbamylation even in the absence of any external inducer.Conclusion
Our in vitro and in silico results together suggest that carbamylation can modulate the aggregation pathway of the amyloidegenic sequences and cause structural changes in fibril assemblies.General significance
Carbamylation acts as a switch, which triggers the aggregation in short amyloidogenic peptide fragments and modulate the structural changes in resulting amyloid fibrils. 相似文献9.
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