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Activating and masking enzymatic activity on demand is of the highest importance in nature. It is achieved by chemical interconversion of enzymes and the corresponding zymogens through, for example, proteolytic processing or reversible phosphorylation, and affords on-demand activation of enzymes, controlled in space and/or time. In stark contrast, examples of chemical zymogens are very few, and in most cases these are based on disulfide chemistry, which is largely indiscriminate as to the nature of the activating thiol. In this work, we address an outstanding challenge of specificity of reactivation of chemical zymogens. We achieve this through engineering affinity between the chemical zymogen and the activator. Additional, higher-level control over zymogen reactivation is installed in a nature-mimicking approach using steroidal hormones. Taken together, the results of this study take a step towards establishing the specificity of reactivation of synthetic, chemical zymogens. We anticipate that the results of this study will contribute significantly to the development of chemical zymogens as tools for diverse use in chemical biology and biotechnology.
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Precursores EnzimáticosRESUMEN
Covalent modification of proteins by reactive pollutants/metabolites might trigger various toxicities resulting from the disruption of protein structures and/or functions, which is critical for understanding the mechanism of pollutants-induced toxicity. However, this mechanism has rarely been touched on due to the lack of a methodology. In this research, the protein modification of bisphenol A (BPA) in rats was characterized using a series of liquid chromatography-tandem mass spectrometry (LC-MS) approaches. BPA-modified cysteine (Cys1) was first released from proteins via enzymatic hydrolysis and identified using LC-MS. Moreover, the positive correlation between Cys1 and hepatotoxicity indicated the involvement of protein modification in BPA toxicity. Then, in vitro incubation of BPA with amino acids and protein confirmed that BPA could specifically modify cysteine residues of proteins after bioactivation and provided four additional modification patterns. Finally, 24 BPA-modified proteins were identified from the liver of BPA-exposed rats using proteomic analysis, and they were mainly enriched in oxidative stress-related pathways. The modification on superoxide dismutases, catalase, and glutathione S-transferases disrupted their enzymatic functions, leading to oxidative damage. These results revealed that the covalent protein modification is an unignorable factor for BPA hepatotoxicity. Moreover, the workflow can be applied to identify protein adducts of other emerging contaminants and possible risk.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Contaminantes Ambientales , Animales , Compuestos de Bencidrilo/toxicidad , Cisteína , Fenoles , Proteínas , Proteómica , RatasRESUMEN
AIMS: 3-n-Butylphthalide (NBP) is widely used for the treatment of cerebral ischaemic stroke but can causeliver injury in clinical practice. This study aims to elucidate the underlying mechanisms and propose potential preventive strategies. MAIN METHODS: NBP and its four major metabolites, 3-hydroxy-NBP (3-OH-NBP), 10-hydroxy-NBP, 10-keto-NBP and NBP-11-oic acid, were synthesized and evaluated in primary human or rat hepatocytes (PHHs, PRHs). NBP-related substances or amino acid adducts were identified and semi-quantitated by ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The target proteins and binding sites were identified by shotgun proteomics based on peptide mass fingerprinting coupled with tandem mass spectrometry and verified by molecular docking. KEY FINDINGS: The toxicity of NBP and its four major metabolites were compared in both PHHs and PRHs, and 3-OH-NBP was found to be the most toxic metabolite. 3-OH-NBP induced remarkable cell death and oxidative stresses in hepatocytes, which correlated well with the levels of glutathione and N-acetylcysteine adducts (3-GSH-NBP and 3-NAC-NBP) in cell supernatants. Additionally, 3-OH-NBP covalently conjugated with intracellular Cys, Lys and Ser, with preferable binding to Cys sites at Myh9 Cys1380, Prdx4 Cys53, Vdac2 Cys48 and Vdac3 Cys36. Furthermore, we found that CYP3A4 induction by rifampicin augmented NBP-induced cell toxicity and supplementing with GSH or NAC alleviated the oxidative stresses and reactive metabolites caused by 3-OH-NBP. SIGNIFICANCE: Our work suggests that glutathione depletion, mitochondrial injury and covalent protein modification are the main causes of NBP-induced hepatotoxicity, which may be prevented by exogenous GSH or NAC supplementation and avoiding concomitant use of CYP3A4 inducers.
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Acetilcisteína/metabolismo , Benzofuranos/metabolismo , Benzofuranos/toxicidad , Glutatión/metabolismo , Hepatocitos/metabolismo , Animales , Sitios de Unión/fisiología , Células Cultivadas , Inductores del Citocromo P-450 CYP3A/metabolismo , Inductores del Citocromo P-450 CYP3A/toxicidad , Relación Dosis-Respuesta a Droga , Hepatocitos/efectos de los fármacos , Humanos , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-DawleyRESUMEN
Plant-derived natural products (NPs) with electrophilic functional groups engage various subsets of the proteome via covalent modification of nucleophilic cysteine residues. This electrophile-nucleophile interaction can change protein conformation, alter protein function, and modulate their biological action. The biological significance of these covalent protein modifications in health and disease is increasingly recognized. One way to understand covalent NP-protein interactions is to utilize traditional proteomics and modern mass spectrometry (MS)-based proteomic strategies. These strategies have proven effective in uncovering specific NP protein targets and are critical first steps that allow for a much deeper understanding of the ability of NPs to modulate cellular processes. Here, plant-derived NPs that covalently modify proteins are reviewed, the biological significance of these covalent modifications, and the different proteomic strategies that have been employed to study these NP-protein interactions.
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Productos Biológicos , Proteómica , Cisteína , Espectrometría de Masas , ProteínasRESUMEN
Endocytosis and intracellular retrograde trafficking from endosomes to the Golgi apparatus are key cellular processes. Endocytosis is directly or indirectly involved in many if not all cellular functions ranging from nutrient uptake and receptor signaling to mitosis, cell division, and migration (Scita, Di Fiore. Nature 463(7280):464-473, 2010; McMahon, Boucrot. Nat Rev Mol Cell Biol 12(8):517-533, 2011). Retrograde trafficking is emerging as a key driver for cell polarity. Robust methods are needed to quantify these processes. At the example of the bacterial Shiga toxin and the endogenous α5ß1 integrin, we here describe generic methods to differentiate (1) internalized from cell surface-accessible cargo proteins and (2) endocytic cargo proteins that have reached the Golgi apparatus via the retrograde route from those that have not. The choice of antibodies or natural ligands allows to adjust these methods to virtually any chosen biological system.
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Endocitosis/genética , Endosomas/genética , Aparato de Golgi/genética , Biología Molecular/métodos , Transporte Biológico/genética , Movimiento Celular/efectos de los fármacos , Polaridad Celular/genética , Células HeLa , Humanos , Proteínas de la Membrana/genética , Redes y Vías Metabólicas/efectos de los fármacos , Toxina Shiga/química , Toxina Shiga/farmacología , Red trans-GolgiRESUMEN
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.
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Proteínas , Triclosán , Animales , Cromatografía Liquida , Imagenología Tridimensional , Espectrometría de Masas , Proteínas/efectos de los fármacos , Proteómica , Ratas , Triclosán/toxicidadRESUMEN
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.
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Protein arginine deiminases (PADs) hydrolyze the side chain of arginine to form citrulline. Aberrant PAD activity is associated with rheumatoid arthritis, multiple sclerosis, lupus, and certain cancers. These pathologies established the PADs as therapeutic targets and multiple PAD inhibitors are known. Herein, we describe the first highly potent PAD1-selective inhibitors (1 and 19). Detailed structure-activity relationships indicate that their potency and selectivity is due to the formation of a halogen bond with PAD1. Importantly, these inhibitors inhibit histone H3 citrullination in HEK293TPAD1 cells and mouse zygotes with excellent potency. Based on this scaffold, we also developed a PAD1-selective activity-based probe that shows remarkable cellular efficacy and proteome selectivity. Based on their potency and selectivity we expect that 1 and 19 will be widely used chemical tools to understand PAD1 biology.