Dataset Information

Chlorpyrifos Oxon-Induced Isopeptide Bond Formation in Human Butyrylcholinesterase.

ABSTRACT: A newly recognized action of organophosphates (OP) is the ability to crosslink proteins through an isopeptide bond. The first step in the mechanism is covalent addition of the OP to the side chain of lysine. This activates OP-lysine for reaction with a nearby glutamic or aspartic acid to make a gamma glutamyl epsilon lysine bond. Crosslinked proteins are high molecular weight aggregates. Our goal was to identify the residues in the human butyrylcholinesterase (HuBChE) tetramer that were crosslinked following treatment with 1.5 mM chlorpyrifos oxon. High molecular weight bands were visualized on an SDS gel. Proteins in the gel bands were digested with trypsin, separated by liquid chromatography and analyzed in an Orbitrap mass spectrometer. MSMS files were searched for crosslinked peptides using the Batch-Tag program in Protein Prospector. MSMS spectra were manually evaluated for the presence of ions that supported the crosslinks. The crosslink between Lys544 in VLEMTGNIDEAEWEWK544AGFHR and Glu542 in VLEMTGNIDEAEWE542WK satisfied our criteria including that of spatial proximity. Distances between Lys544 and Glu542 were 7.4 and 9.5 Å, calculated from the cryo-EM (electron microscopy) structure of the HuBChE tetramer. Paraoxon ethyl, diazoxon, and dichlorvos had less pronounced effects as visualized on SDS gels. Our proof-of-principle study provides evidence that OP have the ability to crosslink proteins. If OP-induced protein crosslinking occurs in the brain, OP exposure could be responsible for some cases of neurodegenerative disease.

SUBMITTER: Biberoglu K

PROVIDER: S-EPMC7037448 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Chlorpyrifos Oxon-Induced Isopeptide Bond Formation in Human Butyrylcholinesterase.

Biberoglu Kevser K Tacal Ozden O Schopfer Lawrence M LM Lockridge Oksana O

Molecules (Basel, Switzerland) 20200125 3

A newly recognized action of organophosphates (OP) is the ability to crosslink proteins through an isopeptide bond. The first step in the mechanism is covalent addition of the OP to the side chain of lysine. This activates OP-lysine for reaction with a nearby glutamic or aspartic acid to make a gamma glutamyl epsilon lysine bond. Crosslinked proteins are high molecular weight aggregates. Our goal was to identify the residues in the human butyrylcholinesterase (HuBChE) tetramer that were crosslin ...[more]

PMID: 31991818

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Project description:Chlorpyrifos (CPF), one of the most widely-used organophosphorus (OP) insecticides in agriculture, is degraded in the field to its oxon form, chlorpyrifos-oxon (CPO), which can represent a significant contaminant in exposures to adults and children. CPO is also responsible for the acetylcholinesterase (AChE) inhibition associated with CPF exposures; CPF is converted by liver CYP450 enzymes to CPO, which binds to and inhibits AChE and other serine active-site esterases, lipases and proteases. Young children represent a particularly susceptible population for exposure to CPF and CPO, in part because levels of the plasma enzyme, paraoxonase (PON1), which hydrolyzes CPO, are very low during early development. While a number of studies have demonstrated developmental neurotoxicity associated with CPF exposure, including effects at or below the threshold levels for AChE inhibition, it is unclear whether these effects were due directly to CPF or to its active metabolite, CPO. PON1 knockout (PON1-/-) mice, which lack PON1, represent a highly sensitive mouse model for toxicity associated with exposure to CPF or CPO. To examine the neurobehavioral consequences of CPO exposure during postnatal development, PON1-/- mice were exposed daily from PND 4 to PND 21 to CPO at 0.15, 0.18, or 0.25 mg/kg/d. A neurobehavioral test battery did not reveal significant effects of CPO on early reflex development, motor coordination, pre-pulse inhibition of startle, startle amplitude, open field behavior, or learning and memory in the contextual fear conditioning, Morris water maze, or water radial-arm maze tests. However, body weight gain and startle latency were significantly affected by exposure to 0.25 mg/kg/d CPO. Additionally, from PNDs 15-20 the mice exposed repeatedly to CPO at all three doses exhibited a dose-related transient hyperkinesis in the 20-min period following CPO administration, suggesting possible effects on catecholaminergic neurotransmission. Our previous study demonstrated wide-ranging effects of neonatal CPO exposure on gene expression in the brain and on brain AChE inhibition, and modulation of both of these effects by the PON1(Q192R) polymorphism. The current study indicates that the neurobehavioral consequences of these effects are more elusive, and suggests that alternative neurobehavioral tests might be warranted, such as tests of social interactions, age-dependent effects on learning and memory, or tests designed specifically to assess dopaminergic or noradrenergic function.

Project description:Many gram-positive pathogens possess external pili or fimbriae with which they adhere to host cells during the infection process. Unusual dual intramolecular isopeptide bonds between Asn and Lys side chains within the N-terminal and C-terminal domains of the pilus subunits have been observed initially in the Streptococcus pyogenes pilin subunit Spy0128 and subsequently in GBS52 from Streptococcus agalactiae, in the BcpA major pilin of Bacillus cereus and in the RrgB pilin of Streptococcus pneumoniae, among others. Within each pilin subunit, intramolecular isopeptide bonds serve to stabilize the protein. These bonds provide a means to withstand large external mechanical forces, as well as possibly assisting in supporting a conformation favored for pilin subunit polymerization via sortase transpeptidases. Genome-wide analyses of pili-containing gram-positive bacteria are known or suspected to contain isopeptide bonds in pilin subunits. For the autocatalytic formation of isopeptide cross-links, a conservation of three amino acids including Asn, Lys, and a catalytically important acidic Glu (or Asp) residue are responsible. However, the chemical mechanism of how isopeptide bonds form within pilin remains poorly understood. Although it is possible that several mechanistic paths could lead to isopeptide bond formation in pili, the requirement of a conserved glutamate and highly organized positioning of residues within the hydrophobic environment of the active site were found in numerous pilin crystal structures such as Spy0128 and RrgB. This suggests a mechanism involving direct coupling of lysine side chain amine to the asparagine carboxamide mediated by critical acid/base or hydrogen bonding interactions with the catalytic glutamate residue. From this mechanistic perspective, we used the QM/MM minimum free-energy path method to examine the reaction details of forming the isopeptide bonds with Spy0128 as a model pilin, specifically focusing on the role of the glutamate in catalysis. It was determined that the reaction mechanism likely consists of two major steps: the nucleophilic attack on C? by nitrogen in the unprotonated Lys ?-amino group and, then two concerted proton transfers occur during the formation of the intramolecular isopeptide bond to subsequently release ammonia. More importantly, within the dual active sites of Spy0128, Glu(117), and Glu(258) residues function as crucial catalysts for each isopeptide bond formation, respectively, by relaying two proton transfers. This work also suggests that domain-domain interactions within Spy0128 may modulate the reactivity of residues within each active site. Our results may hopefully shed light on the molecular mechanisms of pilin biogenesis in gram-positive bacteria.

Dataset Information

Chlorpyrifos Oxon-Induced Isopeptide Bond Formation in Human Butyrylcholinesterase.

Publications

Chlorpyrifos Oxon-Induced Isopeptide Bond Formation in Human Butyrylcholinesterase.

Similar Datasets

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