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The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

ABSTRACT: Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ? dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA.

SUBMITTER: Fenyk S

PROVIDER: S-EPMC4714197 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

Fenyk Stepan S Dixon Christopher H CH Gittens William H WH Townsend Philip D PD Sharples Gary J GJ Pålsson Lars-Olof LO Takken Frank L W FL Cann Martin J MJ

The Journal of biological chemistry 20151124 3

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. ...[more]

PMID: 26601946

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Project description:UnlabelledBacterially induced osteoblast apoptosis may be a major contributor to bone loss during osteomyelitis. We provide evidence for the functional expression in osteoblasts of NLRP3, a member of the NLR family of cytosolic receptors that has been implicated in the initiation of programmed cell death.IntroductionOsteoblasts undergo apoptosis after exposure to intracellular bacterial pathogens commonly associated with osteomyelitis. Death of this bone-forming cell type, in conjunction with increased numbers and activity of osteoclasts, may underlie the destruction of bone tissue at sites of bacterial infection. To date, the mechanisms responsible for bacterially induced apoptotic osteoblast cell death have not been resolved.Materials and methodsWe used flow cytometric techniques to determine whether intracellular invasion is needed for maximal apoptotic cell death in primary osteoblasts after challenge with Salmonella enterica. In addition, we used real-time PCR and immunoblot analyses to assess osteoblast expression of members of the nucleotide-binding domain leucine-rich repeat region-containing family of intracellular receptors (NLRs) that have been predicted to be involved in the induction of programmed cell death. Furthermore, we have used co-immunoprecipitation and siRNA techniques to confirm the functionality of such sensors in this cell type.ResultsIn this study, we showed that invasion of osteoblasts by Salmonella is necessary for maximal induction of apoptosis. We showed that murine and human osteoblasts express NLRP3 (previously known as CIAS1, cryopyrin, PYPAF1, or NALP3) but not NLRC4 (IPAF) and showed that the level of expression of this cytosolic receptor is modulated after bacterial challenge. We showed that osteoblasts express ASC, an adaptor molecule for NLRP3, and that these molecules associate after Salmonella infection. In addition, we showed that a reduction in the expression of NLRP3 attenuates Salmonella-induced reductions in the activity of an anti-apoptotic transcription factor in osteoblasts. Furthermore, we showed that NLRP3 expression is needed for caspase-1 activation and maximal induction of apoptosis in osteoblasts after infection with Salmonella.ConclusionsThe functional expression of NLRP3 in osteoblasts provides a potential mechanism underlying apoptotic cell death of this cell type after challenge with intracellular bacterial pathogens and may be a significant contributory factor to bone loss at sites of infection.

Dataset Information

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

Publications

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

Similar Datasets

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