AtTIP2;2 facilitates resistance to zinc toxicity via promoting zinc immobilization in the root and limiting root-to-shoot zinc translocation in Arabidopsis thaliana.

Zinc (Zn) is an essential micronutrient for plants. However, excess Zn is toxic to non-accumulating plants like Arabidopsis thaliana. To cope with Zn toxicity, non-accumulating plants need to keep excess Zn in the less sensitive root tissues and restrict its translocation to the vulnerable shoot tissues, a process referred to as Zn immobilization in the root. However, the mechanism underlying Zn immobilization is not fully understood. In Arabidopsis, sequestration of excess Zn to the vacuole of root cells is crucial for Zn immobilization, facilitated by distinct tonoplast-localized transporters. As some members of the aquaporin superfamily have been implicated in transporting metal ions besides polar but non-charged small molecules, we tested whether Arabidopsis thaliana tonoplast intrinsic proteins (AtTIPs) could be involved in Zn immobilization and resistance. We found that AtTIP2;2 is involved in retaining excess Zn in the root, limiting its translocation to the shoot, and facilitating its accumulation in the leaf trichome. Furthermore, when expressed in yeast, the tonoplast-localized AtTIP2;2 renders glutathione (GSH)-dependent Zn resistance to yeast cells, suggesting that AtTIP2;2 facilitates the across-tonoplast transport of GSH-Zn complexes. Our findings provide new insights into aquaporins' roles in heavy metal resistance and detoxification in plants.

Nematode Signaling Molecules Are Extensively Metabolized by Animals, Plants, and Microorganisms.

Many bacterivorous and parasitic nematodes secrete signaling molecules called ascarosides that play a central role regulating their behavior and development. Combining stable-isotope labeling and mass spectrometry-based comparative metabolomics, here we show that ascarosides are taken up from the environment and metabolized by a wide range of phyla, including plants, fungi, bacteria, and mammals, as well as nematodes. In most tested eukaryotes and some bacteria, ascarosides are metabolized into derivatives with shortened fatty acid side chains, analogous to ascaroside biosynthesis in nematodes. In plants and C. elegans, labeled ascarosides were additionally integrated into larger, modular metabolites, and use of different ascaroside stereoisomers revealed the stereospecificity of their biosynthesis. The finding that nematodes extensively metabolize ascarosides taken up from the environment suggests that pheromone editing may play a role in conspecific and interspecific interactions. Moreover, our results indicate that plants, animals, and microorganisms may interact with associated nematodes via manipulation of ascaroside signaling.

High CO2 - and pathogen-driven expression of the carbonic anhydrase ßCA3 confers basal immunity in tomato.

Atmospheric CO2 concentrations exert a strong influence on the susceptibility of plants to pathogens. However, the mechanisms involved in the CO2 -dependent regulation of pathogen resistance are largely unknown. Here we show that the expression of tomato (Solanum lycopersicum) ß-CARBONIC ANHYDRASE 3 (ßCA3) is induced by the virulent pathogen Pseudomonas syringae pv. tomato DC3000. The role of ßCA3 in the high CO2 -mediated response in tomato and two other Solanaceae crops is distinct from that in Arabidopsis thaliana. Using ßCA3 knock-out and over-expression plants, we demonstrate that ßCA3 plays a positive role in the activation of basal immunity, particularly under high CO2 . ßCA3 is transcriptionally activated by the transcription factor NAC43 and is also post-translationally regulated by the receptor-like kinase GRACE1. The ßCA3 pathway of basal immunity is independent on stomatal- and salicylic-acid-dependent regulation. Global transcriptome analysis and cell wall metabolite measurement implicate cell wall metabolism/integrity in ßCA3-mediated basal immunity under both CO2 conditions. These data not only highlight the importance of ßCA3 in plant basal immunity under high CO2 in a well-studied susceptible crop-pathogen system, but they also point to new targets for disease management strategies in a changing climate.

Arabidopsis SMALL DEFENSE-ASSOCIATED PROTEIN 1 Modulates Pathogen Defense and Tolerance to Oxidative Stress.

Salicylic acid (SA) and reactive oxygen species (ROS) are known to be key modulators of plant defense. However, mechanisms of molecular signal perception and appropriate physiological responses to SA and ROS during biotic or abiotic stress are poorly understood. Here we report characterization of SMALL DEFENSE-ASSOCIATED PROTEIN 1 (SDA1), which modulates defense against bacterial pathogens and tolerance to oxidative stress. sda1 mutants are compromised in defense gene expression, SA accumulation, and defense against bacterial pathogens. External application of SA rescues compromised defense in sda1 mutants. sda1 mutants are also compromised in tolerance to ROS-generating chemicals. Overexpression of SDA1 leads to enhanced resistance against bacterial pathogens and tolerance to oxidative stress. These results suggest that SDA1 regulates plant immunity via the SA-mediated defense pathway and tolerance to oxidative stress. SDA1 encodes a novel small plant-specific protein containing a highly conserved seven amino acid (S/G)WA(D/E)QWD domain at the N-terminus that is critical for SDA1 function in pathogen defense and tolerance to oxidative stress. Taken together, our studies suggest that SDA1 plays a critical role in modulating both biotic and abiotic stresses in Arabidopsis (Arabidopsis thaliana) and appears to be a plant-specific stress responsive protein.

Plant metabolism of nematode pheromones mediates plant-nematode interactions.

Microorganisms and nematodes in the rhizosphere profoundly impact plant health, and small-molecule signaling is presumed to play a central role in plant rhizosphere interactions. However, the nature of the signals and underlying mechanisms are poorly understood. Here we show that the ascaroside ascr#18, a pheromone secreted by plant-parasitic nematodes, is metabolized by plants to generate chemical signals that repel nematodes and reduce infection. Comparative metabolomics of plant tissues and excretions revealed that ascr#18 is converted into shorter side-chained ascarosides that confer repellency. An Arabidopsis mutant defective in two peroxisomal acyl-CoA oxidases does not metabolize ascr#18 and does not repel nematodes, indicating that plants, like nematodes, employ conserved peroxisomal ß-oxidation to edit ascarosides and change their message. Our results suggest that plant-editing of nematode pheromones serves as a defense mechanism that acts in parallel to conventional pattern-triggered immunity, demonstrating that plants may actively manipulate chemical signaling of soil organisms.

A genome-wide screen for human salicylic acid (SA)-binding proteins reveals targets through which SA may influence development of various diseases.

Salicylic acid (SA) is the major metabolite and active ingredient of aspirin; both compounds reduce pain, fever, and inflammation. Despite over a century of research, aspirin/SA's mechanism(s) of action is still only partially understood. Here we report the results of a genome-wide, high-throughput screen to identify potential SA-binding proteins (SABPs) in human HEK293 cells. Following photo-affinity crosslinking to 4-azidoSA and immuno-selection with an anti-SA antibody, approximately 2,000 proteins were identified. Among these, 95 were enriched more than 10-fold. Pathway enrichment analysis with these 95 candidate SABPs (cSABPs) revealed possible involvement of SA in multiple biological pathways, including (i) glycolysis, (ii) cytoskeletal assembly and/or signaling, and (iii) NF-κB-mediated immune signaling. The two most enriched cSABPs, which corresponded to the glycolytic enzymes alpha-enolase (ENO1) and pyruvate kinase isozyme M2 (PKM2), were assessed for their ability to bind SA and SA's more potent derivative amorfrutin B1 (amoB1). SA and amoB1 bound recombinant ENO1 and PKM2 at low millimolar and micromolar concentrations, respectively, and inhibited their enzymatic activities in vitro. Consistent with these results, low millimolar concentrations of SA suppressed glycolytic activity in HEK293 cells. To provide insights into how SA might affect various human diseases, a cSABP-human disorder/disease network map was also generated.

Mimicking the Host Regulation of Salicylic Acid: A Virulence Strategy by the Clubroot Pathogen Plasmodiophora brassicae.

The plant hormone salicylic acid (SA) plays a critical role in defense against biotrophic pathogens such as Plasmodiophora brassicae, which is an obligate pathogen of crucifer species and the causal agent of clubroot disease of canola (Brassica napus). P. brassicae encodes a protein, predicted to be secreted, with very limited homology to benzoic acid (BA)/SA-methyltransferase, designated PbBSMT. PbBSMT has a SA- and an indole-3-acetic acid-binding domain, which are also present in Arabidopsis thaliana BSMT1 (AtBSMT1) and, like AtBSMT1, has been shown to methylate BA and SA. In support of the hypothesis that P. brassicae uses PbBSMT to overcome SA-mediated defenses by converting SA into inactive methyl salicylate (MeSA), here, we show that PbBSMT suppresses local defense and provide evidence that PbBSMT is much more effective than AtBSMT1 at suppressing the levels of SA and its associated effects. Basal SA levels in Arabidopsis plants that constitutively overexpress PbBSMT compared with those in Arabidopsis wild-type Col-0 (WT) were reduced approximately 80% versus only a 50% reduction in plants overexpressing AtBSMT1. PbBSMT-overexpressing plants were more susceptible to P. brassicae than WT plants; they also were partially compromised in nonhost resistance to Albugo candida. In contrast, AtBSMT1-overexpressing plants were not more susceptible than WT to either P. brassicae or A. candida. Furthermore, transgenic Arabidopsis and tobacco plants overexpressing PbBSMT exhibited increased susceptibility to virulent Pseudomonas syringae pv. tomato DC3000 (DC3000) and virulent Pseudomonas syringae pv. tabaci, respectively. Gene-mediated resistance to DC3000/AvrRpt2 and tobacco mosaic virus (TMV) was also compromised in Arabidopsis and Nicotiana tabacum 'Xanthi-nc' plants overexpressing PbBSMT, respectively. Transient expression of PbBSMT or AtBSMT1 in lower leaves of N. tabacum Xanthi-nc resulted in systemic acquired resistance (SAR)-like enhanced resistance to TMV in the distal systemic leaves. Chimeric grafting experiments revealed that, similar to SAR, the development of a PbBSMT-mediated SAR-like phenotype was also dependent on the MeSA esterase activity of NtSABP2 in the systemic leaves. Collectively, these results strongly suggest that PbBSMT is a novel effector, which is secreted by P. brassicae into its host plant to deplete pathogen-induced SA accumulation.

Systemic Acquired Resistance and Salicylic Acid: Past, Present, and Future.

This article is part of the Distinguished Review Article Series in Conceptual and Methodological Breakthroughs in Molecular Plant-Microbe Interactions. Salicylic acid (SA) is a critical plant hormone that regulates numerous aspects of plant growth and development as well as the activation of defenses against biotic and abiotic stress. Here, we present a historical overview of the progress that has been made to date in elucidating the role of SA in signaling plant immune responses. The ability of plants to develop acquired immunity after pathogen infection was first proposed in 1933. However, most of our knowledge about plant immune signaling was generated over the last three decades, following the discovery that SA is an endogenous defense signal. During this timeframe, researchers have identified i) two pathways through which SA can be synthesized, ii) numerous proteins that regulate SA synthesis and metabolism, and iii) some of the signaling components that function downstream of SA, including a large number of SA targets or receptors. In addition, it has become increasingly evident that SA does not signal immune responses by itself but, rather, as part of an intricate network that involves many other plant hormones. Future efforts to develop a comprehensive understanding of SA-mediated immune signaling will therefore need to close knowledge gaps that exist within the SA pathway itself as well as clarify how crosstalk among the different hormone signaling pathways leads to an immune response that is both robust and optimized for maximal efficacy, depending on the identity of the attacking pathogen.

MORC Proteins: Novel Players in Plant and Animal Health.

Microrchidia (MORC) proteins comprise a family of proteins that have been identified in prokaryotes and eukaryotes. They are defined by two hallmark domains: a GHKL-type ATPase and an S5 fold. MORC proteins in plants were first discovered via a genetic screen for Arabidopsis mutants compromised for resistance to a viral pathogen. Subsequent studies expanded their role in plant immunity and revealed their involvement in gene silencing and transposable element repression. Emerging data suggest that MORC proteins also participate in pathogen-induced chromatin remodeling and epigenetic gene regulation. In addition, biochemical analyses recently demonstrated that plant MORCs have topoisomerase II (topo II)-like DNA modifying activities that may be important for their function. Interestingly, animal MORC proteins exhibit many parallels with their plant counterparts, as they have been implicated in disease development and gene silencing. In addition, human MORCs, like plant MORCs, bind salicylic acid and this inhibits some of their topo II-like activities. In this review, we will focus primarily on plant MORCs, although relevant comparisons with animal MORCs will be provided.

How does the multifaceted plant hormone salicylic acid combat disease in plants and are similar mechanisms utilized in humans?

Salicylic acid (SA) is an important plant hormone that regulates many aspects of plant growth and development, as well as resistance to (a)biotic stress. Efforts to identify SA effector proteins have revealed that SA binds to and alters the activity of multiple plant proteins-this represents a shift from the paradigm that hormones mediate their functions via one or a few receptors. SA and its derivatives also have multiple targets in animals; some of these proteins, like their plant counterparts, are associated with pathological processes. Together, these findings suggest that SA exerts its defense-associated effects in both kingdoms via a large number of targets.

Members of the abscisic acid co-receptor PP2C protein family mediate salicylic acid-abscisic acid crosstalk.

The interplay between abscisic acid (ABA) and salicylic acid (SA) influences plant responses to various (a)biotic stresses; however, the underlying mechanism for this crosstalk is largely unknown. Here, we report that type 2C protein phosphatases (PP2Cs), some of which are negative regulators of ABA signaling, bind SA. SA binding suppressed the ABA-enhanced interaction between these PP2Cs and various ABA receptors belonging to the PYR/PYL/RCAR protein family. Additionally, SA suppressed ABA-enhanced degradation of PP2Cs and ABA-induced stabilization of SnRK2s. Supporting SA's role as a negative regulator of ABA signaling, exogenous SA suppressed ABA-induced gene expression, whereas the SA-deficient sid2-1 mutant displayed heightened PP2C degradation and hypersensitivity to ABA-induced suppression of seed germination. Together, these results suggest a new molecular mechanism through which SA antagonizes ABA signaling. A better understanding of the crosstalk between these hormones is important for improving the sustainability of agriculture in the face of climate change.

Plant and Human MORC Proteins Have DNA-Modifying Activities Similar to Type II Topoisomerases, but Require One or More Additional Factors for Full Activity.

To elucidate one or more mechanisms through which microrchidia (MORC) proteins impact immunity, epigenetic gene silencing, and DNA modifications, the enzymatic activities of plant MORCs were characterized. Previously, we showed that plant MORC1s have ATPase and DNA endonuclease activities. Here, we demonstrate that plant MORCs have topoisomerase type II (topo II)-like activities, as they i) covalently bind DNA, ii) exhibit DNA-stimulated ATPase activity, iii) relax or nick supercoiled DNA, iv) catenate DNA, and v) decatenante kinetoplast DNA. Mutational analysis of tomato SlMORC1 suggests that a K loop-like sequence is required to couple DNA binding to ATPase stimulation as well as for efficient SlMORC1's DNA relaxation and catenation activities and in planta suppression of INF1-induced cell death, which is related to immunity. Human MORCs were found to exhibit the same topo II-like DNA modification activities as their plant counterparts. In contrast to typical topo IIs, SlMORC1 appears to require one or more accessory factors to complete some of its enzymatic activities, since addition of tomato extracts were needed for ATP-dependent, efficient conversion of supercoiled DNA to nicked/relaxed DNA and catenanes and for formation of topoisomer intermediates. Both plant and human MORCs bind salicylic acid; this suppresses their decatenation but not relaxation activity.

DAMPs, MAMPs, and NAMPs in plant innate immunity.

BACKGROUND: Multicellular organisms have evolved systems/mechanisms to detect various forms of danger, including attack by microbial pathogens and a variety of pests, as well as tissue and cellular damage. Detection via cell-surface receptors activates an ancient and evolutionarily conserved innate immune system. RESULT: Potentially harmful microorganisms are recognized by the presence of molecules or parts of molecules that have structures or chemical patterns unique to microbes and thus are perceived as non-self/foreign. They are referred to as Microbe-Associated Molecular Patterns (MAMPs). Recently, a class of small molecules that is made only by nematodes, and that functions as pheromones in these organisms, was shown to be recognized by a wide range of plants. In the presence of these molecules, termed Nematode-Associated Molecular Patterns (NAMPs), plants activate innate immune responses and display enhanced resistance to a broad spectrum of microbial and nematode pathogens. In addition to pathogen attack, the relocation of various endogenous molecules or parts of molecules, generally to the extracellular milieu, as a result of tissue or cellular damage is perceived as a danger signal, and it leads to the induction of innate immune responses. These relocated endogenous inducers are called Damage-Associated Molecular Patterns (DAMPs). CONCLUSIONS: This mini-review is focused on plant DAMPs, including the recently discovered Arabidopsis HMGB3, which is the counterpart of the prototypic animal DAMP HMGB1. The plant DAMPs will be presented in the context of plant MAMPs and NAMPs, as well as animal DAMPs.

Pathogen Infection and MORC Proteins Affect Chromatin Accessibility of Transposable Elements and Expression of Their Proximal Genes in Arabidopsis.

To assess the role of MORC1 in epigenetics in relation to plant immunity, genome-wide chromatin accessibility was compared between mock- or Pseudomonas syringae pv. tomato-inoculated wild type (WT) Arabidopsis, the morc1/2 double mutant, or both. Most changes in chromatin accessibility, scored by DNase I hypersensitive sites (DHSs), were located in the promoters of genes and transposable elements (TEs). Comparisons between morc1/2 and WT receiving the same treatment revealed differential DHSs (dDHSs) predominantly associated with heterochromatic TEs. By contrast, comparisons between mock- and P. syringae pv. tomato-inoculated plants from the same genotype showed dDHSs associated with biotic and abiotic stress-related genes; a smaller but significant population was in TEs. Moreover, many defense genes, including PR-1, PR-2, and PR-5, were proximal to P. syringae pv. tomato-induced, TE-associated dDHSs. A random subset of these defense genes showed moderately delayed or reduced expression or both in P. syringae pv. tomato-infected morc1/2 as compared with WT. MORC1 was physically bound to chromatin in a P. syringae pv. tomato infection-responsive manner at sites dispersed throughout the genome. Notably, silencing of TE-associated dDHSs proximal to these infection-induced, MORC1-interacting sites led to significant suppression of P. syringae pv. tomato-induced transcription of adjacent defense genes, including PR-1. These results provide evidence that MORC1 is associated with TEs and suggest that a subset of these TEs may help regulate their proximal defense genes.

Multiple Targets of Salicylic Acid and Its Derivatives in Plants and Animals.

Salicylic acid (SA) is a critical plant hormone that is involved in many processes, including seed germination, root initiation, stomatal closure, floral induction, thermogenesis, and response to abiotic and biotic stresses. Its central role in plant immunity, although extensively studied, is still only partially understood. Classical biochemical approaches and, more recently, genome-wide high-throughput screens have identified more than two dozen plant SA-binding proteins (SABPs), as well as multiple candidates that have yet to be characterized. Some of these proteins bind SA with high affinity, while the affinity of others exhibit is low. Given that SA levels vary greatly even within a particular plant species depending on subcellular location, tissue type, developmental stage, and with respect to both time and location after an environmental stimulus such as infection, the presence of SABPs exhibiting a wide range of affinities for SA may provide great flexibility and multiple mechanisms through which SA can act. SA and its derivatives, both natural and synthetic, also have multiple targets in animals/humans. Interestingly, many of these proteins, like their plant counterparts, are associated with immunity or disease development. Two recently identified SABPs, high mobility group box protein and glyceraldehyde 3-phosphate dehydrogenase, are critical proteins that not only serve key structural or metabolic functions but also play prominent roles in disease responses in both kingdoms.

Newly Identified Targets of Aspirin and Its Primary Metabolite, Salicylic Acid.

Salicylic acid (SA) is a plant hormone, which influences several physiological processes, and is a critical modulator of multiple levels of immunity in plants. Several high-throughput screens, which were developed to identify SA-binding proteins through which SA mediates its many physiological effects in plants, uncovered several novel targets of aspirin and its primary metabolite, SA, in humans. These include glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and high mobility group box 1 (HMGB1), two proteins associated with some of the most prevalent and devastating human diseases. In addition, natural and synthetic SA derivatives were discovered, which are much more potent than SA at inhibiting the disease-associated activities of these targets.

Activation of Plant Innate Immunity by Extracellular High Mobility Group Box 3 and Its Inhibition by Salicylic Acid.

Damage-associated molecular pattern molecules (DAMPs) signal the presence of tissue damage to induce immune responses in plants and animals. Here, we report that High Mobility Group Box 3 (HMGB3) is a novel plant DAMP. Extracellular HMGB3, through receptor-like kinases BAK1 and BKK1, induced hallmark innate immune responses, including i) MAPK activation, ii) defense-related gene expression, iii) callose deposition, and iv) enhanced resistance to Botrytis cinerea. Infection by necrotrophic B. cinerea released HMGB3 into the extracellular space (apoplast). Silencing HMGBs enhanced susceptibility to B. cinerea, while HMGB3 injection into apoplast restored resistance. Like its human counterpart, HMGB3 binds salicylic acid (SA), which results in inhibition of its DAMP activity. An SA-binding site mutant of HMGB3 retained its DAMP activity, which was no longer inhibited by SA, consistent with its reduced SA-binding activity. These results provide cross-kingdom evidence that HMGB proteins function as DAMPs and that SA is their conserved inhibitor.

Human GAPDH Is a Target of Aspirin's Primary Metabolite Salicylic Acid and Its Derivatives.

The plant hormone salicylic acid (SA) controls several physiological processes and is a key regulator of multiple levels of plant immunity. To decipher the mechanisms through which SA's multiple physiological effects are mediated, particularly in immunity, two high-throughput screens were developed to identify SA-binding proteins (SABPs). Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) from plants (Arabidopsis thaliana) was identified in these screens. Similar screens and subsequent analyses using SA analogs, in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology, established that human GAPDH (HsGAPDH) also binds SA. In addition to its central role in glycolysis, HsGAPDH participates in several pathological processes, including viral replication and neuronal cell death. The anti-Parkinson's drug deprenyl has been shown to suppress nuclear translocation of HsGAPDH, an early step in cell death and the resulting cell death induced by the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Here, we demonstrate that SA, which is the primary metabolite of aspirin (acetyl SA) and is likely responsible for many of its pharmacological effects, also suppresses nuclear translocation of HsGAPDH and cell death. Analysis of two synthetic SA derivatives and two classes of compounds from the Chinese medicinal herb Glycyrrhiza foetida (licorice), glycyrrhizin and the SA-derivatives amorfrutins, revealed that they not only appear to bind HsGAPDH more tightly than SA, but also exhibit a greater ability to suppress translocation of HsGAPDH to the nucleus and cell death.

Conserved nematode signalling molecules elicit plant defenses and pathogen resistance.

Plant-defense responses are triggered by perception of conserved microbe-associated molecular patterns (MAMPs), for example, flagellin or peptidoglycan. However, it remained unknown whether plants can detect conserved molecular patterns derived from plant-parasitic animals, including nematodes. Here we show that several genera of plant-parasitic nematodes produce small molecules called ascarosides, an evolutionarily conserved family of nematode pheromones. Picomolar to micromolar concentrations of ascr#18, the major ascaroside in plant-parasitic nematodes, induce hallmark defense responses including the expression of genes associated with MAMP-triggered immunity, activation of mitogen-activated protein kinases, as well as salicylic acid- and jasmonic acid-mediated defense signalling pathways. Ascr#18 perception increases resistance in Arabidopsis, tomato, potato and barley to viral, bacterial, oomycete, fungal and nematode infections. These results indicate that plants recognize ascarosides as a conserved molecular signature of nematodes. Using small-molecule signals such as ascarosides to activate plant immune responses has potential utility to improve economic and environmental sustainability of agriculture.

Aspirin's Active Metabolite Salicylic Acid Targets High Mobility Group Box 1 to Modulate Inflammatory Responses.

Salicylic acid (SA) and its derivatives have been used for millennia to reduce pain, fever and inflammation. In addition, prophylactic use of acetylsalicylic acid, commonly known as aspirin, reduces the risk of heart attack, stroke and certain cancers. Because aspirin is rapidly de-acetylated by esterases in human plasma, much of aspirin's bioactivity can be attributed to its primary metabolite, SA. Here we demonstrate that human high mobility group box 1 (HMGB1) is a novel SA-binding protein. SA-binding sites on HMGB1 were identified in the HMG-box domains by nuclear magnetic resonance (NMR) spectroscopic studies and confirmed by mutational analysis. Extracellular HMGB1 is a damage-associated molecular pattern molecule (DAMP), with multiple redox states. SA suppresses both the chemoattractant activity of fully reduced HMGB1 and the increased expression of proinflammatory cytokine genes and cyclooxygenase 2 (COX-2) induced by disulfide HMGB1. Natural and synthetic SA derivatives with greater potency for inhibition of HMGB1 were identified, providing proof-of-concept that new molecules with high efficacy against sterile inflammation are attainable. An HMGB1 protein mutated in one of the SA-binding sites identified by NMR chemical shift perturbation studies retained chemoattractant activity, but lost binding of and inhibition by SA and its derivatives, thereby firmly establishing that SA binding to HMGB1 directly suppresses its proinflammatory activities. Identification of HMGB1 as a pharmacological target of SA/aspirin provides new insights into the mechanisms of action of one of the world's longest and most used natural and synthetic drugs. It may also provide an explanation for the protective effects of low-dose aspirin usage.