Insights into assembly of the macromolecular inflammasome complex

This is the published version. It first appeared at http://www.degruyter.com/view/j/infl.2013.1.issue-1/infl-2014-0009/infl-2014-0009.xml.AbstractDramatic advances in our understanding of the ultrastructure of the inflammasome and the molecular interactions involved in its assembly have recently been made. The adaptor protein ASC has been proposed to display prion-like activity that results in the formation of filamentous structures in the cell. These filamentouos structures can subsequently become inflammatory themselves if released into the extracellular space and then phagocytosed. Various groups have now utilised a variety of microscopy and structural approaches in order to visualise components of, and indeed the entire, inflammasome in both endogenous and overexpression systems. In this brief review we draw upon these new pieces of work to describe how our understanding of the global structure of the inflammasome has progressed in light of these new observations. In particular we begin by providing an initial perspective on the possible formation of small circular, wheel-like, oligomers resembling apoptosomes. We then address the current view that inflammasomes result from the formation of a much larger complex which may involve polymeric filaments. We discuss how these developments fit with recent theories of inflammatory signalling, what questions these advances raise, and propose key areas for further investigation.This work was funded by a Wellcome Trust Career Development Fellowship to TPM (WT085090MA )

Insights into the molecular basis of the NOD2 signalling pathway.

The cytosolic pattern recognition receptor NOD2 is activated by the peptidoglycan fragment muramyl dipeptide to generate a proinflammatory immune response. Downstream effects include the secretion of cytokines such as interleukin 8, the upregulation of pro-interleukin 1β, the induction of autophagy, the production of antimicrobial peptides and defensins, and contributions to the maintenance of the composition of the intestinal microbiota. Polymorphisms in NOD2 are the cause of the inflammatory disorder Blau syndrome and act as susceptibility factors for the inflammatory bowel condition Crohn's disease. The complexity of NOD2 signalling is highlighted by the observation that over 30 cellular proteins interact with NOD2 directly and influence or regulate its functional activity. Previously, the majority of reviews on NOD2 function have focused upon the role of NOD2 in inflammatory disease or in its interaction with and response to microbes. However, the functionality of NOD2 is underpinned by its biochemical interactions. Consequently, in this review, we have taken the opportunity to address the more 'basic' elements of NOD2 signalling. In particular, we have focused upon the core interactions of NOD2 with protein factors that influence and modulate the signal transduction pathways involved in NOD2 signalling. Further, where information exists, such as in relation to the role of RIP2, we have drawn comparison with the closely related, but functionally discrete, pattern recognition receptor NOD1. Overall, we provide a comprehensive resource targeted at understanding the complexities of NOD2 signalling.T.P.M. was supported by a Wellcome Trust Career Development Fellowship (WT085090MA). J.P.B. and R.P. were supported by BBSRC Doctoral Training Grants.This is the final published version. It first appeared at: http://rsob.royalsocietypublishing.org/content/4/12/140178

Blau syndrome polymorphisms in NOD2 identify nucleotide hydrolysis and helical domain 1 as signalling regulators.

Understanding how single nucleotide polymorphisms (SNPs) lead to disease at a molecular level provides a starting point for improved therapeutic intervention. SNPs in the innate immune receptor nucleotide oligomerisation domain 2 (NOD2) can cause the inflammatory disorders Blau Syndrome (BS) and early onset sarcoidosis (EOS) through receptor hyperactivation. Here, we show that these polymorphisms cluster into two primary locations: the ATP/Mg(2+)-binding site and helical domain 1. Polymorphisms in these two locations may consequently dysregulate ATP hydrolysis and NOD2 autoinhibition, respectively. Complementary mutations in NOD1 did not mirror the NOD2 phenotype, which indicates that NOD1 and NOD2 are activated and regulated by distinct methods.This work was funded by a Wellcome Trust CDF (WT085090MA).This is the published version. It was originally published by Elsevier on behalf of FEBS Letters, at http://www.sciencedirect.com/science/article/pii/S001457931400578X

Engagement of nucleotide-binding oligomerization domain-containing protein 1 (NOD1) by receptor-interacting protein 2 (RIP2) is insufficient for signal transduction.

Following activation, the cytoplasmic pattern recognition receptor nucleotide-binding oligomerization domain-containing protein 1 (NOD1) interacts with its adaptor protein receptor-interacting protein 2 (RIP2) to propagate immune signaling and initiate a proinflammatory immune response. This interaction is mediated by the caspase recruitment domain (CARD) of both proteins. Polymorphisms in immune proteins can affect receptor function and predispose individuals to specific autoinflammatory disorders. In this report, we show that mutations in helix 2 of the CARD of NOD1 disrupted receptor function but did not interfere with RIP2 interaction. In particular, N43S, a rare polymorphism, resulted in receptor dysfunction despite retaining normal cellular localization, protein folding, and an ability to interact with RIP2. Mutation of Asn-43 resulted in an increased tendency to form dimers, which we propose is the source of this dysfunction. We also demonstrate that mutation of Lys-443 and Tyr-474 in RIP2 disrupted the interaction with NOD1. Mapping the key residues involved in the interaction between NOD1 and RIP2 to the known structures of CARD complexes revealed the likely involvement of both type I and type III interfaces in the NOD1·RIP2 complex. Overall we demonstrate that the NOD1-RIP2 signaling axis is more complex than previously assumed, that simple engagement of RIP2 is insufficient to mediate signaling, and that the interaction between NOD1 and RIP2 constitutes multiple CARD-CARD interfaces.This work was funded by a Wellcome Trust Career Development Fellowship (WT085090MA) to TPM. TAK is supported by the German Research Foundation (DFG), grant SFB670 and acknowledges support by the Koeln Fortune Program / Faculty of Medicine, University of CologneThis is the final published version. It's also available from the Journal of Biological Chemistry website at http://www.jbc.org/content/289/33/22900.abstract

The Metallicity of the Redshift 4.16 Quasar BR2248-1242

We estimate the metallicity in the broad emission-line region of the redshift z=4.16 quasar, BR2248-1242, by comparing line ratios involving nitrogen to theoretical predictions. BR2248-1242 has unusually narrow emission lines with large equivalent widths, thus providing a rare opportunity to measure several line-ratio abundance diagnostics. The combined diagnostics indicate a metallicity of ~2 times solar. This result suggests that an episode of vigorous star formation occurred near BR2248-1242 prior to the observed z=4.16 epoch. The time available for this enrichment episode is only ~1.5 Gyr at z=4.16 (for H_{0}=65 km s^-1 Mpc^-1, Omega_{m}=0.3 and Omega_Lambda ~< 1). This evidence for high metallicities and rapid star formation is consistent with the expected early-epoch evolution of dense galactic nuclei.Comment: 8 pages, 3 figures. Prepared in AAStex. Submitted to the Astrophysical Journal Revised version: added 1 referenc

Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291

Citation: Girinathan, B. P., Monot, M., Boyle, D., McAllister, K. N., Sorg, J. A., Dupuy, B., & Govind, R. (2017). Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291. Msphere, 2(1), 14. doi:10.1128/mSphere.00383-16Clostridium difficile is an important nosocomial pathogen and the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease because it disrupts normally protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. The toxin-encoding genes, tcdA and tcdB, are part of a pathogenicity locus, which also includes the tcdR gene that codes for TcdR, an alternate sigma factor that initiates transcription of tcdA and tcdB genes. We created a tcdR mutant in epidemic-type C. difficile strain R20291 in an attempt to identify the global role of tcdR. A site-directed mutation in tcdR affected both toxin production and sporulation in C. difficile R20291. Spores of the tcdR mutant were more heat sensitive than the wild type (WT). Nearly 3-fold more taurocholate was needed to germinate spores from the tcdR mutant than to germinate the spores prepared from the WT strain. Transmission electron microscopic analysis of the spores also revealed a weakly assembled exosporium on the tcdR mutant spores. Accordingly, comparative transcriptome analysis showed many differentially expressed sporulation genes in the tcdR mutant compared to the WT strain. These data suggest that regulatory networks of toxin production and sporulation in C. difficile strain R20291 are linked with each other. IMPORTANCE C. difficile infects thousands of hospitalized patients every year, causing significant morbidity and mortality. C. difficile spores play a pivotal role in the transmission of the pathogen in the hospital environment. During infection, the spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. Thus, sporulation and toxin production are two important traits of C. difficile. In this study, we showed that a mutation in tcdR, the toxin gene regulator, affects both toxin production and sporulation in epidemic-type C. difficile strain R20291

TgPRELID, a Mitochondrial Protein Linked to Multidrug Resistance in the Parasite Toxoplasma gondii

New drugs to control infection with the protozoan parasite Toxoplasma gondii are needed as current treatments exert toxic side effects on patients. Approaches to develop novel compounds for drug development include screening of compound libraries and targeted inhibition of essential cellular pathways. We identified two distinct compounds that display inhibitory activity against the parasite's replicative stage: F3215-0002, which we previously identified during a compound library screen, and I-BET151, an inhibitor of bromodomains, the "reader" module of acetylated lysines. In independent studies, we sought to determine the targets of these two compounds using forward genetics, generating resistant mutants and identifying the determinants of resistance with comparative genome sequencing. Despite the dissimilarity of the two compounds, we recovered resistant mutants with nonsynonymous mutations in the same domain of the same gene, TGGT1_254250, which we found encodes a protein that localizes to the parasite mitochondrion (designated TgPRELID after the name of said domain). We found that mutants selected with one compound were cross resistant to the other compound, suggesting a common mechanism of resistance. To further support our hypothesis that TgPRELID mutations facilitate resistance to both I-BET151 and F3215-0002, CRISPR (clustered regularly interspaced short palindromic repeat)/CAS9-mediated mutation of TgPRELID directly led to increased F3215-0002 resistance. Finally, all resistance mutations clustered in the same subdomain of TgPRELID. These findings suggest that TgPRELID may encode a multidrug resistance factor or that I-BET151 and F3215-0002 have the same target(s) despite their distinct chemical structures. IMPORTANCE We report the discovery of TgPRELID, a previously uncharacterized mitochondrial protein linked to multidrug resistance in the parasite Toxoplasma gondii. Drug resistance remains a major problem in the battle against parasitic infection, and understanding how TgPRELID mutations augment resistance to multiple, distinct compounds will reveal needed insights into the development of new therapies for toxoplasmosis and other related parasitic diseases

Annual Reports of the Selectmen, Assessors, Overseers of Poor, Treasurer, and Supervisor of Schools, of the Town of Winthrop, for the Year Ending March 14, 1881

Natural materials, such as collagen, can assemble with multiple levels of organization in solution. Achieving a similar degree of control over morphology, stability and hierarchical organization with equilibrium synthetic materials remains elusive. For the assembly of peptidic materials the process is controlled by a complex interplay between hydrophobic interactions, electrostatics and secondary structure formation. Consequently, fine tuning the thermodynamics and kinetics of assembly remains extremely challenging. Here, we synthesized a set of block co polypeptides with varying hydrophobicity and ability to form secondary structure. From this set we select a sequence with balanced interactions that results in the formation of high-aspect ratio thermodynamically favored nanotubes, stable between pH 2 and 12 and up to 80 °C. This stability permits their hierarchical assembly into bundled nanotube fibers by directing the pH and inducing complementary zwitterionic charge behavior. This block co-polypeptide design strategy, using defined sequences, provides a straightforward approach to creating complex hierarchical peptide-based assemblies with tunable interactions

Observations and Theoretical Implications of the Large Separation Lensed Quasar SDSS J1004+4112

We study the recently discovered gravitational lens SDSS J1004+4112, the first quasar lensed by a cluster of galaxies. It consists of four images with a maximum separation of 14.62''. The system has been confirmed as a lensed quasar at z=1.734 on the basis of deep imaging and spectroscopic follow-up observations. We present color-magnitude relations for galaxies near the lens plus spectroscopy of three central cluster members, which unambiguously confirm that a cluster at z=0.68 is responsible for the large image separation. We find a wide range of lens models consistent with the data, but they suggest four general conclusions: (1) the brightest cluster galaxy and the center of the cluster potential well appear to be offset by several kpc; (2) the cluster mass distribution must be elongated in the North--South direction, which is consistent with the observed distribution of cluster galaxies; (3) the inference of a large tidal shear (~0.2) suggests significant substructure in the cluster; and (4) enormous uncertainty in the predicted time delays between the images means that measuring the delays would greatly improve constraints on the models. We also compute the probability of such large separation lensing in the SDSS quasar sample, on the basis of the CDM model. The lack of large separation lenses in previous surveys and the discovery of one in SDSS together imply a mass fluctuation normalization \sigma_8=1.0^{+0.4}_{-0.2} (95% CL), if cluster dark matter halos have an inner slope -1.5. Shallower profiles would require higher values of \sigma_8. Although the statistical conclusion might be somewhat dependent on the degree of the complexity of the lens potential, the discovery is consistent with the predictions of the abundance of cluster-scale halos in the CDM scenario. (Abridged)Comment: 21 pages, 24 figures, 5 tables, accepted for publication in Ap

Mitochondrial Hâ‚‚Oâ‚‚ emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans

High dietary fat intake leads to insulin resistance in skeletal muscle, and this represents a major risk factor for type 2 diabetes and cardiovascular disease. Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the Hâ‚‚Oâ‚‚-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial Hâ‚‚Oâ‚‚ emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial Hâ‚‚Oâ‚‚ emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity. Original version available at http://www.jci.org/articles/view/3704