Novel extracellular and nuclear caspase-1 and inflammasomes propagate inflammation and regulate gene expression: a comprehensive database mining study

Background

Caspase-1 is present in the cytosol as an inactive zymogen and requires the protein complexes named “inflammasomes” for proteolytic activation. However, it remains unclear whether the proteolytic activity of caspase-1 is confined only to the cytosol where inflammasomes are assembled to convert inactive pro-caspase-1 to active caspase-1.

Methods

We conducted meticulous data analysis method?s on proteomic, protein interaction, protein intracellular localization, and gene expressions of 114 experimentally identified caspase-1 substrates and 38 caspase-1 interaction proteins in normal physiological conditions and in various pathologies.

Results

We made the following important findings: (1) Caspase-1 substrates and interaction proteins are localized in various intracellular organelles including nucleus and secreted extracellularly; (2) Caspase-1 may get activated in situ in the nucleus in response to intra-nuclear danger signals; (3) Caspase-1 cleaves its substrates in exocytotic secretory pathways including exosomes to propagate inflammation to neighboring and remote cells; (4) Most of caspase-1 substrates are upregulated in coronary artery disease regardless of their subcellular localization but the majority of metabolic diseases cause no significant expression changes in caspase-1 nuclear substrates; and (5) In coronary artery disease, majority of upregulated caspase-1 extracellular substrate-related pathways are involved in induction of inflammation; and in contrast, upregulated caspase-1 nuclear substrate-related pathways are more involved in regulating cell death and chromatin regulation.

Conclusions

Our identification of novel caspase-1 trafficking sites, nuclear and extracellular inflammasomes, and extracellular caspase-1-based inflammation propagation model provides a list of targets for the future development of new therapeutics to treat cardiovascular diseases, inflammatory diseases, and inflammatory cancers.

     

From Clinging to Digging: The Postembryonic Skeletal Ontogeny of the Indian Purple Frog,Nasikabatrachus sahyadrensis (Anura: Nasikabatrachidae)

The Indian Purple frog, Nasikabatrachus sahyadrensis, occupies a basal phylogenetic position among neobatrachian anurans and has a very unusual life history. Tadpoles have a large ventral oral sucker, which they use to cling to rocks in torrents, whereas metamorphs possess adaptations for life underground. The developmental changes that underlie these shifts in habits and habitats, and especially the internal remodeling of the cranial and postcranial skeleton, are unknown. Using a nearly complete metamorphic series from free-living larva to metamorph, we describe the postembryonic skeletal ontogeny of this ancient and unique monotypic lineage. The torrent-dwelling larva possesses a dorsoventrally flattened body and a head with tiny dorsal eyes, robust lower and upper jaw cartilages, well-developed trabecular horns, and a definable gap between the trabecular horns and the tip of the snout. Unlike tadpoles of many other frogs, those of Nasikabatrachus retain larval mouthparts into late metamorphic stages. This unusual feature enables the larvae to maintain their clinging habit until near the end of metamorphosis. The subsequent ontogenetic shift from clinging to digging is correlated with rapid morphological changes and behavioral modifications. Metamorphs are equipped with a shortened tibiafibula and ossified prehallical elements, which likely facilitate initial digging using the hind limbs. Subsequently, the frogs may shift to headfirst burrowing by using the wedge-shaped skull, anteriorly positioned pectoral girdle, well-developed humeral crests and spatula-shaped forelimbs. The transition from an aquatic life in torrents to a terrestrial life underground entails dramatic changes in skeletal morphology and function that represent an extreme in metamorphic remodeling. Our analysis enhances the scope for detailed comparative studies across anurans, a group renowned for the diversity of its life history strategies.     

Bivalent inhibitors of the tyrosine kinases ABL and SRC: determinants of potency and selectivity

We recently reported a chemical genetic method for generating bivalent inhibitors of protein kinases. This method relies on the use of the DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase (AGT) to display an ATP-competitive inhibitor and a ligand that targets a secondary binding domain. With this method potent and selective inhibitors of the tyrosine kinases SRC and ABL were identified. Here, we dissect the molecular determinants of the potency and selectivity of these bivalent ligands. Systematic analysis of ATP-competitive inhibitors with varying linker lengths revealed that SRC and ABL have differential sensitivities to ligand presentation. Generation of bivalent constructs that contain ligands with differential affinities for the ATP-binding sites and SH3 domains of SRC and ABL demonstrated the modular nature of inhibitors based on the AGT scaffold. Furthermore, these studies revealed that the interaction between the SH3 domain ligand and the kinase SH3 domain is the major selectivity determinant amongst closely-related tyrosine kinases. Finally, the potency of bivalent inhibitors against distinct phospho-isoforms of SRC was determined. Overall, these results provide insight into how individual ligands can be modified to provide more potent and selective bivalent inhibitors of protein kinases.     

Rat brain arachidonic acid metabolism is increased by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide

In a rat model of acute neuroinflammation, produced by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide (LPS), we measured brain activities and protein levels of three phospholipases A2 (PLA2) and of cyclo-oxygenase-1 and -2, and quantified other aspects of brain phospholipid and fatty acid metabolism. The 6-day intracerebral ventricular infusion increased lectin-reactive microglia in the cerebral ventricles, pia mater, and the glial membrane of the cortex and resulted in morphological changes of glial fibrillary acidic protein (GFAP)-positive astrocytes in the cortical mantel and areas surrounding the cerebral ventricles. LPS infusion increased brain cytosolic and secretory PLA2 activities by 71% and 47%, respectively, as well as the brain concentrations of non-esterified linoleic and arachidonic acids, and of prostaglandins E2 and D2. LPS infusion also increased rates of incorporation and turnover of arachidonic acid in phosphatidylethanolamine, plasmenylethanolamine, phosphatidylcholine, and plasmenylcholine by 1.5- to 2.8-fold, without changing these rates in phosphatidylserine or phosphatidylinositol. These observations suggest that selective alterations in brain arachidonic acid metabolism involving cytosolic and secretory PLA2 contribute to early pathology in neuroinflammation.     

Ischemia reperfusion injury, KATP channels, and exercise-induced cardioprotection against apoptosis

Exercise is a potent stimulus against cardiac ischemia reperfusion (IR) injury, although the protective mechanisms are not completely understood. The study purpose was to examine whether the mitochondrial or sarcolemmal ATP-sensitive potassium channel (mito K(ATP) or sarc K(ATP), respectively) mediates exercise-induced cardioprotection against post-IR cell death and apoptosis. Eighty-six, 4-mo-old male Sprague Dawley rats were randomly assigned to treadmill exercise (Ex; 30 m/min, 3 days, 60 min, ～70 maximal oxygen uptake) and sedentary (Sed) treatments. Rats were exposed to regional cardiac ischemia (50 min) and reperfusion (120 min) or Sham (170 min; no ligation) surgeries. Exercise subgroups received placebo (saline), 5-hydroxydecanoate (5HD; 10 mg/kg ip), or HMR1098 (10 mg/kg ip) to inhibit mito K(ATP) or sarc K(ATP) channel. Comprehensive outcome assessments included post-IR ECG arrhythmias, cardiac tissue necrosis, redox perturbations, and autophagy biomarkers. No arrhythmia differences existed between exercised and sedentary hearts following extended-duration IR (P < 0.05). The sarc K(ATP) channel was confirmed essential (P = 0.002) for prevention of antinecrotic tissue death with exercise (percent infarct, Sed = 42%; Ex = 20%; Ex5HD = 16%; ExHMR = 42%), although neither the mito K(ATP) (P = 0.177) nor sarc K(ATP) (P = 0.274) channel provided post-IR protection against apoptosis (terminal deoxynucleotidyl transferase deoxy UTP-mediated nick-end labeling-positive nuclei/mm(2), Sham = 1.8 ± 0.5; Sed = 19.4 ± 6.7; Ex = 7.5 ± 4.6; Ex5HD = 14.0 ± 3.9; ExHMR = 11.1 ± 1.8). Exercise preconditioning also appears to preserve basal autophagy levels, as assessed by Beclin 1 (P ≤ 0.001), microtubule-associated protein-1 light-chain 3B ratios (P = 0.020), and P62 (P ≤ 0.001), in the hours immediately following IR. Further research is needed to better understand these findings and corresponding redox changes in exercised hearts.     

Analysis of the mobilities of band 3 populations associated with ankyrin protein and junctional complexes in intact murine erythrocytes

Current models of the erythrocyte membrane depict three populations of band 3: (i) a population tethered to spectrin via ankyrin, (ii) a fraction attached to the spectrin-actin junctional complex via adducin, and (iii) a freely diffusing population. Because many studies of band 3 diffusion also distinguish three populations of the polypeptide, it has been speculated that the three populations envisioned in membrane models correspond to the three fractions observed in diffusion analyses. To test this hypothesis, we characterized band 3 diffusion by single-particle tracking in wild-type and ankyrin- and adducin-deficient erythrocytes. We report that ～40% of total band 3 in wild-type murine erythrocytes is attached to ankyrin, whereas ～33% is immobilized by adducin, and ～27% is not attached to any cytoskeletal anchor. More detailed analyses reveal that mobilities of individual ankyrin- and adducin-tethered band 3 molecules are heterogeneous, varying by nearly 2 orders of magnitude and that there is considerable overlap in diffusion coefficients for adducin and ankyrin-tethered populations. Taken together, the data suggest that although the ankyrin- and adducin-immobilized band 3 can be monitored separately, significant heterogeneity still exists within each population, suggesting that structural and compositional properties likely vary considerably within each band 3 complex.     

Identification of Contact Sites between Ankyrin and Band 3 in the Human Erythrocyte Membrane

The red cell membrane is stabilized by a spectrin/actin-based cortical cytoskeleton connected to the phospholipid bilayer via multiple protein bridges. By virtue of its interaction with ankyrin and adducin, the anion transporter, band 3 (AE1), contributes prominently to these bridges. In a previous study, we demonstrated that an exposed loop comprising residues 175-185 of the cytoplasmic domain of band 3 (cdB3) constitutes a critical docking site for ankyrin on band 3. In this paper, we demonstrate that an adjacent loop, comprising residues 63-73 of cdB3, is also essential for ankyrin binding. Data that support this hypothesis include the following. (1) Deletion or mutation of residues within the latter loop abrogates ankyrin binding without affecting cdB3 structure or its other functions. (2) Association of cdB3 with ankyrin is inhibited by competition with the loop peptide. (3) Resealing of the loop peptide into erythrocyte ghosts alters membrane morphology and stability. To characterize cdB3-ankyrin interaction further, we identified their interfacial contact sites using molecular docking software and the crystal structures of D(3)D(4)-ankyrin and cdB3. The best fit for the interaction reveals multiple salt bridges and hydrophobic contacts between the two proteins. The most important ion pair interactions are (i) cdB3 K69-ankyrin E645, (ii) cdB3 E72-ankyrin K611, and (iii) cdB3 D183-ankyrin N601 and Q634. Mutation of these four residues on ankyrin yielded an ankyrin with a native CD spectrum but little or no affinity for cdB3. These data define the docking interface between cdB3 and ankyrin in greater detail.     

Apyrase inhibitors enhance the ability of diverse fungicides to inhibit the growth of different plant‐pathogenic fungi

A previous study has demonstrated that the treatment of Arabidopsis plants with chemical inhibitors of apyrase enzymes increases their sensitivity to herbicides. In this study, we found that the addition of the same or related apyrase inhibitors could potentiate the ability of different fungicides to inhibit the growth of five different pathogenic fungi in plate growth assays. The growth of all five fungi was partially inhibited by three commonly used fungicides: copper octanoate, myclobutanil and propiconazole. However, when these fungicides were individually tested in combination with any one of four different apyrase inhibitors (AI.1, AI.10, AI.13 or AI.15), their potency to inhibit the growth of five fungal pathogens was increased significantly relative to their application alone. The apyrase inhibitors were most effective in potentiating the ability of copper octanoate to inhibit fungal growth, and least effective in combination with propiconazole. Among the five pathogens assayed, that most sensitive to the fungicide‐potentiating effects of the inhibitors was Sclerotinia sclerotiorum. Overall, among the 60 treatment combinations tested (five pathogens, four apyrase inhibitors, three fungicides), the addition of apyrase inhibitors increased significantly the sensitivity of fungi to the fungicide treatments in 53 of the combinations. Consistent with their predicted mode of action, inhibitors AI.1, AI.10 and AI.13 each increased the level of propiconazole retained in one of the fungi, suggesting that they could partially block the ability of efflux transporters to remove propiconazole from these fungi.     

Frankixalus,a New Rhacophorid Genus of Tree Hole Breeding Frogs with Oophagous Tadpoles

Despite renewed interest in the biogeography and evolutionary history of Old World tree frogs (Rhacophoridae), this family still includes enigmatic frogs with ambiguous phylogenetic placement. During fieldwork in four northeastern states of India, we discovered several populations of tree hole breeding frogs with oophagous tadpoles. We used molecular data, consisting of two nuclear and three mitochondrial gene fragments for all known rhacophorid genera, to investigate the phylogenetic position of these new frogs. Our analyses identify a previously overlooked, yet distinct evolutionary lineage of frogs that warrants recognition as a new genus and is here described as Frankixalus gen. nov. This genus, which contains the enigmatic ‘Polypedates’ jerdonii described by Günther in 1876, forms the sister group of a clade containing Kurixalus, Pseudophilautus, Raorchestes, Mercurana and Beddomixalus. The distinctiveness of this evolutionary lineage is also corroborated by the external morphology of adults and tadpoles, adult osteology, breeding ecology, and life history features.