Stimulation of transit-peptide release and ATP hydrolysis by a cochaperone during protein import into chloroplasts

Three components of the chloroplast protein translocon, Tic110, Hsp93 (ClpC), and Tic40, have been shown to be important for protein translocation across the inner envelope membrane into the stroma. We show the molecular interactions among these three components that facilitate processing and translocation of precursor proteins. Transit-peptide binding by Tic110 recruits Tic40 binding to Tic110, which in turn causes the release of transit peptides from Tic110, freeing the transit peptides for processing. The Tic40 C-terminal domain, which is homologous to the C terminus of cochaperones Sti1p/Hop and Hip but with no known function, stimulates adenosine triphosphate hydrolysis by Hsp93. Hsp93 dissociates from Tic40 in the presence of adenosine diphosphate, suggesting that Tic40 functions as an adenosine triphosphatase activation protein for Hsp93. Our data suggest that chloroplasts have evolved the Tic40 cochaperone to increase the efficiency of precursor processing and translocation

Morus alba and active compound oxyresveratrol exert anti-inflammatory activity via inhibition of leukocyte migration involving MEK/ERK signaling

Background: Morus alba has long been used in traditional Chinese medicine to treat inflammatory diseases;however, the scientific basis for such usage and the mechanism of action are not well understood. This studyinvestigated the action of M. alba on leukocyte migration, one key step in inflammation.Methods: Gas chromatography-mass spectrometry (GC-MS) and cluster analyses of supercritical CO2 extractsof three Morus species were performed for chemotaxonomy-aided plant authentication. Phytochemistry andCXCR4-mediated chemotaxis assays were used to characterize the chemical and biological properties of M. albaand its active compound, oxyresveratrol. fluorescence-activated cell sorting (FACS) and Western blot analyses wereconducted to determine the mode of action of oxyresveratrol.Results: Chemotaxonomy was used to help authenticate M. alba. Chemotaxis-based isolation identifiedoxyresveratrol as an active component in M. alba. Phytochemical and chemotaxis assays showed that the crudeextract, ethyl acetate fraction and oxyresveratrol from M. alba suppressed cell migration of Jurkat T cells in responseto SDF-1. Mechanistic study indicated that oxyresveratrol diminished CXCR4-mediated T-cell migration via inhibitionof the MEK/ERK signaling cascade.Conclusions: A combination of GC-MS and cluster analysis techniques are applicable for authentication of theMorus species. Anti-inflammatory benefits of M. alba and its active compound, oxyresveratrol, may involve theinhibition of CXCR-4-mediated chemotaxis and MEK/ERK pathway in T and other immune cells

Evaluation of Antioxidant and Free Radical Scavenging Capacities of Polyphenolics from Pods of Caesalpinia pulcherrima

Thirteen polyphenolics were isolated from fresh pods of Caesalpinia pulcherrima using various methods of column chromatography. The structures of these polyphenolics were elucidated as gallic acid (1), methyl gallate (2), 6-O-galloyl-d-glucoside (3), methyl 6-O-galloyl-β-d-glucoside (4), methyl 3,6-di-O-galloyl-α-d-glucopyranoside (5), gentisic acid 5-O-α-d-(6′-O-galloyl)glucopyranoside (6), guaiacylglycerol 4-O-β-d-(6′-O-galloyl)glucopyranoside (7), 3-methoxy-4-hydroxyphenol 1-O-β-d-(6′-O-galloyl) glucopyranoside (8), (+)-gallocatechin (9), (+)-catechin (10), (+)-gallocatechin 3-O-gallate (11), myricetin 3-rhamnoside (12), and ampelopsin (13). All isolated compounds were tested for their antioxidant activities in the 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and peroxynitrite radicals scavenging assays. Among those compounds, 11, 12, and 2 exhibited the best DPPH-, hydroxyl-, and peroxynitrite radical-scavenging activities, respectively. Compound 7 is a new compound, and possesses better scavenging activities towards DPPH but has equivalent hydroxyl radical scavenging activity when compared to BHT. The paper is the first report on free radical scavenging properties of components of the fresh pods of Caesalpinia pulcherrima. The results obtained from the current study indicate that the free radical scavenging property of fresh pods of Caesalpinia pulcherrima may be one of the mechanisms by which this herbal medicine is effective in several free radical mediated diseases

Src-family kinase-Cbl axis negatively regulates NLRP3 inflammasome activation.

Activation of the NLRP3 inflammasome is crucial for immune defense, but improper and excessive activation causes inflammatory diseases. We previously reported that Pyk2 is essential for NLRP3 inflammasome activation. Here we show that the Src-family kinases (SFKs)-Cbl axis plays a pivotal role in suppressing NLRP3 inflammasome activation in response to stimulation by nigericin or ATP, as assessed using gene knockout and gene knockdown cells, dominant active/negative mutants, and pharmacological inhibition. We reveal that the phosphorylation of Cbl is regulated by SFKs, and that phosphorylation of Cbl at Tyr371 suppresses NLRP3 inflammasome activation. Mechanistically, Cbl decreases the level of phosphorylated Pyk2 (p-Pyk2) through ubiquitination-mediated proteasomal degradation and reduces mitochondrial ROS (mtROS) production by contributing to the maintenance of mitochondrial size. The lower levels of p-Pyk2 and mtROS dampen NLRP3 inflammasome activation. In vivo, inhibition of Cbl with an analgesic drug, hydrocotarnine, increases inflammasome-mediated IL-18 secretion in the colon, and protects mice from dextran sulphate sodium-induced colitis. Together, our novel findings provide new insights into the role of the SFK-Cbl axis in suppressing NLRP3 inflammasome activation and identify a novel clinical utility of hydrocortanine for disease treatment

Pyk2 activates the NLRP3 inflammasome by directly phosphorylating ASC and contributes to inflammasome-dependent peritonitis

The inflammasome adaptor protein, ASC, contributes to both innate immune responses and inflammatory diseases via self-oligomerization, which leads to the activation of the protease, caspase-1. Here, we report that the cytosolic tyrosine kinases, FAK and Pyk2, are differentially involved in NLRP3 and AIM2 inflammasome activation. The inhibition of FAK and Pyk2 with RNA interference or chemical inhibitors dramatically abolished ASC oligomerization, caspase-1 activation, and IL-1β secretion in response to NLRP3 or AIM2 stimulation. Pyk2 is phosphorylated by the kinase Syk and relocalizes to the ASC specks upon NLRP3 inflammasome activation. Pyk2, but not FAK, could directly phosphorylate ASC at Tyr146, and only the phosphorylated ASC could participate in speck formation and trigger IL-1β secretion. Moreover, the clinical-trial-tested Pyk2/FAK dual inhibitor PF-562271 reduced monosodium urate-mediated peritonitis, a disease model used for studying the consequences of NLRP3 activation. Our results suggest that although Pyk2 and FAK are involved in inflammasome activation, only Pyk2 directly phosphorylates ASC and brings ASC into an oligomerization-competent state by allowing Tyr146 phosphorylation to participate ASC speck formation and subsequent NLRP3 inflammation

Cbl negatively regulates nlrp3 inflammasome activation through glut1-dependent glycolysis inhibition

Activation of the nod-like receptor 3 (NLRP3) inflammasomes is crucial for immune defense, but improper and excessive activation causes inflammatory diseases. We previously reported that Cbl plays a pivotal role in suppressing NLRP3 inflammasome activation by inhibiting Pyk2-mediated apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization. Here, we showed that Cbl dampened NLRP3 inflammasome activation by inhibiting glycolysis, as demonstrated with Cbl knockout cells and treatment with the Cbl inhibitor hydrocotarnine. We revealed that the inhibition of Cbl promoted caspase-1 cleavage and interleukin (IL)-1β secretion through a glycolysis-dependent mechanism. Inhibiting Cbl increased cellular glucose uptake, glycolytic capacity, and mitochondrial oxidative phosphorylation capacity. Upon NLRP3 inflammasome activation, inhibiting Cbl increased glycolysis-dependent activation of mitochondrial respiration and increased the production of reactive oxygen species, which contributes to NLRP3 inflammasome activation and IL-1β secretion. Mechanistically, inhibiting Cbl increased surface expression of glucose transporter 1 (GLUT1) protein through post-transcriptional regulation, which increased cellular glucose uptake and consequently raised glycolytic capacity, and in turn enhanced NLRP3 inflammasome activation. Together, our findings provide new insights into the role of Cbl in NLRP3 inflammasome regulation through GLUT1 downregulation. We also show that a novel Cbl inhibitor, hydrocortanine, increased NLRP3 inflammasome activity via its effect on glycolysis