IITZ-01 activates NLRP3 inflammasome by inducing mitochondrial damage

NLRP3 inflammasome can be activated by a variety of pathogen activators (including components of bacteria, viruses and fungi) or “danger signals” (including abnormal metabolites and environmental components), so its activation mechanism is extremely complex. IITZ-01 is a lysosomotropic molecule that can disrupt lysosomal functions. We found that IITZ-01 can activate inflammasome at a low concentration. Then, we determined that IITZ-01 is a specific activator of NLRP3 inflammasome through inflammasome stimulation, ELISA, Western blot and other experiments. Mechanistically, NLRP3 inflammasome activation induced by IITZ-01 is independent of direct binding and ion flow but dependent on mitochondrial damage and mROS accumulation. This study suggests that a lysosomotropic compound can activate NLRP3 inflammasome by impairing mitochondrial functions.     

Calcium content of mitochondria and endoplasmic reticulum in liver frozen rapidly in vivo

The recognition that the endoplasmic reticulum (ER), rather than the mitochondria, is the main organelle regulating the cytoplasmic Ca2+ concentration in non-muscle cells supports the notion that an alternative physiological role of mitochondrial Ca transport is the modulation of Ca-sensitive mitochondrial enzymes through small (micromolar) fluctuations in the concentration of mitochondrial matrix Ca2+. The latter mechanism could operate only if the mitochondrial Ca concentration were low, as it is in muscle and retinal rods, below the levels saturating the regulated enzymes. In contrast, if the ER serves as an intracellular Ca store, its Ca content would be expected to be high. In view of the major metabolic function of the liver, the question of whether hepatic mitochondrial matrix Ca2+ regulates metabolism is particularly important, but the range of Ca concentrations reported for isolated liver mitochondria is too wide to provide a conclusive answer. Therefore, we have used electron probe X-ray microanalysis (EPMA) to measure the subcellular distribution of Ca in liver snap-frozen in vivo, and report here that the endoplasmic reticulum is a major intracellular store of Ca, while the concentration of Ca in mitochondria is low and compatible with the regulation of mitochondrial enzymes.     

NLRP6 negatively regulates innate immunity and host defence against bacterial pathogens

Members of the intracellular nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family contribute to immune responses through activation of nuclear factor-κB (NF-κB), type I interferon and inflammasome signalling. Mice lacking the NLR family member NLRP6 were recently shown to be susceptible to colitis and colorectal tumorigenesis, but the role of NLRP6 in microbial infections and the nature of the inflammatory signalling pathways regulated by NLRP6 remain unclear. Here we show that Nlrp6-deficient mice are highly resistant to infection with the bacterial pathogens Listeria monocytogenes, Salmonella typhimurium and Escherichia coli. Infected Nlrp6-deficient mice had increased numbers of monocytes and neutrophils in circulation, and NLRP6 signalling in both haematopoietic and radioresistant cells contributed to increased susceptibility. Nlrp6 deficiency enhanced activation of mitogen-activated protein kinase (MAPK) and the canonical NF-κB pathway after Toll-like receptor ligation, but not cytosolic NOD1/2 ligation, in vitro. Consequently, infected Nlrp6-deficient cells produced increased levels of NF-κB- and MAPK-dependent cytokines and chemokines. Thus, our results reveal NLRP6 as a negative regulator of inflammatory signalling, and demonstrate a role for this NLR in impeding clearance of both Gram-positive and -negative bacterial pathogens.     

Role of Bax and Bak in mitochondrial morphogenesis

Bcl-2 family proteins are potent regulators of programmed cell death. Although their intracellular localization to mitochondria and the endoplasmic reticulum has focused research on these organelles, how they function remains unknown. Two members of the Bcl-2 family, Bax and Bak, change intracellular location early in the promotion of apoptosis to concentrate in focal clusters at sites of mitochondrial division. Here we report that in healthy cells Bax or Bak is required for normal fusion of mitochondria into elongated tubules. Bax seems to induce mitochondrial fusion by activating assembly of the large GTPase Mfn2 and changing its submitochondrial distribution and membrane mobility-properties that correlate with different GTP-bound states of Mfn2. Our results show that Bax and Bak regulate mitochondrial dynamics in healthy cells and indicate that Bcl-2 family members may also regulate apoptosis through organelle morphogenesis machineries.     

COPS3的稳定干涉细胞系建立及其在NLRP3炎症小体激活中的作用

COPS3(COP9 Signalosome Subunit 3)作为COP9信号复合体的第三个亚基,在多种恶性肿瘤中高表达。本实验室通过酵母双杂交系统筛选,发现COPS3与NLRP3 (NOD-like receptor family, pyrin domain containing 3)存在相互作用。目前关于COPS3与NLRP3炎症小体的相关性未见文献报道。为了研究COPS3对NLRP3炎症小体的影响,我们利用慢病毒感染系统建立了稳定干涉COPS3的THP1细胞系,首先将COPS3的不同干涉序列片段稳定整合到THP1细胞中,通过嘌呤霉素压力筛选,应用实时定量荧光PCR(Real time PCR)手段检测COPS3的干涉效果,最终确定成功建立干涉COPS3的稳定细胞株。在稳定敲低COPS3的细胞株中,我们发现NLRP3 炎症小体的激活受到明显抑制。综上提示,COPS3能够正向调节NLRP3炎症小体的激活。     

Novel role of PKR in inflammasome activation and HMGB1 release

The inflammasome regulates the release of caspase activation-dependent cytokines, including interleukin (IL)-1β, IL-18 and high-mobility group box 1 (HMGB1). By studying HMGB1 release mechanisms, here we identify a role for double-stranded RNA-dependent protein kinase (PKR, also known as EIF2AK2) in inflammasome activation. Exposure of macrophages to inflammasome agonists induced PKR autophosphorylation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminium, rotenone, live Escherichia coli, anthrax lethal toxin, DNA transfection and Salmonella typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1β, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with several inflammasome components, including NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), NLRP1, NLR family CARD domain-containing protein 4 (NLRC4), absent in melanoma 2 (AIM2), and broadly regulates inflammasome activation. PKR autophosphorylation in a cell-free system with recombinant NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC, also known as PYCARD) and pro-caspase-1 reconstitutes inflammasome activity. These results show a crucial role for PKR in inflammasome activation, and indicate that it should be possible to pharmacologically target this molecule to treat inflammation.     

Distribution of two distinct Ca2+-ATPase-like proteins and their relationships to the agonist-sensitive calcium store in adrenal chromaffin cells

Many cellular functions are regulated by activation of cell-surface receptors that mobilize calcium from internal stores sensitive to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). The nature of these internal calcium stores and their localization in cells is not clear and has been a subject of debate. It was originally suggested that the Ins(1,4,5)P3-sensitive store is the endoplasmic reticulum, but a new organelle, the calciosome, identified by its possession of the calcium-binding protein, calsequestrin, and a Ca2+-ATPase-like protein of relative molecular mass 100,000 (100K), has been described as a potential Ins(1,4,5)P3-sensitive calcium store. Direct evidence on whether the calciosome is the Ins(1,4,5)P3-sensitive store is lacking. Using monoclonal antibodies raised against the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, we show that bovine adrenal chromaffin cells contain two Ca2+-ATPase-like proteins with distinct subcellular distributions. A 100K Ca2+-ATPase-like protein is diffusely distributed, whereas a 140K Ca2+-ATPase-like protein is restricted to a region in close proximity to the nucleus. In addition, Ins(1,4,5)P3-generating agonists result in a highly localized rise in cytosolic calcium concentration ([Ca2+]i) initiated in a region close to the nucleus, whereas caffeine results in a rise in [Ca2+]i throughout the cytoplasm. Our results indicate that chromaffin cells possess two calcium stores with distinct Ca2+-ATPases and that the organelle with the 100K Ca2+-ATPase is not the Ins(1,4,5)P3-sensitive store.     

TLR signalling augments macrophage bactericidal activity through mitochondrial ROS

Reactive oxygen species (ROS) are essential components of the innate immune response against intracellular bacteria and it is thought that professional phagocytes generate ROS primarily via the phagosomal NADPH oxidase machinery. However, recent studies have suggested that mitochondrial ROS (mROS) also contribute to mouse macrophage bactericidal activity, although the mechanisms linking innate immune signalling to mitochondria for mROS generation remain unclear. Here we demonstrate that engagement of a subset of Toll-like receptors (TLR1, TLR2 and TLR4) results in the recruitment of mitochondria to macrophage phagosomes and augments mROS production. This response involves translocation of a TLR signalling adaptor, tumour necrosis factor receptor-associated factor 6 (TRAF6), to mitochondria, where it engages the protein ECSIT (evolutionarily conserved signalling intermediate in Toll pathways), which is implicated in mitochondrial respiratory chain assembly. Interaction with TRAF6 leads to ECSIT ubiquitination and enrichment at the mitochondrial periphery, resulting in increased mitochondrial and cellular ROS generation. ECSIT- and TRAF6-depleted macrophages have decreased levels of TLR-induced ROS and are significantly impaired in their ability to kill intracellular bacteria. Additionally, reducing macrophage mROS levels by expressing catalase in mitochondria results in defective bacterial killing, confirming the role of mROS in bactericidal activity. These results reveal a novel pathway linking innate immune signalling to mitochondria, implicate mROS as an important component of antibacterial responses and further establish mitochondria as hubs for innate immune signalling.     

Inflammasomes in health and disease

Inflammasomes are a group of protein complexes built around several proteins, including NLRP3, NLRC4, AIM2 and NLRP6. Recognition of a diverse range of microbial, stress and damage signals by inflammasomes results in direct activation of caspase-1, which subsequently induces secretion of potent pro-inflammatory cytokines and a form of cell death called pyroptosis. Inflammasome-mediated processes are important during microbial infections and also in regulating both metabolic processes and mucosal immune responses. We review the functions of the different inflammasome complexes and discuss how aberrations in them are implicated in the pathogenesis of human diseases.     

NLRP3炎症小体在牛种布鲁氏菌2308和RB51侵染人巨噬细胞THP-1过程中作用的初步研究

为了探索NLRP3炎症小体在布鲁氏菌侵染宿主细胞过程中的作用,以牛种布鲁氏菌强毒株2308、弱毒株RB51侵染人巨噬细胞THP-1,用实时荧光定量PCR方法探索布鲁氏菌引起宿主细胞中NLRP3炎症小体及相关细胞因子的变化情况。结果表明:在布鲁氏菌侵染THP-1细胞后0-24 h,NLRP3炎症小体在转录水平上总体呈现先下降后上升的趋势,THP-1细胞形态随着侵染时间的延长变得不规则,且出现聚集现象。强毒株2308对NLRP3炎症小体相关基因转录水平和THP-1细胞形态变化的影响均强于弱毒株RB51。这表明布鲁氏菌在感染初期有短暂抑制炎症小体的能力,这可能是布鲁氏菌逃避巨噬细胞杀灭作用的一种策略。     

沙冬青（Ammopiptanthus mongolicus）小孢子发育的超微结构研究

本文对沙冬青小孢子发育的超微结构进行了观察，主要结果为：１）小孢子母细胞早期壁上有胞间连丝，随着发育，壁上出现了胞质通道．母细胞中内质网、线粒体和质体丰富，细胞处于明显的代谢活跃状态．四分体时期内质网消失．２）在小孢子发育各期质体和线粒体一直存在，细胞质中分布着小液泡，质膜上有泡状内陷．小孢子核靠壁后发生有丝分裂形成两个大小悬殊的细胞，此时，可见到大量的质体集中分布在营养细胞质中靠近生殖细胞壁的附近．３）在小孢子早期，绒毡层中内质网、线粒体和质体丰富，内质网持续产生大量小泡并在其中沉积电子致密物形成球状体（Ｏｒｂｉｃｕｌｅ）．小孢子后期，绒毡层细胞中内质网消失，出现大量的多泡体、球状体和脂体，绒毡层开始解体．在２－细胞花粉时绒毡层的解体产物大量流入花药腔中．     

Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production

Systems for protein degradation are essential for tight control of the inflammatory immune response. Autophagy, a bulk degradation system that delivers cytoplasmic constituents into autolysosomes, controls degradation of long-lived proteins, insoluble protein aggregates and invading microbes, and is suggested to be involved in the regulation of inflammation. However, the mechanism underlying the regulation of inflammatory response by autophagy is poorly understood. Here we show that Atg16L1 (autophagy-related 16-like 1), which is implicated in Crohn's disease, regulates endotoxin-induced inflammasome activation in mice. Atg16L1-deficiency disrupts the recruitment of the Atg12-Atg5 conjugate to the isolation membrane, resulting in a loss of microtubule-associated protein 1 light chain 3 (LC3) conjugation to phosphatidylethanolamine. Consequently, both autophagosome formation and degradation of long-lived proteins are severely impaired in Atg16L1-deficient cells. Following stimulation with lipopolysaccharide, a ligand for Toll-like receptor 4 (refs 8, 9), Atg16L1-deficient macrophages produce high amounts of the inflammatory cytokines IL-1beta and IL-18. In lipopolysaccharide-stimulated macrophages, Atg16L1-deficiency causes Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-dependent activation of caspase-1, leading to increased production of IL-1beta. Mice lacking Atg16L1 in haematopoietic cells are highly susceptible to dextran sulphate sodium-induced acute colitis, which is alleviated by injection of anti-IL-1beta and IL-18 antibodies, indicating the importance of Atg16L1 in the suppression of intestinal inflammation. These results demonstrate that Atg16L1 is an essential component of the autophagic machinery responsible for control of the endotoxin-induced inflammatory immune response.     

Studies on Locusts‘ Pathogenesis and Energy Metabolic Inhibition Induced by the Insecticidal Protein Purified from Pseudomonas Pseudoalcaligenes

The insecticidal protein produced by Pseudomonas pseudoalcaligenes is purified from the suspension of the bacterial culture. As an intact molecule, the protein acts on the foregut, midgut, hindgut, vasa Malpighii and fat body, and kills locusts. The disinsection rates of feeding and injection are 63.3% and 65.7%, respectively. After 24h to 48h, it is observed that all these tissues and cells show pathological changes in varying degrees, and so did the host cellular organs of these cells, such as cytoblast, mitochondria, endoplasmic reticulum and ribosome. Particularly, the changes of the mitochondria are much more serious than those of others. Detection of oxygen electrode shows the efficiency of oxidative phosphorylation and the ATP synthesis decreases, while the activity of mitochondrial ATPase is almost not affected. That means the energy utilization of locusts is in gear, but the shortage of the supplying of energy results in their death. This research forms the substantial basis for controlling locusts.     

Complete replacement of mitochondrial DNA in Drosophila

The introduction of foreign mitochondria or mitochondrial DNA into a cell is a useful technique for clarifying the molecular mechanisms responsible for the maintenance of mitochondria. Novel combinations of mitochondrial and nuclear genomes have been studied in mammalian cells in culture and in yeast. In Drosophila, we have recently constructed heteroplasmic flies possessing both endogenous mitochondrial DNA and foreign mitochondrial DNA by intra- and interspecific transplantation of germ plasm. During the maintenance of these heteroplasmic lines, flies of D. melanogaster are produced that no longer possess their own mitochondrial DNA but retain the foreign mitochondrial DNA from D. mauritiana. .These flies are fertile and the foreign mitochondrial DNA is stably maintained in their offspring. Here we report the complete replacement of endogenous mitochondrial DNA with that from another multicellular species. Molecular and genetic analysis of this replacement in Drosophila should provide new insight into the functional interaction between nuclear and organelle genomes.     

Matrix proteins can generate the higher order architecture of the Golgi apparatus

The Golgi apparatus in animal cells comprises a reticulum of linked stacks in the pericentriolar and often in the juxtanuclear regions of the cell. The unique architecture of this organelle is thought to depend on the cytoskeleton and cytoplasmic matrix proteins--the best characterized being the golgin family of fibrous, coiled-coil proteins and the GRASP family of stacking proteins. Here we show that these matrix proteins can be separated from oligosaccharide-modifying enzymes in the Golgi stack without affecting their ability to form a ribbon-like reticulum in the correct location near to the nucleus. Our data suggest that the Golgi is a structural scaffold that can exist independently of, but is normally populated by, the enzyme-containing membranes that modify transiting cargo. This new concept of the Golgi further indicates that the Golgi may be an autonomous organelle rather than one that is in simple dynamic equilibrium with the endoplasmic reticulum.     

细胞应激条件下嗜热四膜虫线粒体膜电位变化研究

单细胞线虫纲原生动物四膜虫(Tetrahymena),在H2O2的诱导下可发生凋亡样死亡.另外,最新研究表明细胞中活性氧簇(ROS)的积累可有效地诱导细胞自噬途径的发生.通过流式细胞技术和荧光显微技术,检测了经饥饿和ROS诱导剂处理后,线粒体内膜电位变化及细胞内ROS的积累.此外,应用两种抗氧化剂氮乙酰半胱氨酸(NAC)和过氧化氢酶(Catalase)分别对四膜虫细胞进行处理,检测了在氧化应激条件下的抗氧化作用效果.结果表明,Oligomycin和Menadione可有效抑制线粒体膜电位的维持,从而导致细胞内ROS积累.同时,H2O2处理和饥饿处理可以导致嗜热四膜虫细胞线粒体膜电位丧失以及细胞质ROS积累.另外,N-acetylcystine和Catalase可有效地降低四膜虫细胞内ROS的积累,以及保持四膜虫细胞线粒体内膜电位,维持通透性.     

Essential role for Nix in autophagic maturation of erythroid cells

Erythroid cells undergo enucleation and the removal of organelles during terminal differentiation. Although autophagy has been suggested to mediate the elimination of organelles for erythroid maturation, the molecular mechanisms underlying this process remain undefined. Here we report a role for a Bcl-2 family member, Nix (also called Bnip3L), in the regulation of erythroid maturation through mitochondrial autophagy. Nix(-/-) mice developed anaemia with reduced mature erythrocytes and compensatory expansion of erythroid precursors. Erythrocytes in the peripheral blood of Nix(-/-) mice exhibited mitochondrial retention and reduced lifespan in vivo. Although the clearance of ribosomes proceeded normally in the absence of Nix, the entry of mitochondria into autophagosomes for clearance was defective. Deficiency in Nix inhibited the loss of mitochondrial membrane potential (DeltaPsi(m)), and treatment with uncoupling chemicals or a BH3 mimetic induced the loss of DeltaPsi(m) and restored the sequestration of mitochondria into autophagosomes in Nix(-/-) erythroid cells. These results suggest that Nix-dependent loss of DeltaPsi(m) is important for targeting the mitochondria into autophagosomes for clearance during erythroid maturation, and interference with this function impairs erythroid maturation and results in anaemia. Our study may also provide insights into molecular mechanisms underlying mitochondrial quality control involving mitochondrial autophagy.     

Non-mitochondrial complex I proteins in a hydrogenosomal oxidoreductase complex

Trichomonas vaginalis is a unicellular microaerophilic eukaryote that lacks mitochondria yet contains an alternative organelle, the hydrogenosome, involved in pyruvate metabolism. Pathways between the two organelles differ substantially: in hydrogenosomes, pyruvate oxidation is catalysed by pyruvate:ferredoxin oxidoreductase (PFOR), with electrons donated to an [Fe]-hydrogenase which produces hydrogen. ATP is generated exclusively by substrate-level phosphorylation in hydrogenosomes, as opposed to oxidative phosphorylation in mitochondria. PFOR and hydrogenase are found in eubacteria and amitochondriate eukaryotes, but not in typical mitochondria. Analyses of mitochondrial genomes indicate that mitochondria have a single endosymbiotic origin from an alpha-proteobacterial-type progenitor. The absence of a genome in trichomonad hydrogenosomes precludes such comparisons, leaving the endosymbiotic history of this organelle unclear. Although phylogenetic reconstructions of a few proteins indicate that trichomonad hydrogenosomes share a common origin with mitochondria, others do not. Here we describe a novel NADH dehydrogenase module of respiratory complex I that is coupled to the central hydrogenosomal fermentative pathway to form a hydrogenosomal oxidoreductase complex that seems to function independently of quinones. Phylogenetic analyses of hydrogenosomal complex I-like proteins Ndh51 and Ndh24 reveal that neither has a common origin with mitochondrial homologues. These studies argue against a vertical origin of trichomonad hydrogenosomes from the proto-mitochondrial endosymbiont.     

Reconstitution of a phospholipid flippase from rat liver microsomes

The endoplasmic reticulum is the principal site of synthesis and initial incorporation of membrane lipids in eukaryotic cells; the enzymes of glycerolipid biosynthesis are exclusively located on its cytoplasmic surface. To maintain a phospholipid bilayer in this organelle, newly synthesized phospholipids must be translocated to the lumenal surface. Consistent with this are measurements indicating that movement of phospholipids across microsomal membranes is rapid, with a half-time less than 5 min (refs 3 and 4). Rapid movement of phospholipids has also been detected across the plasma membrane of Bacillus megaterium, another site of de novo lipid biosynthesis. The rapid transmembrane movement of phosphatidylcholine has not been detected, however, in vesicles prepared from microsomal lipids. These latter data suggest involvement in the endoplasmic reticulum of a phospholipid-translocating protein, as was first proposed by Bretscher who called it 'flippase'. Here we report reconstitution of a phospholipid flippase from rat liver microsomes into lipid vesicles.     

三唑磷蓄积对日本(虫寻)4种组织超微结构的影响

首先根据急性毒性试验确定了三唑磷对日本?的安全浓度为 0.27 mg/L,然后采用透射电镜技术研究了该浓度下三唑磷蓄积对日本(虫寻)4种组织细胞(肌肉、鳃、肝胰脏和心脏)超微结构的影响.结果表明 :三唑磷对肌肉损伤的主要靶部位是线粒体和肌原纤维,表现为线粒体内嵴肿胀、空泡化,线粒体溶解,肌原纤维断裂,明暗带不清楚;鳃细胞的损害表现为鳃丝水肿,细胞器溶解,角质层变薄,线粒体、内质网的肿胀、解体,细胞核不规则,核膜肿胀突起;肝胰脏细胞的主要损害特征为肝管微绒毛减少、线粒体水肿解体、内质网扩张、脂肪滴增加;心脏细胞的毒理变化为线粒体内嵴肿胀、瓦解,肌原纤维不规则,细胞核膜肿胀、突起,内质网溶解.鳃和肝胰脏中的变化要比肌肉和心脏明显.