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Sample records for macrophage lipid chaperone

  1. Lipid Chaperones and Metabolic Inflammation

    PubMed Central

    Furuhashi, Masato; Ishimura, Shutaro; Ota, Hideki; Miura, Tetsuji

    2011-01-01

    Over the past decade, a large body of evidence has emerged demonstrating an integration of metabolic and immune response pathways. It is now clear that obesity and associated disorders such as insulin resistance and type 2 diabetes are associated with a metabolically driven, low-grade, chronic inflammatory state, referred to as “metaflammation.” Several inflammatory cytokines as well as lipids and metabolic stress pathways can activate metaflammation, which targets metabolically critical organs and tissues including adipocytes and macrophages to adversely affect systemic homeostasis. On the other hand, inside the cell, fatty acid-binding proteins (FABPs), a family of lipid chaperones, as well as endoplasmic reticulum (ER) stress, and reactive oxygen species derived from mitochondria play significant roles in promotion of metabolically triggered inflammation. Here, we discuss the molecular and cellular basis of the roles of FABPs, especially FABP4 and FABP5, in metaflammation and related diseases including obesity, diabetes, and atherosclerosis. PMID:22121495

  2. ER stress is associated with reduced ABCA-1 protein levels in macrophages treated with advanced glycated albumin - reversal by a chemical chaperone.

    PubMed

    Castilho, Gabriela; Okuda, Ligia S; Pinto, Raphael S; Iborra, Rodgiro T; Nakandakare, Edna R; Santos, Celio X; Laurindo, Francisco R; Passarelli, Marisa

    2012-07-01

    ATP-binding cassette transporter A1 mediates the export of excess cholesterol from macrophages, contributing to the prevention of atherosclerosis. Advanced glycated albumin (AGE-alb) is prevalent in diabetes mellitus and is associated with the development of atherosclerosis. Independently of changes in ABCA-1 mRNA levels, AGE-alb induces oxidative stress and reduces ABCA-1 protein levels, which leads to macrophage lipid accumulation. These metabolic conditions are known to elicit endoplasmic reticulum (ER) stress. We sought to determine if AGE-alb induces ER stress and unfolded protein response (UPR) in macrophages and how disturbances to the ER could affect ABCA-1 content and cholesterol efflux in macrophages. AGE-alb induced a time-dependent increase in ER stress and UPR markers. ABCA-1 content and cellular cholesterol efflux were reduced by 33% and 47%, respectively, in macrophages treated with AGE-alb, and both were restored by treatment with 4-phenyl butyric acid (a chemical chaperone that alleviates ER stress), but not MG132 (a proteasome inhibitor). Tunicamycin, a classical ER stress inductor, also impaired ABCA-1 expression and cholesterol efflux (showing a decrease of 61% and 82%, respectively), confirming the deleterious effect of ER stress in macrophage cholesterol accumulation. Glycoxidation induces macrophage ER stress, which relates to the reduction in ABCA-1 and in reverse cholesterol transport, endorsing the adverse effect of macrophage ER stress in atherosclerosis. Thus, chemical chaperones that alleviate ER stress may represent a useful tool for the prevention and treatment of atherosclerosis in diabetes.

  3. Inhibiting the HSP90 chaperone destabilizes macrophage migration inhibitory factor and thereby inhibits breast tumor progression

    PubMed Central

    Schulz, Ramona; Marchenko, Natalia D.; Holembowski, Lena; Fingerle-Rowson, Günter; Pesic, Marina; Zender, Lars; Dobbelstein, Matthias

    2012-01-01

    Intracellular macrophage migration inhibitory factor (MIF) often becomes stabilized in human cancer cells. MIF can promote tumor cell survival, and elevated MIF protein correlates with tumor aggressiveness and poor prognosis. However, the molecular mechanism facilitating MIF stabilization in tumors is not understood. We show that the tumor-activated HSP90 chaperone complex protects MIF from degradation. Pharmacological inhibition of HSP90 activity, or siRNA-mediated knockdown of HSP90 or HDAC6, destabilizes MIF in a variety of human cancer cells. The HSP90-associated E3 ubiquitin ligase CHIP mediates the ensuing proteasome-dependent MIF degradation. Cancer cells contain constitutive endogenous MIF–HSP90 complexes. siRNA-mediated MIF knockdown inhibits proliferation and triggers apoptosis of cultured human cancer cells, whereas HSP90 inhibitor-induced apoptosis is overridden by ectopic MIF expression. In the ErbB2 transgenic model of human HER2-positive breast cancer, genetic ablation of MIF delays tumor progression and prolongs overall survival of mice. Systemic treatment with the HSP90 inhibitor 17AAG reduces MIF expression and blocks growth of MIF-expressing, but not MIF-deficient, tumors. Together, these findings identify MIF as a novel HSP90 client and suggest that HSP90 inhibitors inhibit ErbB2-driven breast tumor growth at least in part by destabilizing MIF. PMID:22271573

  4. Alteration of macrophage membrane lipids following processing of bacterial peptidoglycan

    SciTech Connect

    Polanski, M.; Gray, G.R.

    1986-03-01

    As part of the continuing investigation into the role played by macrophages in antigen presentation and bacterial adjuvant activation, the authors have examined the metabolites produced by macrophages after encounter with peptidoglycan. Peptidoglycan was chosen because it contains N-acetyl-muramyl-L-alanyl-D-isoglutamine (muramyl dipeptide), a known adjuvant whose primary target cell is the macrophage. In previous work, the authors established that a series of muramyl dipeptide-like glycopeptides was released into the medium following phagocytosis of peptidoglycan by a macrophage cell line. Here the authors report on the finding that, additionally, a membrane lipid has been covalently altered by the addition of a peptidoglycan fragment. Bacillus subtilis cell walls which had been radiolabeled in their muramic acid, glucosamine and alanine residues, were incubated with the murine macrophage cell line RAW264. Using standard lipid extraction procedures, a lipid was isolated and found to contain equal molar ratios of alanine, glutamic acid and diaminopimelic acid. Since lipidated peptidoglycan peptides have been shown to be immunoactivators, the isolated lipid derivative may serve as a signal for interactions with other lymphocytes.

  5. Lipid profiling of polarized human monocyte-derived macrophages.

    PubMed

    Montenegro-Burke, J Rafael; Sutton, Jessica A; Rogers, Lisa M; Milne, Ginger L; McLean, John A; Aronoff, David M

    2016-12-01

    The highly orchestrated transcriptional and metabolic reprogramming during activation drastically transforms the main functions and physiology of human macrophages across the polarization spectrum. Lipids, for example, can modify protein function by acting remotely as signaling molecules but also locally by altering the physical properties of cellular membranes. These changes play key roles in the functions of highly plastic immune cells due to their involvement in inflammation, immune responses, phagocytosis and wound healing processes. We report an analysis of major membrane lipids of distinct phenotypes of resting (M0), classically activated (M1), alternatively activated (M2a) and deactivated (M2c) human monocyte derived macrophages from different donors. Samples were subjected to supercritical fluid chromatography-ion mobility-mass spectrometry analysis, which allowed separations based on lipid class, facilitating the profiling of their fatty acid composition. Different levels of arachidonic acid mobilization as well as other fatty acid changes were observed for different lipid classes in the distinct polarization phenotypes, suggesting the activation of highly orchestrated and specific enzymatic processes in the biosynthesis of lipid signaling molecules and cell membrane remodeling. Thromboxane A2 production appeared to be a specific marker of M1 polarization. These alterations to the global composition of lipid bi-layer membranes in the cell provide a potential methodology for the definition and determination of cellular and tissue activation states.

  6. Native low density lipoprotein promotes lipid raft formation in macrophages

    PubMed Central

    SONG, JIAN; PING, LING-YAN; DUONG, DUC M.; GAO, XIAO-YAN; HE, CHUN-YAN; WEI, LEI; WU, JUN-ZHU

    2016-01-01

    Oxidized low-density lipoprotein (LDL) has an important role in atherogenesis; however, the mechanisms underlying cell-mediated LDL oxidation remain to be elucidated. The present study investigated whether native-LDL induced lipid raft formation, in order to gain further insight into LDL oxidation. Confocal microscopic analysis revealed that lipid rafts were aggregated or clustered in the membrane, which were colocalized with myeloperoxidase (MPO) upon native LDL stimulation; however, in the presence of methyl-β-cyclodextrin (MβCD), LDL-stimulated aggregation, translocation, and colocalization of lipid rafts components was abolished.. In addition, lipid raft disruptors MβCD and filipin decreased malondialdehyde expression levels. Density gradient centrifugation coupled to label-free quantitative proteomic analysis identified 1,449 individual proteins, of which 203 were significantly upregulated following native-LDL stimulation. Functional classification of the proteins identified in the lipid rafts revealed that the expression levels of translocation proteins were upregulated. In conclusion, the results of the present study indicated that native-LDL induced lipid raft clustering in macrophages, and the expression levels of several proteins were altered in the stimulated macrophages, which provided novel insights into the mechanism underlying LDL oxidation. PMID:26781977

  7. Endoplasmic reticulum chaperone gp96 in macrophages is essential for protective immunity during Gram-negative pneumonia.

    PubMed

    Anas, Adam A; de Vos, Alex F; Hoogendijk, Arie J; van Lieshout, Miriam H P; van Heijst, Jeroen W J; Florquin, Sandrine; Li, Zihai; van 't Veer, Cornelis; van der Poll, Tom

    2016-01-01

    Klebsiella pneumoniae is among the most common Gram-negative bacteria that cause pneumonia. Gp96 is an endoplasmic reticulum chaperone that is essential for the trafficking and function of Toll-like receptors (TLRs) and integrins. To determine the role of gp96 in myeloid cells in host defence during Klebsiella pneumonia, mice homozygous for the conditional Hsp90b1 allele encoding gp96 were crossed with mice expressing Cre-recombinase under control of the LysM promoter to generate LysMcre-Hsp90b1-flox mice. LysMcre-Hsp90b1-flox mice showed absence of gp96 protein in macrophages and partial depletion in monocytes and granulocytes. This was accompanied by almost complete absence of TLR2 and TLR4 on macrophages. Likewise, integrin subunits CD11b and CD18 were not detectable on macrophages, while being only slightly reduced on monocytes and granulocytes. Gp96-deficient macrophages did not release pro-inflammatory cytokines in response to Klebsiella and displayed reduced phagocytic capacity independent of CD18. LysMcre-Hsp90b1-flox mice were highly vulnerable to lower airway infection induced by K. pneumoniae, as reflected by enhanced bacterial growth and a higher mortality rate. The early inflammatory response in Hsp90b1-flox mice was characterized by strongly impaired recruitment of granulocytes into the lungs, accompanied by attenuated production of pro-inflammatory cytokines, while the inflammatory response during late-stage pneumonia was not dependent on the presence of gp96. Blocking CD18 did not reproduce the impaired host defence of LysMcre-Hsp90b1-flox mice during Klebsiella pneumonia. These data indicate that macrophage gp96 is essential for protective immunity during Gram-negative pneumonia by regulating TLR expression.

  8. Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids.

    PubMed

    Cambier, C J; Takaki, Kevin K; Larson, Ryan P; Hernandez, Rafael E; Tobin, David M; Urdahl, Kevin B; Cosma, Christine L; Ramakrishnan, Lalita

    2014-01-09

    The evolutionary survival of Mycobacterium tuberculosis, the cause of human tuberculosis, depends on its ability to invade the host, replicate, and transmit infection. At its initial peripheral infection site in the distal lung airways, M. tuberculosis infects macrophages, which transport it to deeper tissues. How mycobacteria survive in these broadly microbicidal cells is an important question. Here we show in mice and zebrafish that M. tuberculosis, and its close pathogenic relative Mycobacterium marinum, preferentially recruit and infect permissive macrophages while evading microbicidal ones. This immune evasion is accomplished by using cell-surface-associated phthiocerol dimycoceroserate (PDIM) lipids to mask underlying pathogen-associated molecular patterns (PAMPs). In the absence of PDIM, these PAMPs signal a Toll-like receptor (TLR)-dependent recruitment of macrophages that produce microbicidal reactive nitrogen species. Concordantly, the related phenolic glycolipids (PGLs) promote the recruitment of permissive macrophages through a host chemokine receptor 2 (CCR2)-mediated pathway. Thus, we have identified coordinated roles for PDIM, known to be essential for mycobacterial virulence, and PGL, which (along with CCR2) is known to be associated with human tuberculosis. Our findings also suggest an explanation for the longstanding observation that M. tuberculosis initiates infection in the relatively sterile environment of the lower respiratory tract, rather than in the upper respiratory tract, where resident microflora and inhaled environmental microbes may continually recruit microbicidal macrophages through TLR-dependent signalling.

  9. Incorporation of bacterial peptidoglycan constituents into macrophage lipids during phagocytosis.

    PubMed

    Polanski, M; Gray, G R

    1989-10-15

    It has previously been established that several glycopeptides of peptidoglycan origin are formed as a result of processing of Bacillus subtilis cell walls by the macrophage-like cell line RAW264. Although the formation of these glycopeptides could account for the humoral immune responses characteristic of bacterial peptidoglycans, their formation does not account for the cellular-mediated immune responses observed for water-in-oil emulsions of peptidoglycan or for lipophilic derivatives of glycopeptide fragments thereof. Therefore, the processing of peptidoglycan by macrophages was reexamined to establish whether the lipophilic derivative of any peptidoglycan-derived glycopeptide was formed. The experiments were performed by incubating B. subtilis cell walls radiolabeled in muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid residues in the presence of the macrophage-like cell line RAW264. The crude lipid fraction derived from the macrophages was further fractionated and analyzed, revealing the presence of two lipophilic glycopeptides that contained glucosamine, muramic acid, and alanine of bacterial origin.

  10. Changes in macrophage function modulated by the lipid environment

    PubMed Central

    Williams, Michael R; Cauvi, David M; Rivera, Isabel; Hawisher, Dennis; De Maio, Antonio

    2016-01-01

    Macrophages (Mϕs) play a critical role in the defense against pathogens, orchestrating the inflammatory response during injury and maintaining tissue homeostasis. During these processes, macrophages encounter a variety of environmental conditions that are likely to change their gene expression pattern, which modulates their function. In this study, we found that murine Mϕs displayed two different subpopulations characterized by differences in morphologies, expression of surface markers and phagocytic capacity under non-stimulated conditions. These two subpopulations could be recapitulated by changes in the culture conditions. Thus, Mϕs grown in suspension in the presence of serum were highly phagocytic, whereas subtraction of serum resulted in rapid attachment and reduced phagocytic activity. The difference in phagocytosis between these subpopulations was correlated with the expression levels of FcγR. These two cell subpopulations also differed in their responses to LPS and the expression of surface markers, including CD14, CD86, scavenger receptor A1, TLR4 and low-density lipoprotein receptor. Moreover, we found that the lipid/cholesterol content in the culture medium mediated the differences between these two cell subpopulations. Thus, we described a mechanism that modulates Mϕ function depending on the exposure to lipids within their surrounding microenvironment. PMID:26951856

  11. Enhanced rifampicin delivery to alveolar macrophages by solid lipid nanoparticles

    NASA Astrophysics Data System (ADS)

    Chuan, Junlan; Li, Yanzhen; Yang, Likai; Sun, Xun; Zhang, Qiang; Gong, Tao; Zhang, Zhirong

    2013-05-01

    The present study aimed at developing a drug delivery system targeting the densest site of tuberculosis infection, the alveolar macrophages (AMs). Rifampicin (RFP)-loaded solid lipid nanoparticles (RFP-SLNs) with an average size of 829.6 ± 16.1 nm were prepared by a modified lipid film hydration method. The cytotoxicity of RFP-SLNs to AMs and alveolar epithelial type II cells (AECs) was examined using MTT assays. The viability of AMs and AECs was above 80 % after treatment with RFP-SLNs, which showed low toxicity to both AMs and AECs. Confocal Laser Scanning Microscopy was employed to observe the interaction between RFP-SLNs and both AMs and AECs. After incubating the cells with RFP-SLNs for 2 h, the fluorescent intensity in AMs was more and remained longer (from 0.5 to 12 h) when compared with that in AECs (from 0.5 to 8 h). In vitro uptake characteristics of RFP-SLNs in AMs and AECs were also investigated by detection of intracellular RFP by High performance liquid chromatography. Results showed that RFP-SLNs delivered markedly higher RFP into AMs (691.7 ng/mg in cultured AMs, 662.6 ng/mg in primary AMs) than that into AECs (319.2 ng/mg in cultured AECs, 287.2 ng/mg in primary AECs). Subsequently, in vivo delivery efficiency and the selectivity of RFP-SLNs were further verified in Sprague-Dawley rats. Under pulmonary administration of RFP-SLNs, the amount of RFP in AMs was significantly higher than that in AECs at each time point. Our results demonstrated that solid lipid nanoparticles are a promising strategy for the delivery of rifampicin to alveolar macrophages selectively.

  12. Incorporation of bacterial peptidoglycan constituents into macrophage lipids during phagocytosis. [Bacillus subtillis

    SciTech Connect

    Polanski, M.

    1987-01-01

    Bacillus subtilis radiolabeled cell walls were incubated with the macrophage cell line RAW264 in order to determine whether a peptidoglycan fragment were subsequently maintained on a macrophage lipid. Specifically, cell walls were radiolabeled in their glucosamine, muramic acid and alanine residues with D-(1-/sup 3/H) glucosamine and L(U-/sup 14/C)alanine. Following encounter with these radiolabeled cell walls, macrophages were collected and subjected to lipid extraction procedures. Further fractionation produced a phosphatidylethanolamine co-migrating lipid which upon hydrolysis and amino acid analysis revealed radiolabeled muramic acid, glucosamine, and alanine residues. These residues were shown to form a common fragment since the aqueous soluble material obtained after saponification of the crude lipid extract eluted as a single peak following gel permeation chromatography. Saponification destroyed the TLC mobility of the lipid showing that the fragment was covalently attached to the lipid.

  13. Vascular lipid accumulation, lipoprotein oxidation and macrophage lipid uptake in hypercholesterolemic zebrafish

    PubMed Central

    Stoletov, Konstantin; Fang, Longhou; Choi, Soo-Ho; Hartvigsen, Karsten; Hansen, Lotte F.; Hall, Chris; Pattison, Jennifer; Juliano, Joseph; Miller, Elizabeth R.; Almazan, Felicidad; Crosier, Phil; Witztum, Joseph L.; Klemke, Richard L.; Miller, Yury I.

    2010-01-01

    Lipid accumulation in arteries induces vascular inflammation and atherosclerosis, the major cause of heart attack and stroke in humans. Extreme hyperlipidemia induced in mice and rabbits enables modeling many aspects of human atherosclerosis, but microscopic examination of plaques is possible only postmortem. Here we report that feeding adult zebrafish (Danio rerio) a high-cholesterol diet (HCD) resulted in hypercholesterolemia, remarkable lipoprotein oxidation and fatty streak formation in the arteries. Feeding an HCD supplemented with a fluorescent cholesteryl ester to optically transparent fli1:EGFP zebrafish larvae in which endothelial cells (EC) express GFP, and using confocal microscopy enabled monitoring vascular lipid accumulation and the EC layer disorganization and thickening in a live animal. The HCD feeding also increased leakage of a fluorescent dextran from the blood vessels. Administering ezetimibe significantly diminished the HCD-induced EC layer thickening and improved its barrier function. Feeding HCD to lyz:DsRed2 larvae in which macrophages and granulocytes express DsRed, resulted in the accumulation of fluorescent myeloid cells in the vascular wall. Using a fluorogenic substrate for phospholipase A2 (PLA2), we observed an increased vascular PLA2 activity in live HCD-fed larvae compared to control larvae. Furthermore, by transplanting genetically modified murine cells into HCD-fed larvae, we demonstrated that toll-like receptor-4 (TLR4) was required for efficient in vivo lipid uptake by macrophages. These results suggest that the novel zebrafish model is suitable for studying temporal characteristics of certain inflammatory processes of early atherogenesis and the in vivo function of vascular cells. PMID:19265037

  14. miRNA-133a attenuates lipid accumulation via TR4-CD36 pathway in macrophages.

    PubMed

    Peng, Xiao-Ping; Huang, Lei; Liu, Zhi-Hong

    2016-08-01

    lipid metabolism is the major causes of atherosclerosis. There is increasing evidence that miR-133a plays an important role in atherosclerosis. However, the regulatory mechanism of miR-133a in macrophages is still unclear. Several lines of evidence indicate that loss of TR4 leads to reduce lipid accumulation in liver and adipose tissues, etc, and lesional macrophages-derived TR4 can greatly increase the foam cell formation through increasing the CD36-mediated the uptake of ox-LDL. Interestingly, computational analysis suggests that TR4 may be a target gene of miR-133a. Here, we examined whether miR-133a regulates TR4 expression in ox-LDL-induced mouse RAW 264.7 macrophages, thereby affecting lipid accumulation. Using ox-LDL-treatment RAW 264.7 macrophages transfected with miR-133a mimics or inhibitors, we have showed that miR-133a can directly regulate the expression of TR4 in RAW 264.7 cells, thereby attenuates CD36-medide lipid accumulation. Furthermore, our studies suggest an additional explanation for the regulatory mechanism of miR-133a regulation to its functional target, TR4 in RAW 264.7 macrophages. Thus, our findings suggest that miR-133a may regulate lipid accumulation in ox-LDL-stimulated RAW 264.7 macrophages via TR4-CD36 pathway.

  15. Automobile diesel exhaust particles induce lipid droplet formation in macrophages in vitro.

    PubMed

    Cao, Yi; Jantzen, Kim; Gouveia, Ana Cecilia Damiao; Skovmand, Astrid; Roursgaard, Martin; Loft, Steffen; Møller, Peter

    2015-07-01

    Exposure to diesel exhaust particles (DEP) has been associated with adverse cardiopulmonary health effects, which may be related to dysregulation of lipid metabolism and formation of macrophage foam cells. In this study, THP-1 derived macrophages were exposed to an automobile generated DEP (A-DEP) for 24h to study lipid droplet formation and possible mechanisms. The results show that A-DEP did not induce cytotoxicity. The production of reactive oxygen species was only significantly increased after exposure for 3h, but not 24h. Intracellular level of reduced glutathione was increased after 24h exposure. These results combined indicate an adaptive response to oxidative stress. Exposure to A-DEP was associated with significantly increased formation of lipid droplets, as well as changes in lysosomal function, assessed as reduced LysoTracker staining. In conclusion, these results indicated that exposure to A-DEP may induce formation of lipid droplets in macrophages in vitro possibly via lysosomal dysfunction.

  16. Phosphoethanolamine Modification of Neisseria gonorrhoeae Lipid A Reduces Autophagy Flux in Macrophages

    PubMed Central

    Zughaier, Susu M.; Kandler, Justin L.; Balthazar, Jacqueline T.; Shafer, William M.

    2015-01-01

    Autophagy, an ancient homeostasis mechanism for macromolecule degradation, performs an important role in host defense by facilitating pathogen elimination. To counteract this host defense strategy, bacterial pathogens have evolved a variety of mechanisms to avoid or otherwise dysregulate autophagy by phagocytic cells so as to enhance their survival during infection. Neisseria gonorrhoeae is a strictly human pathogen that causes the sexually transmitted infection, gonorrhea. Phosphoethanolamine (PEA) addition to the 4' position of the lipid A (PEA-lipid A) moiety of the lipooligosaccharide (LOS) produced by gonococci performs a critical role in this pathogen’s ability to evade innate defenses by conferring decreased susceptibility to cationic antimicrobial (or host-defense) peptides, complement-mediated killing by human serum and intraleukocytic killing by human neutrophils compared to strains lacking this PEA decoration. Heretofore, however, it was not known if gonococci can evade autophagy and if so, whether PEA-lipid A contributes to this ability. Accordingly, by using murine macrophages and human macrophage-like phagocytic cell lines we investigated if PEA decoration of gonococcal lipid A modulates autophagy formation. We report that infection with PEA-lipid A-producing gonococci significantly reduced autophagy flux in murine and human macrophages and enhanced gonococcal survival during their association with macrophages compared to a PEA-deficient lipid A mutant. Our results provide further evidence that PEA-lipid A produced by gonococci is a critical component in the ability of this human pathogen to evade host defenses. PMID:26641098

  17. RORα and 25-Hydroxycholesterol Crosstalk Regulates Lipid Droplet Homeostasis in Macrophages

    PubMed Central

    Tuong, Zewen Kelvin; Lau, Patrick; Du, Ximing; Condon, Nicholas D.; Goode, Joel M.; Oh, Tae Gyu; Yeo, Jeremy C.; Muscat, George E. O.; Stow, Jennifer L.

    2016-01-01

    Nuclear hormone receptors have important roles in the regulation of metabolic and inflammatory pathways. The retinoid-related orphan receptor alpha (Rorα)-deficient staggerer (sg/sg) mice display several phenotypes indicative of aberrant lipid metabolism, including dyslipidemia, and increased susceptibility to atherosclerosis. In this study we demonstrate that macrophages from sg/sg mice have increased ability to accumulate lipids and accordingly exhibit larger lipid droplets (LD). We have previously shown that BMMs from sg/sg mice have significantly decreased expression of cholesterol 25-hydroxylase (Ch25h) mRNA, the enzyme that produces the oxysterol, 25-hydroxycholesterol (25HC), and now confirm this at the protein level. 25HC functions as an inverse agonist for RORα. siRNA knockdown of Ch25h in macrophages up-regulates Vldlr mRNA expression and causes increased accumulation of LDs. Treatment with physiological concentrations of 25HC in sg/sg macrophages restored lipid accumulation back to normal levels. Thus, 25HC and RORα signify a new pathway involved in the regulation of lipid homeostasis in macrophages, potentially via increased uptake of lipid which is suggested by mRNA expression changes in Vldlr and other related genes. PMID:26812621

  18. Augmentation of macrophage growth-stimulating activity of lipids by their peroxidation

    SciTech Connect

    Yui, S.; Yamazaki, M. )

    1990-02-15

    Previously, we reported that some kinds of lipids (cholesterol esters, triglycerides, and some negatively charged phospholipids) that are constituents of lipoproteins or cell membranes induce growth of peripheral macrophages in vitro. In this paper, we examined the effect of peroxidation of lipids on their macrophage growth-stimulating activity because lipid peroxidation is observed in many pathological states such as inflammation. When phosphatidylserine, one of the phospholipids with growth-stimulating activity, was peroxidized by UV irradiation, its macrophage growth-stimulating activity was augmented in proportion to the extent of its peroxidation. The activity of phosphatidylethanolamine was also increased by UV irradiation. On the other hand, phosphatidylcholine or highly unsaturated free fatty acids, such as arachidonic acid and eicosapentaenoic acid, did not induce macrophage growth irrespective of whether they were peroxidized. The augmented activity of UV-irradiated phosphatidylserine was not affected by the coexistence of an antioxidant, vitamin E or BHT. These results suggest that some phospholipids included in damaged cells or denatured lipoproteins which are scavenged by macrophages in vivo may induce growth of peripheral macrophages more effectively when they are peroxidized by local pathological processes.

  19. Macrophage Differentiation from Monocytes Is Influenced by the Lipid Oxidation Degree of Low Density Lipoprotein

    PubMed Central

    Seo, Jin-Won; Yang, Eun-Jeong; Yoo, Kyung-Hwa; Choi, In-Hong

    2015-01-01

    LDL plays an important role in atherosclerotic plaque formation and macrophage differentiation. However, there is no report regarding the oxidation degree of LDL and macrophage differentiation. Our study has shown that the differentiation into M1 or M2 macrophages is related to the lipid oxidation level of LDL. Based on the level of lipid peroxidation, LDL is classified into high-oxidized LDL (hi-oxLDL) and low-oxidized LDL (low-oxLDL). The differentiation profiles of macrophages were determined by surface receptor expression and cytokine secretion profiles. Low-oxLDL induced CD86 expression and production of TNF-α and IL-12p40 in THP-1 cells, indicating an M1 macrophage phenotype. Hi-oxLDL induced mannose receptor expression and production of IL-6 and monocyte chemoattractant protein-1, which mostly match the phenotype of M2 macrophages. Further supporting evidence for an M2 polarization by hi-oxLDL was the induction of LOX-1 in THP-1 cells treated with hi-oxLDL but not with low-oxLDL. Similar results were obtained in primary human monocytes. Therefore, our results strongly suggest that the oxidation degree of LDL influences the differentiation of monocytes into M1 or M2 macrophages and determines the inflammatory fate in early stages of atherosclerosis. PMID:26294848

  20. 15(S)-Lipoxygenase-1 associates with neutral lipid droplets in macrophage foam cells: evidence of lipid droplet metabolism

    PubMed Central

    Weibel, Ginny L.; Joshi, Michelle R.; Wei, Cong; Bates, Sandra R.; Blair, Ian A.; Rothblat, George H.

    2009-01-01

    15(S)-lipoxygenase-1 (15-LO-1) was present in the whole-cell homogenate of an acute human monocytic leukemia cell line (THP-1). Additionally, 15-LO-1 was detected on neutral lipid droplets isolated from THP-1 foam cells. To investigate if 15-LO-1 is active on lipid droplets, we used the mouse leukemic monocytic macrophage cell line (RAW 264.7), which are stably transfected with human 15-LO-1. The RAW 15-LO-1 cells were incubated with acetylated low density lipoprotein to generate foam cells. 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], the major 15-LO-1 metabolite of arachidonic acid, was produced in the 15-LO-1 RAW but not in the mock transfected cells when incubated with arachidonic acid. Lipid droplets were isolated from the cells and incubated with arachidonic acid, and production of 15(S)-HETE was measured over 2 h. 15(S)-HETE was produced in the incubations with the lipid droplets, and this production was attenuated when the lipid droplet fraction was subjected to enzyme inactivation through heating. Efflux of 15(S)-HETE from cholesteryl ester-enriched 15-LO RAW cells, when lipid droplets are present, was significantly reduced compared with that from cells enriched with free cholesterol (lipid droplets are absent). We propose that 15-LO-1 is present and functional on cytoplasmic neutral lipid droplets in macrophage foam cells, and these droplets may act to accumulate the anti-inflammatory lipid mediator 15(S)-HETE. PMID:19528634

  1. Monitoring intra-cellular lipid metabolism in macrophages by Raman- and CARS-microscopy

    NASA Astrophysics Data System (ADS)

    Matthäus, Christian; Bergner, Gero; Krafft, Christoph; Dietzek, Benjamin; Lorkowski, Stefan; Popp, Jürgen

    2010-04-01

    Monocyte-derived macrophages play a key role in lipid metabolism in vessel wall tissues. Macrophages can take up lipids by various mechanisms. As phagocytes, macrophages are important for the decomposition of lipid plaques within arterial walls that contribute to arteriosclerosis. Of special interest are uptake dynamics and intra-cellular fate of different individual types of lipids as, for example, fatty acids, triglycerides or free and esterified cholesterol. Here we utilize Raman microscopy to image the metabolism of such lipids and follow subsequent storage or degradation patterns. The combination of optical microscopy with Raman spectroscopy allows visualization at the diffraction limit of the employed laser light and biochemical characterization through the associated spectral information. Relatively long measuring times, due to the weakness of Raman scattering can be overcome by non-linear effects such as coherent anti-Stokes Raman scattering (CARS). With this contribution we introduce first results to monitor the incorporation of lipid components into individual cells employing Raman and CARS microscopy.

  2. Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages.

    PubMed

    Daniel, Jaiyanth; Maamar, Hédia; Deb, Chirajyoti; Sirakova, Tatiana D; Kolattukudy, Pappachan E

    2011-06-01

    Two billion people are latently infected with Mycobacterium tuberculosis (Mtb). Mtb-infected macrophages are likely to be sequestered inside the hypoxic environments of the granuloma and differentiate into lipid-loaded macrophages that contain triacylglycerol (TAG)-filled lipid droplets which may provide a fatty acid-rich host environment for Mtb. We report here that human peripheral blood monocyte-derived macrophages and THP-1 derived macrophages incubated under hypoxia accumulate Oil Red O-staining lipid droplets containing TAG. Inside such hypoxic, lipid-loaded macrophages, nearly half the Mtb population developed phenotypic tolerance to isoniazid, lost acid-fast staining and accumulated intracellular lipid droplets. Dual-isotope labeling of macrophage TAG revealed that Mtb inside the lipid-loaded macrophages imports fatty acids derived from host TAG and incorporates them intact into Mtb TAG. The fatty acid composition of host and Mtb TAG were nearly identical suggesting that Mtb utilizes host TAG to accumulate intracellular TAG. Utilization of host TAG by Mtb for lipid droplet synthesis was confirmed when fluorescent fatty acid-labeled host TAG was utilized to accumulate fluorescent lipid droplets inside the pathogen. Deletion of the Mtb triacylglycerol synthase 1 (tgs1) gene resulted in a drastic decrease but not a complete loss in both radiolabeled and fluorescent TAG accumulation by Mtb suggesting that the TAG that accumulates within Mtb is generated mainly by the incorporation of fatty acids released from host TAG. We show direct evidence for the utilization of the fatty acids from host TAG for lipid metabolism inside Mtb. Taqman real-time PCR measurements revealed that the mycobacterial genes dosR, hspX, icl1, tgs1 and lipY were up-regulated in Mtb within hypoxic lipid loaded macrophages along with other Mtb genes known to be associated with dormancy and lipid metabolism.

  3. Cellular uptake and metabolism of curcuminoids in monocytes/macrophages: regulatory effects on lipid accumulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We previously showed that curcumin (CUR) may increase lipid accumulation in cultured THP-1 monocytes/macrophages, but tetrahydrocurcumin (THC), an in vivo metabolite of CUR, had no such effect. In the present study, we have hypothesized that different cellular uptake and/or metabolism of CUR and THC...

  4. Mycobacterium tuberculosis-driven targeted recalibration of macrophage lipid homeostasis promotes the foamy phenotype.

    PubMed

    Singh, Varshneya; Jamwal, Shilpa; Jain, Ritu; Verma, Priyanka; Gokhale, Rajesh; Rao, Kanury V S

    2012-11-15

    Upon infection, Mycobacterium tuberculosis (Mtb) metabolically alters the macrophage to create a niche that is ideally suited to its persistent lifestyle. Infected macrophages acquire a "foamy" phenotype characterized by the accumulation of lipid bodies (LBs), which serve as both a source of nutrients and a secure niche for the bacterium. While the functional significance of the foamy phenotype is appreciated, the biochemical pathways mediating this process are understudied. We found that Mtb induces the foamy phenotype via targeted manipulation of host cellular metabolism to divert the glycolytic pathway toward ketone body synthesis. This dysregulation enabled feedback activation of the anti-lipolytic G protein-coupled receptor GPR109A, leading to perturbations in lipid homeostasis and consequent accumulation of LBs in the macrophage. ESAT-6, a secreted Mtb virulence factor, mediates the enforcement of this feedback loop. Finally, we demonstrate that pharmacological targeting of pathways mediating this host-pathogen metabolic crosstalk provides a potential strategy for developing tuberculosis chemotherapy.

  5. Disruption of Lipid Rafts Interferes with the Interaction of Toxoplasma gondii with Macrophages and Epithelial Cells

    PubMed Central

    Cruz, Karla Dias; Cruz, Thayana Araújo; Veras de Moraes, Gabriela; Paredes-Santos, Tatiana Christina; Attias, Marcia; de Souza, Wanderley

    2014-01-01

    The intracellular parasite Toxoplasma gondii can penetrate any warm-blooded animal cell. Conserved molecular assemblies of host cell plasma membranes should be involved in the parasite-host cell recognition. Lipid rafts are well-conserved membrane microdomains that contain high concentrations of cholesterol, sphingolipids, glycosylphosphatidylinositol, GPI-anchored proteins, and dually acylated proteins such as members of the Src family of tyrosine kinases. Disturbing lipid rafts of mouse peritoneal macrophages and epithelial cells of the lineage LLC-MK2 with methyl-beta cyclodextrin (MβCD) and filipin, which interfere with cholesterol or lidocaine, significantly inhibited internalization of T. gondii in both cell types, although adhesion remained unaffected in macrophages and decreased only in LLC-MK2 cells. Scanning and transmission electron microscopy confirmed these observations. Results are discussed in terms of the original role of macrophages as professional phagocytes versus the LLC-MK2 cell lineage originated from kidney epithelial cells. PMID:24734239

  6. Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction.

    PubMed

    Fantin, Alessandro; Vieira, Joaquim M; Gestri, Gaia; Denti, Laura; Schwarz, Quenten; Prykhozhij, Sergey; Peri, Francesca; Wilson, Stephen W; Ruhrberg, Christiana

    2010-08-05

    Blood vessel networks expand in a 2-step process that begins with vessel sprouting and is followed by vessel anastomosis. Vessel sprouting is induced by chemotactic gradients of the vascular endothelial growth factor (VEGF), which stimulates tip cell protrusion. Yet it is not known which factors promote the fusion of neighboring tip cells to add new circuits to the existing vessel network. By combining the analysis of mouse mutants defective in macrophage development or VEGF signaling with live imaging in zebrafish, we now show that macrophages promote tip cell fusion downstream of VEGF-mediated tip cell induction. Macrophages therefore play a hitherto unidentified and unexpected role as vascular fusion cells. Moreover, we show that there are striking molecular similarities between the pro-angiogenic tissue macrophages essential for vascular development and those that promote the angiogenic switch in cancer, including the expression of the cell-surface proteins TIE2 and NRP1. Our findings suggest that tissue macrophages are a target for antiangiogenic therapies, but that they could equally well be exploited to stimulate tissue vascularization in ischemic disease.

  7. A Pneumocyte–Macrophage Paracrine Lipid Axis Drives the Lung toward Fibrosis

    PubMed Central

    Romero, Freddy; Shah, Dilip; Duong, Michelle; Penn, Raymond B.; Fessler, Michael B.; Madenspacher, Jennifer; Stafstrom, William; Kavuru, Mani; Lu, Bo; Kallen, Caleb B.; Walsh, Kenneth

    2015-01-01

    Lipid-laden macrophages, or “foam cells,” are observed in the lungs of patients with fibrotic lung disease, but their contribution to disease pathogenesis remains unexplored. Here, we demonstrate that fibrosis induced by bleomycin, silica dust, or thoracic radiation promotes early and sustained accumulation of foam cells in the lung. In the bleomycin model, we show that foam cells arise from neighboring alveolar epithelial type II cells, which respond to injury by dumping lipids into the distal airspaces of the lungs. We demonstrate that oxidized phospholipids accumulate within alveolar macrophages (AMs) after bleomycin injury and that murine and human AMs treated with oxidized phosphatidylcholine (oxPc) become polarized along an M2 phenotype and display enhanced production of transforming growth factor-β1. The direct instillation of oxPc into the mouse lung induces foam cell formation and triggers a severe fibrotic reaction. Further, we show that reducing pulmonary lipid clearance by targeted deletion of the lipid efflux transporter ATP-binding cassette subfamily G member 1 increases foam cell formation and worsens lung fibrosis after bleomycin. Conversely, we found that treatment with granulocyte-macrophage colony-stimulating factor attenuates fibrotic responses, at least in part through its ability to decrease AM lipid accumulation. In summary, this work describes a novel mechanism leading to foam cell formation in the mouse lung and suggests that strategies aimed at blocking foam cell formation might be effective for treating fibrotic lung disorders. PMID:25409201

  8. Differential lipid metabolism in monocytes and macrophages: influence of cholesterol loading[S

    PubMed Central

    Fernandez-Ruiz, Irene; Puchalska, Patrycja; Narasimhulu, Chandrakala Aluganti; Sengupta, Bhaswati; Parthasarathy, Sampath

    2016-01-01

    The influence of the hypercholesterolemia associated with atherosclerosis on monocytes is poorly understood. Monocytes are exposed to high concentrations of lipids, particularly cholesterol and lysophosphatidylcholine (lyso-PC). Indeed, in line with recent reports, we found that monocytes accumulate cholesteryl esters (CEs) in hypercholesterolemic mice, demonstrating the need for studies that analyze the effects of lipid accumulation on monocytes. Here we analyze the effects of cholesterol and lyso-PC loading in human monocytes and macrophages. We found that cholesterol acyltransferase and CE hydrolase activities are lower in monocytes. Monocytes also showed a different expression profile of cholesterol influx and efflux genes in response to lipid loading and a different pattern of lyso-PC metabolism. In monocytes, increased levels of CE slowed the conversion of lyso-PC into PC. Interestingly, although macrophages accumulated glycerophosphocholine, phosphocholine was the main water-soluble choline metabolite being generated in monocytes, suggesting a role for mono- and diacylglycerol in the chemoattractability of these cells. In summary, monocytes and macrophages show significant differences in lipid metabolism and gene expression profiles in response to lipid loading. These findings provide new insights into the mechanisms of atherosclerosis and suggest potentials for targeting monocyte chemotactic properties not only in atherosclerosis but also in other diseases. PMID:26839333

  9. Lipid raft-dependent uptake, signaling, and intracellular fate of Porphyromonas gingivalis in mouse macrophages

    PubMed Central

    Wang, Min; Hajishengallis, George

    2009-01-01

    Summary Lipid rafts are cholesterol-enriched microdomains involved in cellular trafficking and implicated as portals for certain pathogens. We sought to determine whether the oral pathogen Porphyromonas gingivalis enters macrophages via lipid rafts, and if so, to examine the impact of raft entry on its intracellular fate. Using J774A.1 mouse macrophages, we found that P. gingivalis colocalizes with lipid rafts in a cholesterol-dependent way. Depletion of cellular cholesterol using methyl-β-cyclodextrin resulted in about 50% inhibition of P. gingivalis uptake, although this effect was reversed by cholesterol reconstitution. The intracellular survival of P. gingivalis was dramatically inhibited in cholesterol-depleted cells relative to untreated or cholesterol-reconstituted cells, even when infections were adjusted to allow equilibration of the initial intracellular bacterial load. P. gingivalis thus appeared to exploit raft-mediated uptake for promoting its survival. Consistent with this, lipid raft disruption enhanced the colocalization of internalized P. gingivalis with lysosomes. In contrast, raft disruption did not affect the expression of host receptors interacting with P. gingivalis, although it significantly inhibited signal transduction. In summary, P. gingivalis uses macrophage lipid rafts as signaling and entry platforms, which determine its intracellular fate to the pathogen’s own advantage. PMID:18547335

  10. Ocimum basilicum ethanolic extract decreases cholesterol synthesis and lipid accumulation in human macrophages.

    PubMed

    Bravo, Elena; Amrani, Souliman; Aziz, Mohammed; Harnafi, Hicham; Napolitano, Mariarosaria

    2008-12-01

    Macrophage lipid accumulation induced by low density lipoproteins (LDL) plays a pivotal role in atherosclerotic plaque development. Previous work showed that Ocimum basilicum extract, used as hypocholesterolemic agent by traditional medicine in Morocco, has hypolipidemic activity in rat acute hyperlipimidemia. This study investigated the effects of ethanolic extract of O. basilicum on lipid accumulation in human macrophages. As modification of LDL increase atherogenicity of the particles we evaluated the effects of the extract on LDL oxidation. The extract caused a dose-related increase of LDL-resistance to Cu(2+)-induced oxidation. Furthermore, at the dose of 60 microg/ml, significantly decreases the accumulation of macrophage lipid droplets induced by modified LDL evaluated as by red-oil staining. Cholesterol esterification and triacylglycerol synthesis in the cells were not affected. Macrophage treatment with 60 microg/ml, but not 20 microg/ml, of the extract reduced newly synthesized unesterified cholesterol by about 60% and decreased scavenger receptors activity by about 20-30%, evaluated by the internalization of cholesterol carried by [(3)H]CE-aggregated-LDL. The results suggest that O. basilicum ethanolic extract has the capability to reduce foam cell formation through the reduction of cholesterol synthesis and the modulation of the activity of surface scavenger receptors.

  11. Chaperoning erythropoiesis

    PubMed Central

    dos Santos, Camila O.

    2009-01-01

    Multisubunit complexes containing molecular chaperones regulate protein production, stability, and degradation in virtually every cell type. We are beginning to recognize how generalized and tissue-specific chaperones regulate specialized aspects of erythropoiesis. For example, chaperones intersect with erythropoietin signaling pathways to protect erythroid precursors against apoptosis. Molecular chaperones also participate in hemoglobin synthesis, both directly and indirectly. Current knowledge in these areas only scratches the surface of what is to be learned. Improved understanding of how molecular chaperones regulate erythropoietic development and hemoglobin homeostasis should identify biochemical pathways amenable to pharmacologic manipulation in a variety of red blood cell disorders including thalassemia and other anemias associated with hemoglobin instability. PMID:19109556

  12. Regulation of adhesion behavior of murine macrophage using supported lipid membranes displaying tunable mannose domains

    NASA Astrophysics Data System (ADS)

    Kaindl, T.; Oelke, J.; Pasc, A.; Kaufmann, S.; Konovalov, O. V.; Funari, S. S.; Engel, U.; Wixforth, A.; Tanaka, M.

    2010-07-01

    Highly uniform, strongly correlated domains of synthetically designed lipids can be incorporated into supported lipid membranes. The systematic characterization of membranes displaying a variety of domains revealed that the equilibrium size of domains significantly depends on the length of fluorocarbon chains, which can be quantitatively interpreted within the framework of an equivalent dipole model. A mono-dispersive, narrow size distribution of the domains enables us to treat the inter-domain correlations as two-dimensional colloidal crystallization and calculate the potentials of mean force. The obtained results demonstrated that both size and inter-domain correlation can precisely be controlled by the molecular structures. By coupling α-D-mannose to lipid head groups, we studied the adhesion behavior of the murine macrophage (J774A.1) on supported membranes. Specific adhesion and spreading of macrophages showed a clear dependence on the density of functional lipids. The obtained results suggest that such synthetic lipid domains can be used as a defined platform to study how cells sense the size and distribution of functional molecules during adhesion and spreading.

  13. Clofazimine modulates the expression of lipid metabolism proteins in Mycobacterium leprae-infected macrophages.

    PubMed

    Degang, Yang; Akama, Takeshi; Hara, Takeshi; Tanigawa, Kazunari; Ishido, Yuko; Gidoh, Masaichi; Makino, Masahiko; Ishii, Norihisa; Suzuki, Koichi

    2012-01-01

    Mycobacterium leprae (M. leprae) lives and replicates within macrophages in a foamy, lipid-laden phagosome. The lipids provide essential nutrition for the mycobacteria, and M. leprae infection modulates expression of important host proteins related to lipid metabolism. Thus, M. leprae infection increases the expression of adipophilin/adipose differentiation-related protein (ADRP) and decreases hormone-sensitive lipase (HSL), facilitating the accumulation and maintenance of lipid-rich environments suitable for the intracellular survival of M. leprae. HSL levels are not detectable in skin smear specimens taken from leprosy patients, but re-appear shortly after multidrug therapy (MDT). This study examined the effect of MDT components on host lipid metabolism in vitro, and the outcome of rifampicin, dapsone and clofazimine treatment on ADRP and HSL expression in THP-1 cells. Clofazimine attenuated the mRNA and protein levels of ADRP in M. leprae-infected cells, while those of HSL were increased. Rifampicin and dapsone did not show any significant effects on ADRP and HSL expression levels. A transient increase of interferon (IFN)-β and IFN-γ mRNA was also observed in cells infected with M. leprae and treated with clofazimine. Lipid droplets accumulated by M. leprae-infection were significantly decreased 48 h after clofazimine treatment. Such effects were not evident in cells without M. leprae infection. In clinical samples, ADRP expression was decreased and HSL expression was increased after treatment. These results suggest that clofazimine modulates lipid metabolism in M. leprae-infected macrophages by modulating the expression of ADRP and HSL. It also induces IFN production in M. leprae-infected cells. The resultant decrease in lipid accumulation, increase in lipolysis, and activation of innate immunity may be some of the key actions of clofazimine.

  14. High-polarity Mycobacterium avium-derived lipids interact with murine macrophage lipid rafts.

    PubMed

    Maldonado-García, G; Chico-Ortiz, M; Lopez-Marin, L M; Sánchez-García, F J

    2004-11-01

    Cholesterol- and sphingolipid-rich membrane microdomains (lipid rafts) are widely recognized as portals for pathogenic micro-organisms. A growing body of evidence demonstrates mobilization of host plasma cell membrane lipid rafts towards the site of contact with several pathogens as well as a strict dependence on cholesterol for appropriate internalization. The fate of lipid rafts once the pathogen has been internalized and the nature of the pathogen components that interact with them is however less understood. To address both these issues, infection of the J774 murine cell line with Mycobacterium avium was used as a model. After demonstrating that M. avium induces lipid raft mobilization and that M. avium infects J774 by a cholesterol-dependent mechanism, it is shown here that mycobacterial phagosomes harbour lipid rafts, which are, at least in part, of plasma cell membrane origin. On the other hand, by using latex microbeads coated with any of the three fractions of M. avium-derived lipids of different polarity, we provide evidence that high-polarity, in contrast to low-polarity and intermediate-polarity, mycobacterial lipids or uncoated latex beads have a strong capacity to induce lipid raft mobilization. These results suggest that high-polarity mycobacterial lipid(s) interact with host cell cholesterol-enriched microdomains which may in turn influence the course of infection.

  15. Induction of DKK1 by ox-LDL negatively regulates intracellular lipid accumulation in macrophages.

    PubMed

    Zhang, Yu; Ge, Cheng; Wang, Lin; Liu, Xinxin; Chen, Yifei; Li, Mengmeng; Zhang, Mei

    2015-01-02

    Dickkopf1 (DKK1), a canonical Wnt/β-catenin pathway antagonist, is closely associated with cardiovascular disease and adipogenesis. We performed an in vitro study to determine whether oxidized low-density lipoprotein (ox-LDL) increased the expression of DKK1 in macrophages and whether β-catenin and liver X receptor α (LXRα) were involved in this regulation. Induction of DKK1 expression by ox-LDL decreased the level of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) via a Wnt/β-catenin pathway and increased ATP-binding cassette transporter A/G1 (ABCA/G1) levels via a signal transducer and activator of transcription 3 (STAT3) pathway. Lower LOX-1 and higher ABCA/G1 levels inhibited cholesterol loading in macrophages. In conclusion, ox-LDL may induce DKK1 expression in macrophages to inhibit the accumulation of lipids through a mechanism that involves downregulation of LOX-1-mediated lipid uptake and upregulation of ABCA/G1-dependent cholesterol efflux.

  16. Cytokines, macrophage lipid metabolism and foam cells: implications for cardiovascular disease therapy.

    PubMed

    McLaren, James E; Michael, Daryn R; Ashlin, Tim G; Ramji, Dipak P

    2011-10-01

    Cardiovascular disease is the biggest killer globally and the principal contributing factor to the pathology is atherosclerosis; a chronic, inflammatory disorder characterized by lipid and cholesterol accumulation and the development of fibrotic plaques within the walls of large and medium arteries. Macrophages are fundamental to the immune response directed to the site of inflammation and their normal, protective function is harnessed, detrimentally, in atherosclerosis. Macrophages contribute to plaque development by internalizing native and modified lipoproteins to convert them into cholesterol-rich foam cells. Foam cells not only help to bridge the innate and adaptive immune response to atherosclerosis but also accumulate to create fatty streaks, which help shape the architecture of advanced plaques. Foam cell formation involves the disruption of normal macrophage cholesterol metabolism, which is governed by a homeostatic mechanism that controls the uptake, intracellular metabolism, and efflux of cholesterol. It has emerged over the last 20 years that an array of cytokines, including interferon-γ, transforming growth factor-β1, interleukin-1β, and interleukin-10, are able to manipulate these processes. Foam cell targeting, anti-inflammatory therapies, such as agonists of nuclear receptors and statins, are known to regulate the actions of pro- and anti-atherogenic cytokines indirectly of their primary pharmacological function. A clear understanding of macrophage foam cell biology will hopefully enable novel foam cell targeting therapies to be developed for use in the clinical intervention of atherosclerosis.

  17. Metformin reduces lipid accumulation in macrophages by inhibiting FOXO1-mediated transcription of fatty acid-binding protein 4

    SciTech Connect

    Song, Jun; Ren, Pingping; Zhang, Lin; Wang, Xing Li; Chen, Li; Shen, Ying H.

    2010-02-26

    Objective: The accumulation of lipids in macrophages contributes to the development of atherosclerosis. Strategies to reduce lipid accumulation in macrophages may have therapeutic potential for preventing and treating atherosclerosis and cardiovascular complications. The antidiabetic drug metformin has been reported to reduce lipid accumulation in adipocytes. In this study, we examined the effects of metformin on lipid accumulation in macrophages and investigated the mechanisms involved. Methods and results: We observed that metformin significantly reduced palmitic acid (PA)-induced intracellular lipid accumulation in macrophages. Metformin promoted the expression of carnitine palmitoyltransferase I (CPT-1), while reduced the expression of fatty acid-binding protein 4 (FABP4) which was involved in PA-induced lipid accumulation. Quantitative real-time PCR showed that metformin regulates FABP4 expression at the transcriptional level. We identified forkhead transcription factor FOXO1 as a positive regulator of FABP4 expression. Inhibiting FOXO1 expression with FOXO1 siRNA significantly reduced basal and PA-induced FABP4 expression. Overexpression of wild-type FOXO1 and constitutively active FOXO1 significantly increased FABP4 expression, whereas dominant negative FOXO1 dramatically decreased FABP4 expression. Metformin reduced FABP4 expression by promoting FOXO1 nuclear exclusion and subsequently inhibiting its activity. Conclusions: Taken together, these results suggest that metformin reduces lipid accumulation in macrophages by repressing FOXO1-mediated FABP4 transcription. Thus, metformin may have a protective effect against lipid accumulation in macrophages and may serve as a therapeutic agent for preventing and treating atherosclerosis in metabolic syndrome.

  18. Activation of autophagy in macrophages by pro-resolving lipid mediators

    PubMed Central

    Prieto, Patricia; Rosales-Mendoza, César Eduardo; Terrón, Verónica; Toledano, Víctor; Cuadrado, Antonio; López-Collazo, Eduardo; Bannenberg, Gerard; Martín-Sanz, Paloma; Fernández-Velasco, María; Boscá, Lisardo

    2015-01-01

    The resolution of inflammation is an active process driven by specialized pro-resolving lipid mediators, such as 15-epi-LXA4 and resolvin D1 (RvD1), that promote tissue regeneration. Macrophages regulate the innate immune response being key players during the resolution phase to avoid chronic inflammatory pathologies. Their half-life is tightly regulated to accomplish its phagocytic function, allowing the complete cleaning of the affected area. The balance between apoptosis and autophagy appears to be essential to control the survival of these immune cells within the inflammatory context. In the present work, we demonstrate that 15-epi-LXA4 and RvD1 at nanomolar concentrations promote autophagy in murine and human macrophages. Both compounds induced the MAP1LC3-I to MAP1LC3-II processing and the degradation of SQSTM1 as well as the formation of MAP1LC3+ autophagosomes, a typical signature of autophagy. Furthermore, 15-epi-LXA4 and RvD1 treatment favored the fusion of the autophagosomes with lysosomes, allowing the final processing of the autophagic vesicles. This autophagic response involves the activation of MAPK1 and NFE2L2 pathways, but by an MTOR-independent mechanism. Moreover, these pro-resolving lipids improved the phagocytic activity of macrophages via NFE2L2. Therefore, 15-epi-LXA4 and RvD1 improved both survival and functionality of macrophages, which likely supports the recovery of tissue homeostasis and avoiding chronic inflammatory diseases. PMID:26506892

  19. Activation of autophagy in macrophages by pro-resolving lipid mediators.

    PubMed

    Prieto, Patricia; Rosales-Mendoza, César Eduardo; Terrón, Verónica; Toledano, Víctor; Cuadrado, Antonio; López-Collazo, Eduardo; Bannenberg, Gerard; Martín-Sanz, Paloma; Fernández-Velasco, María; Boscá, Lisardo

    2015-01-01

    The resolution of inflammation is an active process driven by specialized pro-resolving lipid mediators, such as 15-epi-LXA4 and resolvin D1 (RvD1), that promote tissue regeneration. Macrophages regulate the innate immune response being key players during the resolution phase to avoid chronic inflammatory pathologies. Their half-life is tightly regulated to accomplish its phagocytic function, allowing the complete cleaning of the affected area. The balance between apoptosis and autophagy appears to be essential to control the survival of these immune cells within the inflammatory context. In the present work, we demonstrate that 15-epi-LXA4 and RvD1 at nanomolar concentrations promote autophagy in murine and human macrophages. Both compounds induced the MAP1LC3-I to MAP1LC3-II processing and the degradation of SQSTM1 as well as the formation of MAP1LC3(+) autophagosomes, a typical signature of autophagy. Furthermore, 15-epi-LXA4 and RvD1 treatment favored the fusion of the autophagosomes with lysosomes, allowing the final processing of the autophagic vesicles. This autophagic response involves the activation of MAPK1 and NFE2L2 pathways, but by an MTOR-independent mechanism. Moreover, these pro-resolving lipids improved the phagocytic activity of macrophages via NFE2L2. Therefore, 15-epi-LXA4 and RvD1 improved both survival and functionality of macrophages, which likely supports the recovery of tissue homeostasis and avoiding chronic inflammatory diseases.

  20. The interplay of lung surfactant proteins and lipids assimilates the macrophage clearance of nanoparticles.

    PubMed

    Ruge, Christian A; Schaefer, Ulrich F; Herrmann, Jennifer; Kirch, Julian; Cañadas, Olga; Echaide, Mercedes; Pérez-Gil, Jesús; Casals, Cristina; Müller, Rolf; Lehr, Claus-Michael

    2012-01-01

    The peripheral lungs are a potential entrance portal for nanoparticles into the human body due to their large surface area. The fact that nanoparticles can be deposited in the alveolar region of the lungs is of interest for pulmonary drug delivery strategies and is of equal importance for toxicological considerations. Therefore, a detailed understanding of nanoparticle interaction with the structures of this largest and most sensitive part of the lungs is important for both nanomedicine and nanotoxicology. Astonishingly, there is still little known about the bio-nano interactions that occur after nanoparticle deposition in the alveoli. In this study, we compared the effects of surfactant-associated protein A (SP-A) and D (SP-D) on the clearance of magnetite nanoparticles (mNP) with either more hydrophilic (starch) or hydrophobic (phosphatidylcholine) surface modification by an alveolar macrophage (AM) cell line (MH-S) using flow cytometry and confocal microscopy. Both proteins enhanced the AM uptake of mNP compared with pristine nanoparticles; for the hydrophilic ST-mNP, this effect was strongest with SP-D, whereas for the hydrophobic PL-mNP it was most pronounced with SP-A. Using gel electrophoretic and dynamic light scattering methods, we were able to demonstrate that the observed cellular effects were related to protein adsorption and to protein-mediated interference with the colloidal stability. Next, we investigated the influence of various surfactant lipids on nanoparticle uptake by AM because lipids are the major surfactant component. Synthetic surfactant lipid and isolated native surfactant preparations significantly modulated the effects exerted by SP-A and SP-D, respectively, resulting in comparable levels of macrophage interaction for both hydrophilic and hydrophobic nanoparticles. Our findings suggest that because of the interplay of both surfactant lipids and proteins, the AM clearance of nanoparticles is essentially the same, regardless of different

  1. Lipid Droplet Formation, Their Localization and Dynamics during Leishmania major Macrophage Infection

    PubMed Central

    Rabhi, Sameh; Rabhi, Imen; Trentin, Bernadette; Piquemal, David; Regnault, Béatrice; Goyard, Sophie; Lang, Thierry; Descoteaux, Albert; Enninga, Jost; Guizani-Tabbane, Lamia

    2016-01-01

    Leishmania, the causative agent of vector-borne diseases, known as leishmaniases, is an obligate intracellular parasite within mammalian hosts. The outcome of infection depends largely on the activation status of macrophages, the first line of mammalian defense and the major target cells for parasite replication. Understanding the strategies developed by the parasite to circumvent macrophage defense mechanisms and to survive within those cells help defining novel therapeutic approaches for leishmaniasis. We previously showed the formation of lipid droplets (LDs) in L. major infected macrophages. Here, we provide novel insights on the origin of the formed LDs by determining their cellular distribution and to what extent these high-energy sources are directed to the proximity of Leishmania parasites. We show that the ability of L. major to trigger macrophage LD accumulation is independent of parasite viability and uptake and can also be observed in non-infected cells through paracrine stimuli suggesting that LD formation is from cellular origin. The accumulation of LDs is demonstrated using confocal microscopy and live-cell imagin in parasite-free cytoplasmic region of the host cell, but also promptly recruited to the proximity of Leishmania parasites. Indeed LDs are observed inside parasitophorous vacuole and in parasite cytoplasm suggesting that Leishmania parasites besides producing their own LDs, may take advantage of these high energy sources. Otherwise, these LDs may help cells defending against parasitic infection. These metabolic changes, rising as common features during the last years, occur in host cells infected by a large number of pathogens and seem to play an important role in pathogenesis. Understanding how Leishmania parasites and different pathogens exploit this LD accumulation will help us define the common mechanism used by these different pathogens to manipulate and/or take advantage of this high-energy source. PMID:26871576

  2. Lipid Droplet Formation, Their Localization and Dynamics during Leishmania major Macrophage Infection.

    PubMed

    Rabhi, Sameh; Rabhi, Imen; Trentin, Bernadette; Piquemal, David; Regnault, Béatrice; Goyard, Sophie; Lang, Thierry; Descoteaux, Albert; Enninga, Jost; Guizani-Tabbane, Lamia

    2016-01-01

    Leishmania, the causative agent of vector-borne diseases, known as leishmaniases, is an obligate intracellular parasite within mammalian hosts. The outcome of infection depends largely on the activation status of macrophages, the first line of mammalian defense and the major target cells for parasite replication. Understanding the strategies developed by the parasite to circumvent macrophage defense mechanisms and to survive within those cells help defining novel therapeutic approaches for leishmaniasis. We previously showed the formation of lipid droplets (LDs) in L. major infected macrophages. Here, we provide novel insights on the origin of the formed LDs by determining their cellular distribution and to what extent these high-energy sources are directed to the proximity of Leishmania parasites. We show that the ability of L. major to trigger macrophage LD accumulation is independent of parasite viability and uptake and can also be observed in non-infected cells through paracrine stimuli suggesting that LD formation is from cellular origin. The accumulation of LDs is demonstrated using confocal microscopy and live-cell imagin in parasite-free cytoplasmic region of the host cell, but also promptly recruited to the proximity of Leishmania parasites. Indeed LDs are observed inside parasitophorous vacuole and in parasite cytoplasm suggesting that Leishmania parasites besides producing their own LDs, may take advantage of these high energy sources. Otherwise, these LDs may help cells defending against parasitic infection. These metabolic changes, rising as common features during the last years, occur in host cells infected by a large number of pathogens and seem to play an important role in pathogenesis. Understanding how Leishmania parasites and different pathogens exploit this LD accumulation will help us define the common mechanism used by these different pathogens to manipulate and/or take advantage of this high-energy source.

  3. Reduction of NO Synthase Expression and Tumor Necrosis Factor Alpha Production in Macrophages by Amphotericin B Lipid Carriers

    PubMed Central

    Larabi, Malika; Legrand, Philippe; Appel, Martine; Gil, Sophie; Lepoivre, Michel; Devissaguet, Jean-Philippe; Puisieux, Francis; Barratt, Gillian

    2001-01-01

    The present study compared the abilities of different lipid carriers of amphotericin B (AMB) to activate murine peritoneal macrophages, as assessed by their capacities to produce nitric oxide (NO) and tumor necrosis factor alpha (TNF-α). Although AMB alone did not induce NO production, synergy was observed with gamma interferon but not with lipopolysaccharide. This synergy could not be explained by the mobilization of the nuclear activation factor NF-κB by AMB. On the other hand, AMB induced TNF-α production without a costimulator and no synergy was observed. Anti-TNF-α antibodies did not influence NO production, and an inhibitor of NO synthase did not affect TNF-α production, indicating that the production of one of these effector molecules was independent of that of the other. The incorporation of AMB into lipid carriers reduced NO and TNF-α production with all formulations but more so with liposomes than with lipid complexes. NO production was correlated with the induction of NO synthase II, revealed by Western blotting. The extent of association of AMB with macrophages depended on the formulation, especially on the AMB/lipids ratio: the higher the ratio was, the greater the AMB association with macrophages. However, there was no clear correlation between AMB association with macrophages, whether internalized or bound to the membrane, and immunostimulating effects. These results may explain the reduced toxicities of lipid-based formulations of AMB. PMID:11158754

  4. Lipid IVa incompletely activates MyD88-independent Toll-like receptor 4 signaling in mouse macrophage cell lines.

    PubMed

    Ogura, Norihiko; Muroi, Masashi; Sugiura, Yuka; Tanamoto, Ken-ichi

    2013-04-01

    We investigated the difference in the effect of synthetic lipid A compounds on MyD88-dependent and -independent Toll-like receptor 4 (TLR4) signaling in mouse macrophage cells. At higher concentrations, Escherichia coli-type hexa-acylated lipid A 506, Salmonella-type hepta-acylated lipid A 516, the lipid A precursor lipid IVa and monophosphoryl lipid A induced similar levels of production of the MyD88-dependent cytokine IL-1β although their potencies varied, whereas the maximum production of the MyD88-independent cytokine RANTES induced by lipid IVa was less than 50% that of other lipid A compounds. A maximum level of NF-κB activation, which is involved in IL-1β gene transcription, was also induced to a similar level by these four lipid A compounds, while the maximum level of IFN-β promoter activity induced during MyD88-independent signaling was also less than 50% for lipid IVa stimulation compared with other lipid A compounds. Early IκBα phosphorylation activated by MyD88-dependent signaling was similarly induced by 506 and lipid IVa, whereas lipid IVa barely stimulated the phosphorylation of IRF3, a MyD88-independent transcription factor, although efficient phosphorylation was observed with 506 stimulation. These results indicate that lipid IVa has limited activity toward MyD88-independent signaling of TLR4, in macrophage cell lines, despite having efficient activity in the MyD88-dependent pathway.

  5. Hypericin-mediated sonodynamic therapy induces autophagy and decreases lipids in THP-1 macrophage by promoting ROS-dependent nuclear translocation of TFEB

    PubMed Central

    Li, Xuesong; Zhang, Xin; Zheng, Longbin; Kou, Jiayuan; Zhong, Zhaoyu; Jiang, Yueqing; Wang, Wei; Dong, Zengxiang; Liu, Zhongni; Han, Xiaobo; Li, Jing; Tian, Ye; Zhao, Yajun; Yang, Liming

    2016-01-01

    Lipid catabolism disorder is the primary cause of atherosclerosis. Transcription factor EB (TFEB) prevents atherosclerosis by activating macrophage autophagy to promote lipid degradation. Hypericin-mediated sonodynamic therapy (HY-SDT) has been proved non-invasively inducing THP-1-derived macrophage apoptosis; however, it is unknown whether macrophage autophagy could be triggered by HY-SDT to influence cellular lipid catabolism via regulating TFEB. Here, we report that HY-SDT resulted in the time-dependent THP-1-derived macrophage autophagy activation through AMPK/AKT/mTOR pathway. Besides, TFEB nuclear translocation in macrophage was triggered by HY-SDT to promote autophagy activation and lysosome regeneration which enhanced lipid degradation in response to atherogenic lipid stressors. Moreover, following HY-SDT, the ABCA1 expression level was increased to promote lipid efflux in macrophage, and the expression levels of CD36 and SR-A were decreased to inhibit lipid uptake, both of which were prevented by TFEB knockdown. These results indicated that TFEB nuclear translocation activated by HY-SDT was not only the key regulator of autophagy activation and lysosome regeneration in macrophage to promote lipolysis, but also had a crucial role in reverse cholesterol transporters to decrease lipid uptake and increase lipid efflux. Reactive oxygen species (ROS) were adequately generated in macrophage by HY-SDT. Further, ROS scavenger N-acetyl-l-cysteine abolished HY-SDT-induced TFEB nuclear translocation and autophagy activation, implying that ROS were the primary upstream factors responsible for these effects during HY-SDT. In summary, our data indicate that HY-SDT decreases lipid content in macrophage by promoting ROS-dependent nuclear translocation of TFEB to influence consequent autophagy activation and cholesterol transporters. Thus, HY-SDT may be beneficial for atherosclerosis via TFEB regulation to ameliorate lipid overload in atherosclerotic plaques. PMID:28005078

  6. Chronic Alcohol Ingestion in Rats Alters Lung Metabolism, Promotes Lipid Accumulation, and Impairs Alveolar Macrophage Functions

    PubMed Central

    Romero, Freddy; Shah, Dilip; Duong, Michelle; Stafstrom, William; Hoek, Jan B.; Kallen, Caleb B.; Lang, Charles H.

    2014-01-01

    Chronic alcoholism impairs pulmonary immune homeostasis and predisposes to inflammatory lung diseases, including infectious pneumonia and acute respiratory distress syndrome. Although alcoholism has been shown to alter hepatic metabolism, leading to lipid accumulation, hepatitis, and, eventually, cirrhosis, the effects of alcohol on pulmonary metabolism remain largely unknown. Because both the lung and the liver actively engage in lipid synthesis, we hypothesized that chronic alcoholism would impair pulmonary metabolic homeostasis in ways similar to its effects in the liver. We reasoned that perturbations in lipid metabolism might contribute to the impaired pulmonary immunity observed in people who chronically consume alcohol. We studied the metabolic consequences of chronic alcohol consumption in rat lungs in vivo and in alveolar epithelial type II cells and alveolar macrophages (AMs) in vitro. We found that chronic alcohol ingestion significantly alters lung metabolic homeostasis, inhibiting AMP-activated protein kinase, increasing lipid synthesis, and suppressing the expression of genes essential to metabolizing fatty acids (FAs). Furthermore, we show that these metabolic alterations promoted a lung phenotype that is reminiscent of alcoholic fatty liver and is characterized by marked accumulation of triglycerides and free FAs within distal airspaces, AMs, and, to a lesser extent, alveolar epithelial type II cells. We provide evidence that the metabolic alterations in alcohol-exposed rats are mechanistically linked to immune impairments in the alcoholic lung: the elevations in FAs alter AM phenotypes and suppress both phagocytic functions and agonist-induced inflammatory responses. In summary, our work demonstrates that chronic alcohol ingestion impairs lung metabolic homeostasis and promotes pulmonary immune dysfunction. These findings suggest that therapies aimed at reversing alcohol-related metabolic alterations might be effective for preventing and

  7. Essential role of hormone-sensitive lipase (HSL) in the maintenance of lipid storage in Mycobacterium leprae-infected macrophages.

    PubMed

    Tanigawa, Kazunari; Degang, Yang; Kawashima, Akira; Akama, Takeshi; Yoshihara, Aya; Ishido, Yuko; Makino, Masahiko; Ishii, Norihisa; Suzuki, Koichi

    2012-05-01

    Mycobacterium leprae (M. leprae), the causative agent of leprosy, parasitizes within the foamy or enlarged phagosome of macrophages where rich lipids accumulate. Although the mechanisms for lipid accumulation in the phagosome have been clarified, it is still unclear how such large amounts of lipids escape degradation. To further explore underlying mechanisms involved in lipid catabolism in M. leprae-infected host cells, we examined the expression of hormone-sensitive lipase (HSL), a key enzyme in fatty acid mobilization and lipolysis, in human macrophage THP-1 cells. We found that infection by live M. leprae significantly suppressed HSL expression levels. This suppression was not observed with dead M. leprae or latex beads. Macrophage activation by peptidoglycan (PGN), the ligand for toll-like receptor 2 (TLR2), increased HSL expression; however, live M. leprae suppressed this increase. HSL expression was abolished in the slit-skin smear specimens from patients with lepromatous and borderline leprosy. In addition, the recovery of HSL expression was observed in patients who experienced a lepra reaction, which is a cell-mediated, delayed-type hypersensitivity immune response, or in patients who were successfully treated with multi-drug therapy. These results suggest that M. leprae suppresses lipid degradation through inhibition of HSL expression, and that the monitoring of HSL mRNA levels in slit-skin smear specimens may be a useful indicator of patient prognosis.

  8. The impact of membrane lipid composition on macrophage activation in the immune defense against Rhodococcus equi and Pseudomonas aeruginosa.

    PubMed

    Schoeniger, Axel; Adolph, Stephanie; Fuhrmann, Herbert; Schumann, Julia

    2011-01-01

    Nutritional fatty acids are known to have an impact on membrane lipid composition of body cells, including cells of the immune system, thus providing a link between dietary fatty acid uptake, inflammation and immunity. In this study we reveal the significance of macrophage membrane lipid composition on gene expression and cytokine synthesis thereby highlighting signal transduction processes, macrophage activation as well as macrophage defense mechanisms. Using RAW264.7 macrophages as a model system, we identified polyunsaturated fatty acids (PUFA) of both the n-3 and the n-6 family to down-regulate the synthesis of: (i) the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α; (ii) the co-stimulatory molecule CD86; as well as (iii) the antimicrobial polypeptide lysozyme. The action of the fatty acids partially depended on the activation status of the macrophages. It is particularly important to note that the anti-inflammatory action of the PUFA could also be seen in case of infection of RAW264.7 with viable microorganisms of the genera R. equi and P. aeruginosa. In summary, our data provide strong evidence that PUFA from both the n-3 and the n-6 family down-regulate inflammation processes in context of chronic infections caused by persistent pathogens.

  9. Macrophages enhance the radiosensitizing activity of lipid A: A novel role for immune cells in tumor cell radioresponse

    SciTech Connect

    Ridder, Mark de . E-mail: Mark.De.Ridder@vub.ac.be; Verovski, Valeri N.; Darville, Martine I.; Berge, Dirk L. van den; Monsaert, Christinne; Eizirik, Decio L.; Storme, Guy A.

    2004-10-01

    Purpose: This study examines whether activated macrophages may radiosensitize tumor cells through the release of proinflammatory mediators. Methods and materials: RAW 264.7 macrophages were activated by lipid A, and the conditioned medium (CM) was analyzed for the secretion of cytokines and the production of nitric oxide (NO) through inducible nitric oxide synthase (iNOS). EMT-6 tumor cells were exposed to CM and analyzed for hypoxic cell radiosensitivity. The role of nuclear factor (NF)-{kappa}B in the transcriptional activation of iNOS was examined by luciferase reporter gene assay. Results: Clinical immunomodulator lipid A, at a plasma-relevant concentration of 3 {mu}g/mL, stimulated RAW 264.7 macrophages to release NO, tumor necrosis factor (TNF)-{alpha}, and other cytokines. This in turn activated iNOS-mediated NO production in EMT-6 tumor cells and drastically enhanced their radiosensitivity. Radiosensitization was abrogated by the iNOS inhibitor aminoguanidine but not by a neutralizing anti-TNF-{alpha} antibody. The mechanism of iNOS induction was linked to NF-{kappa}B but not to JAK/STAT signaling. Interferon-{gamma} further increased the NO production by macrophages to a level that caused radiosensitization of EMT-6 cells through the bystanding effect of diffused NO. Conclusions: We demonstrate for the first time that activated macrophages may radiosensitize tumor cells through the induction of NO synthesis, which occurs in both tumor and immune cells.

  10. In Vitro Evaluation of the Biological Responses of Canine Macrophages Challenged with PLGA Nanoparticles Containing Monophosphoryl Lipid A

    PubMed Central

    Guldner, Delphine; Hwang, Julianne K.; Cardieri, Maria Clara D.; Eren, Meaghan; Ziaei, Parissa; Norton, M. Grant; Souza, Cleverson D.

    2016-01-01

    Poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) have been considerably studied as a promising biodegradable delivery system to induce effective immune responses and to improve stability, safety, and cost effectiveness of vaccines. The study aimed at evaluating early inflammatory effects and cellular safety of PLGA NPs, co-encapsulating ovalbumin (PLGA/OVA NPs), as a model antigen and the adjuvant monophosphoryl lipid A (PLGA/MPLA NPs) as an adjuvant, on primary canine macrophages. The PLGA NPs constructs were prepared following the emulsion-solvent evaporation technique and further physic-chemically characterized. Peripheral blood mononuclear cells were isolated from canine whole blood by magnetic sorting and further cultured to generate macrophages. The uptake of PLGA NP constructs by macrophages was demonstrated by flow cytometry, transmission electron microscopy and confocal microscopy. Macrophage viability and morphology were evaluated by trypan blue exclusion and light microscopy. Macrophages were immunophenotyped for the expression of MHC-I and MHC-II and gene expression of Interleukin-10 (IL-10), Interleukin-12 (IL-12p40), and tumor necrosis factor alpha (TNF-α) were measured. The results showed that incubation of PLGA NP constructs with macrophages revealed effective early uptake of the PLGA NPs without altering the viability of macrophages. PLGA/OVA/MPLA NPs strongly induced TNF-α and IL-12p40 expression by macrophages as well as increase relative expression of MHC-I but not MHC-II molecules. Taken together, these results indicated that PLGA NPs with addition of MPLA represent a good model, when used as antigen carrier, for further, in vivo, work aiming to evaluate their potential to induce strong, specific, immune responses in dogs. PMID:27835636

  11. CD169+ MACROPHAGES PRESENT LIPID ANTIGENS TO MEDIATE EARLY ACTIVATION OF INVARIANT NKT CELLS IN LYMPH NODES

    PubMed Central

    Barral, Patricia; Polzella, Paolo; Bruckbauer, Andreas; van Rooijen, Nico; Besra, Gurdyal S.; Cerundolo, Vincenzo; Batista, Facundo D.

    2010-01-01

    Invariant NKT (iNKT) cells are involved in host defence against microbial infections. While it is known that iNKT cells recognize glycolipids presented by CD1d, how and where they encounter antigen in vivo remains unclear. We used multi-photon microscopy to visualize the dynamics and activation of iNKT cells in lymph nodes. Following antigen administration, iNKT cells become confined in a CD1d-dependent manner in close proximity to subcapsular sinus CD169+ macrophages. These macrophages retain, internalize and present lipid antigen, and are required for iNKT cell activation, cytokine production and expansion. Thus, CD169+ macrophages can act as bona fide antigen presenting cells controlling early iNKT cell activation and favouring fast initiation of immune responses. PMID:20228797

  12. CD169(+) macrophages present lipid antigens to mediate early activation of iNKT cells in lymph nodes.

    PubMed

    Barral, Patricia; Polzella, Paolo; Bruckbauer, Andreas; van Rooijen, Nico; Besra, Gurdyal S; Cerundolo, Vincenzo; Batista, Facundo D

    2010-04-01

    Invariant natural killer T cells (iNKT cells) are involved in the host defense against microbial infection. Although it is known that iNKT cells recognize glycolipids presented by CD1d, how and where they encounter antigen in vivo remains unclear. Here we used multiphoton microscopy to visualize the dynamics and activation of iNKT cells in lymph nodes. After antigen administration, iNKT cells became confined in a CD1d-dependent manner in close proximity to subcapsular sinus CD169(+) macrophages. These macrophages retained, internalized and presented lipid antigen and were required for iNKT cell activation, cytokine production and population expansion. Thus, CD169(+) macrophages can act as true antigen-presenting cells controlling early iNKT cell activation and favoring the fast initiation of immune responses.

  13. Alpinetin enhances cholesterol efflux and inhibits lipid accumulation in oxidized low-density lipoprotein-loaded human macrophages.

    PubMed

    Jiang, Zhengming; Sang, Haiqiang; Fu, Xin; Liang, Ying; Li, Ling

    2015-01-01

    Alpinetin is a natural flavonoid abundantly present in the ginger family. Here, we investigated the effect of alpinetin on cholesterol efflux and lipid accumulation in oxidized low-density lipoprotein (ox-LDL)-treated THP-1 macrophages and human peripheral blood monocyte-derived macrophages (HMDMs). After exposing THP-1 macrophages to alpinetin, cholesterol efflux was determined by liquid scintillator. The mRNA and protein levels of peroxisome proliferator-activated receptor gamma (PPAR-γ), liver X receptor alpha (LXR-α), ATP-binding cassette transporter A1 (ABCA1), and ABCG1 and scavenger receptor class B member 1 were determined by reverse-transcriptase PCR (RT-PCR) and Western blot analysis, respectively. Alpinetin promoted apolipoprotein A-I- and high-density-lipoprotein-mediated cholesterol efflux and elevated PPAR-γ and LXR-α mRNA and protein expression in a dose-dependent fashion in ox-LDL-treated THP-1 macrophages and HMDMs. Small interfering RNA-mediated silencing of PPAR-γ or LXR-α dose dependently reversed alpinetin-increased cholesterol efflux in THP-1 macrophages, indicating the involvement of PPAR-γ and LXR-α in alpinetin-promoted cholesterol efflux. Alpinetin inhibited ox-LDL-induced lipid accumulation and enhanced the expression of ABCA1 and ABCG1 mRNA and protein, which was reversed by specific knockdown of PPAR-γ or LXR-α. Taken together, our results reveal that alpinetin exhibits positive effects on cholesterol efflux and inhibits ox-LDL-induced lipid accumulation, which might be through PPAR-γ/LXR-α/ABCA1/ABCG1 pathway.

  14. Enhancement of macrophage survival and DNA synthesis by oxidized-low-density-lipoprotein (LDL)-derived lipids and by aggregates of lightly oxidized LDL.

    PubMed Central

    Hamilton, J A; Jessup, W; Brown, A J; Whitty, G

    2001-01-01

    Human atherosclerotic plaque contains a partially characterized range of normal and oxidized lipids formed mainly from free and esterified cholesterol and phospholipids, some of which can be located in macrophage-derived "foam" cells. Oxidation of low-density lipoprotein (LDL) is often considered as an important event leading to subsequent foam-cell development, which may also include enhanced cell survival and/or proliferation. The active component(s) in oxidized LDL (ox.LDL) causing macrophage proliferation is debated. We report here that the lipid component of ox.LDL can promote macrophage survival and DNA synthesis, the latter response showing a synergistic effect in the presence of low concentrations of macrophage colony-stimulating factor. 7-Ketocholesterol showed some stimulation of macrophage DNA synthesis whereas hypochlorite-oxidized (i.e. apolipoprotein B-oxidized) LDL did not. Plaque-derived lipids could enhance macrophage survival. It has not been proven that LDL in lesions is oxidized sufficiently to be the dominant source of sterols in vivo or to be able to induce macrophage growth in vitro or in vivo; it has been suggested that aggregation of modified LDL in vivo is an important step in the deposition of intracellular lipid. We found that aggregation of lightly oxidized LDL potentiated dramatically its ability to stimulate macrophage DNA synthesis, indicating that extensive oxidation of LDL is not required for this response in vitro and perhaps in vivo. PMID:11256965

  15. Culture of mouse peritoneal macrophages with mouse serum induces lipid bodies that associate with the parasitophorous vacuole and decrease their microbicidal capacity against Toxoplasma gondii

    PubMed Central

    Mota, Laura Azeredo Miranda; Roberto, João; Monteiro, Verônica Gomes; Lobato, Caroliny Samary Silva; de Oliveira, Marco Antonio; da Cunha, Maura; D’Ávila, Heloisa; Seabra, Sérgio Henrique; Bozza, Patrícia Torres; DaMatta, Renato Augusto

    2014-01-01

    Lipid bodies [lipid droplets (LBs)] are lipid-rich organelles involved in lipid metabolism, signalling and inflammation. Recent findings suggest a role for LBs in host response to infection; however, the potential functions of this organelle in Toxoplasma gondii infection and how it alters macrophage microbicidal capacity during infection are not well understood. Here, we investigated the role of host LBs in T. gondii infection in mouse peritoneal macrophages in vitro. Macrophages cultured with mouse serum (MS) had higher numbers of LBs than those cultured in foetal bovine serum and can function as a model to study the role of LBs during intracellular pathogen infection. LBs were found in association with the parasitophorous vacuole, suggesting that T. gondii may benefit from this lipid source. Moreover, increased numbers of macrophage LBs correlated with high prostaglandin E2 (PGE2) production and decreased nitric oxide (NO) synthesis. Accordingly, LB-enriched macrophages cultured with MS were less efficient at controlling T. gondii growth. Treatment of macrophages cultured with MS with indomethacin, an inhibitor of PGE2 production, increased the microbicidal capacity against T. gondii. Collectively, these results suggest that culture with MS caused a decrease in microbicidal activity of macrophages against T. gondii by increasing PGE2 while lowering NO production. PMID:25317704

  16. Balancing the effect of corona on therapeutic efficacy and macrophage uptake of lipid nanocapsules.

    PubMed

    Sánchez-Moreno, P; Buzón, P; Boulaiz, H; Peula-García, J M; Ortega-Vinuesa, J L; Luque, I; Salvati, A; Marchal, J A

    2015-08-01

    Several studies have shown the potential of biocompatible lipid nanocapsules as hydrophobic drug delivery systems. Understanding the factors that determine the interactions of these oil-in-water nanoemulsions with cells is a necessary step to guide the design of the most effective formulations. The aim of this study was to probe the ability of two surfactants with a markedly different nature, a non-ionic poloxamer, and a charged phospholipid, to prepare formulations with shells of different composition and different surface properties. Thus we determined their effects on the interaction with biological environments. In particular, we investigated how the shell formulation affected the adsorption of biomolecules from the surrounding biological fluids on the nanocapsule surface (corona formation). A complete physicochemical characterization including an isothermal titration calorimetry (ITC) study revealed that the use of poloxamer led to nanocapsules with a marked reduction in the number of protein-binding sites. Surface hydrophilicity and changes in corona formation strongly correlated to changes in uptake by cancer cells and by macrophages. Our results indicate that the nature and concentration of surfactants in the nanocapsules can be easily manipulated to effectively modulate their surface architecture with the aim of controlling the environmental interactions, thus optimizing functionality for in vivo applications. In particular, addition of surfactants that reduce protein binding can modulate nanoparticle clearance by the immune system, but also screens the desired interactions with cells, leading to lower uptake, thus lower therapeutic efficacy. The two effects need to be balanced in order to obtain successful formulations.

  17. Mitofusin 2 decreases intracellular lipids in macrophages by regulating peroxisome proliferator-activated receptor-γ

    SciTech Connect

    Liu, Chun; Ge, Beihai; He, Chao; Zhang, Yi; Liu, Xiaowen; Liu, Kejian; Qian, Cuiping; Zhang, Yu; Peng, Wenzhong; Guo, Xiaomei

    2014-07-18

    Highlights: • Mfn2 decreases cellular lipid accumulation by activating cholesterol transporters. • PPARγ is involved in the Mfn2-mediated increase of cholesterol transporter expressions. • Inactivation of ERK1/2 and p38 is involved in Mfn2-induced PPARγ expression. - Abstract: Mitofusin 2 (Mfn2) inhibits atherosclerotic plaque formation, but the underlying mechanism remains elusive. This study aims to reveal how Mfn2 functions in the atherosclerosis. Mfn2 expression was found to be significantly reduced in arterial atherosclerotic lesions of both mice and human compared with healthy counterparts. Here, we observed that Mfn2 increased cellular cholesterol transporter expression in macrophages by upregulating peroxisome proliferator-activated receptor-γ, an effect achieved at least partially by inhibiting extracellular signal-regulated kinase1/2 (ERK1/2) and p38 mitogen-activated protein kinases (MAPKs) pathway. These findings provide insights into potential mechanisms of Mfn2-mediated alterations in cholesterol transporter expression, which may have significant implications for the treatment of atherosclerotic heart disease.

  18. The Anti-Inflammatory Effect of Algae-Derived Lipid Extracts on Lipopolysaccharide (LPS)-Stimulated Human THP-1 Macrophages.

    PubMed

    Robertson, Ruairi C; Guihéneuf, Freddy; Bahar, Bojlul; Schmid, Matthias; Stengel, Dagmar B; Fitzgerald, Gerald F; Ross, R Paul; Stanton, Catherine

    2015-08-20

    Algae contain a number of anti-inflammatory bioactive compounds such as omega-3 polyunsaturated fatty acids (n-3 PUFA) and chlorophyll a, hence as dietary ingredients, their extracts may be effective in chronic inflammation-linked metabolic diseases such as cardiovascular disease. In this study, anti-inflammatory potential of lipid extracts from three red seaweeds (Porphyra dioica, Palmaria palmata and Chondrus crispus) and one microalga (Pavlova lutheri) were assessed in lipopolysaccharide (LPS)-stimulated human THP-1 macrophages. Extracts contained 34%-42% total fatty acids as n-3 PUFA and 5%-7% crude extract as pigments, including chlorophyll a, β-carotene and fucoxanthin. Pretreatment of the THP-1 cells with lipid extract from P. palmata inhibited production of the pro-inflammatory cytokines interleukin (IL)-6 (p < 0.05) and IL-8 (p < 0.05) while that of P. lutheri inhibited IL-6 (p < 0.01) production. Quantitative gene expression analysis of a panel of 92 genes linked to inflammatory signaling pathway revealed down-regulation of the expression of 14 pro-inflammatory genes (TLR1, TLR2, TLR4, TLR8, TRAF5, TRAF6, TNFSF18, IL6R, IL23, CCR1, CCR4, CCL17, STAT3, MAP3K1) by the lipid extracts. The lipid extracts effectively inhibited the LPS-induced pro-inflammatory signaling pathways mediated via toll-like receptors, chemokines and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling molecules. These results suggest that lipid extracts from P. lutheri, P. palmata, P. dioica and C. crispus can inhibit LPS-induced inflammatory pathways in human macrophages. Therefore, algal lipid extracts should be further explored as anti-inflammatory ingredients for chronic inflammation-linked metabolic diseases.

  19. Apoptosis inhibitor of macrophage (AIM) diminishes lipid droplet-coating proteins leading to lipolysis in adipocytes

    SciTech Connect

    Iwamura, Yoshihiro; Mori, Mayumi; Nakashima, Katsuhiko; Mikami, Toshiyuki; Murayama, Katsuhisa; Arai, Satoko; Miyazaki, Toru

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer AIM induces lipolysis in a distinct manner from that of hormone-dependent lipolysis. Black-Right-Pointing-Pointer AIM ablates activity of peroxisome proliferator-activated receptor in adipocytes. Black-Right-Pointing-Pointer AIM reduces mRNA levels of lipid-droplet coating proteins leading to lipolysis. -- Abstract: Under fasting conditions, triacylglycerol in adipose tissue undergoes lipolysis to supply fatty acids as energy substrates. Such lipolysis is regulated by hormones, which activate lipases via stimulation of specific signalling cascades. We previously showed that macrophage-derived soluble protein, AIM induces obesity-associated lipolysis, triggering chronic inflammation in fat tissue which causes insulin resistance. However, the mechanism of how AIM mediates lipolysis remains unknown. Here we show that AIM induces lipolysis in a manner distinct from that of hormone-dependent lipolysis, without activation or augmentation of lipases. In vivo and in vitro, AIM did not enhance phosphorylation of hormone-sensitive lipase (HSL) in adipocytes, a hallmark of hormone-dependent lipolysis activation. Similarly, adipose tissue from obese AIM-deficient and wild-type mice showed comparable HSL phosphorylation. Consistent with the suppressive effect of AIM on fatty acid synthase activity, the amount of saturated and unsaturated fatty acids was reduced in adipocytes treated with AIM. This response ablated transcriptional activity of peroxisome proliferator-activated receptor (PPAR{gamma}), leading to diminished gene expression of lipid-droplet coating proteins including fat-specific protein 27 (FSP27) and Perilipin, which are indispensable for triacylglycerol storage in adipocytes. Accordingly, the lipolytic effect of AIM was overcome by a PPAR{gamma}-agonist or forced expression of FSP27, while it was synergized by a PPAR{gamma}-antagonist. Overall, distinct modes of lipolysis appear to take place in different physiological

  20. Receptors for oxidized low-density lipoprotein on elicited mouse peritoneal macrophages can recognize both the modified lipid moieties and the modified protein moieties: Implications with respect to macrophage recognition of apoptotic cells

    PubMed Central

    Bird, David A.; Gillotte, Kristin L.; Hörkkö, Sohvi; Friedman, Peter; Dennis, Edward A.; Witztum, Joseph L.; Steinberg, Daniel

    1999-01-01

    It has been shown previously that the binding of oxidized low-density lipoprotein (OxLDL) to resident mouse peritoneal macrophages can be inhibited (up to 70%) by the apoprotein B (apoB) isolated from OxLDL, suggesting that macrophage recognition of OxLDL is primarily dependent on its modified protein moiety. However, recent experiments have demonstrated that the lipids isolated from OxLDL and reconstituted into a microemulsion can also strongly inhibit uptake of OxLDL (up to 80%). The present studies show that lipid microemulsions prepared from OxLDL bind to thioglycollate-elicited macrophages at 4°C in a saturable fashion and inhibit the binding of intact OxLDL and also of the apoB from OxLDL. Reciprocally, the binding of the OxLDL-lipid microemulsions was strongly inhibited by intact OxLDL. A conjugate of synthetic 1-palmitoyl 2(5-oxovaleroyl) phosphatidylcholine (an oxidation product of 1-palmitoyl 2-arachidonoyl phosphatidylcholine) with serum albumin, shown previously to inhibit macrophage binding of intact OxLDL, also inhibited the binding of both the apoprotein and the lipid microemulsions prepared from OxLDL. Finally, a monoclonal antibody against oxidized phospholipids, one that inhibits binding of intact OxLDL to macrophages, also inhibited the binding of both the resolubilized apoB and the lipid microemulsions prepared from OxLDL. These studies support the conclusions that: (i) at least some of the macrophage receptors for oxidized LDL can recognize both the lipid and the protein moieties; and (ii) oxidized phospholipids, in the lipid phase of the lipoprotein and/or covalently linked to the apoB of OxLDL, likely play a role in that recognition. PMID:10339590

  1. Dissociation of lipopolysaccharide (LPS)-inducible gene expression in murine macrophages pretreated with smooth LPS versus monophosphoryl lipid A.

    PubMed Central

    Henricson, B E; Manthey, C L; Perera, P Y; Hamilton, T A; Vogel, S N

    1993-01-01

    Lipopolysaccharide (LPS) and the nontoxic derivative of lipid A, monophosphoryl lipid A (MPL), were employed to assess the relationship between expression of LPS-inducible inflammatory genes and the induction of tolerance to LPS in murine macrophages. Both LPS and MPL induced expression (as assessed by increased steady-state mRNA levels) of a panel of seven "early" inflammatory genes including the tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, type 2 TNF receptor (TNFR-2), IP-10, D3, D8, and D2 genes (the last four represent LPS-inducible early genes whose functions remain unknown). In addition, LPS and MPL were both capable of inducing tolerance to LPS. The two stimuli differed in the relative concentration required to induce various outcome measures, with LPS being 100- to 1,000-fold more potent on a mass concentration basis. Characterization of the tolerant state identified three distinct categories of responsiveness. Two genes (IP-10 and D8) exhibited strong desensitization in macrophages pretreated with tolerance-inducing concentrations of either LPS or MPL. In macrophages rendered tolerant by pretreatment with LPS or MPL, a second group of inducible mRNAs (TNF-alpha, interleukin-1 beta, and D3) showed moderate suppression of response to secondary stimulation by LPS. The third category of inducible genes (TNFR-2 and D2) showed increased expression in macrophages pretreated with tolerance-inducing concentrations of either LPS or MPL. All of the LPS-inducible genes examined exhibited modest superinduction with less than tolerance-inducing concentrations of either stimulus, suggesting a priming effect of these adjuvants at low concentration. The differential behavior of the members of this panel of endotoxin-responsive genes thus offers insight into molecular events associated with acquisition of transient tolerance to LPS. PMID:8388859

  2. Surfactant, but not the size of solid lipid nanoparticles (SLN) influences viability and cytokine production of macrophages.

    PubMed

    Schöler, N; Olbrich, C; Tabatt, K; Müller, R H; Hahn, H; Liesenfeld, O

    2001-06-19

    After intravenous (i.v.) injection, solid lipid nanoparticles (SLN) interact with mononuclear cells. Murine peritoneal macrophages were incubated with SLN formulations consisting of Dynasan 114 coated with different surfactants. The present study was performed to examine the impact of surfactants, which are important surface defining components of SLN, on viability and cytokine production by macrophages. Cytotoxicity, as assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test, was strongly influenced by the surfactant used being marked with cetylpyridinium chloride- (CPC-) coated SLN at a concentration of 0.001% and further increased at SLN concentrations of 0.01 and 0.1%. All other SLN formulations -- containing Poloxamine 908 (P908), Poloxamer 407 (P407), Poloxamer 188 (P188), Solutol HS15 (HS15), Tween 80 (T80), Lipoid S75 (S75), sodium cholate (SC), or sodium dodecylsulfate (SDS) -- when used at the same concentrations reduced cell viability only slightly. None of the SLN formulations tested induced cytokine production but a concentration-dependent decrease of IL-6 production was observed, which appeared to be associated with cytotoxic effects. IL-12 and TNF-alpha were detected neither in supernatants of macrophages treated with SLN at any concentration nor in those of untreated cells. In contrast to the type of surfactant, the size of SLN was found neither to affect cytotoxicity of SLN nor to result in induction or digression of cytokine production by macrophages. In conclusion, testing the effects of surfactants on SLN on activity of macrophages is a prerequisite prior to in vivo use of SLN.

  3. Exposure to atheroma-relevant 7-oxysterols causes proteomic alterations in cell death, cellular longevity, and lipid metabolism in THP-1 macrophages

    PubMed Central

    Ljunggren, Stefan A.; Karlsson, Helen; Li, Wei; Yuan, Xi-Ming

    2017-01-01

    The 7-oxysterols are recognised as strong enhancers of inflammatory processes in foamy macrophages. Atheroma-relevant 7-oxysterol mixtures induce a mixed type of cell death in macrophages, and trigger cellular oxidative stress responses, which mimic oxidative exposures observed in atherosclerotic lesions. However, the macrophage proteome has not previously been determined in the 7-oxysterol treated cell model. The aim of the present study was to determine the specific effects of an atheroma-relevant 7-oxysterol mixture on human macrophage proteome. Human THP-1 macrophages were exposed to an atheroma-relevant mixture of 7β-hydroxycholesterol and 7-ketocholesterol. Two-dimensional gel electrophoresis and mass spectrometry techniques were used to analyse the alterations in macrophage proteome, which resulted in the identification of 19 proteins with significant differential expression upon oxysterol loading; 8 increased and 11 decreased. The expression patterns of 11 out of 19 identified significant proteins were further confirmed by tandem-mass spectrometry, including further validation of increased histone deacetylase 2 and macrophage scavenger receptor types I and II expressions by western blot analysis. Identified proteins with differential expression in the cell model have been associated with i) signalling imbalance in cell death and cellular longevity; ii) lipid uptake and metabolism in foam cells; and iii) inflammatory proteins. The presented findings highlight a new proteomic platform for further studies into the functional roles of macrophages in atherosclerosis, and present a cell model for future studies to modulate the macrophage proteome by potential anti-atherosclerotic agents. PMID:28350877

  4. Reversible lipid accumulation and associated division arrest of Mycobacterium avium in lipoprotein-induced foamy macrophages may resemble key events during latency and reactivation of tuberculosis.

    PubMed

    Caire-Brändli, Irène; Papadopoulos, Alexia; Malaga, Wladimir; Marais, David; Canaan, Stéphane; Thilo, Lutz; de Chastellier, Chantal

    2014-02-01

    During the dormant phase of tuberculosis, Mycobacterium tuberculosis persists in lung granulomas by residing in foamy macrophages (FM) that contain abundant lipid bodies (LB) in their cytoplasm, allowing bacilli to accumulate lipids as intracytoplasmic lipid inclusions (ILI). An experimental model of FM is presented where bone marrow-derived mouse macrophages are infected with M. avium and exposed to very-low-density lipoprotein (VLDL) as a lipid source. Quantitative analysis of detailed electron microscope observations showed the following results. (i) Macrophages became foamy, and mycobacteria formed ILI, for which host triacylglycerides, rather than cholesterol, was essential. (ii) Lipid transfer occurred via mycobacterium-induced fusion between LB and phagosomes. (iii) Mycobacteria showed a thinned cell wall and became elongated but did not divide. (iv) Upon removal of VLDL, LB and ILI declined within hours, and simultaneous resumption of mycobacterial division restored the number of mycobacteria to the same level as that found in untreated control macrophages. This showed that the presence of ILI resulted in a reversible block of division without causing a change in the mycobacterial replication rate. Fluctuation between ILI either partially or fully extending throughout the mycobacterial cytoplasm was suggestive of bacterial cell cycle events. We propose that VLDL-driven FM constitute a well-defined cellular system in which to study changed metabolic states of intracellular mycobacteria that may relate to persistence and reactivation of tuberculosis.

  5. Macrophage-Derived upd3 Cytokine Causes Impaired Glucose Homeostasis and Reduced Lifespan in Drosophila Fed a Lipid-Rich Diet

    PubMed Central

    Woodcock, Katie J.; Kierdorf, Katrin; Pouchelon, Clara A.; Vivancos, Valérie; Dionne, Marc S.; Geissmann, Frédéric

    2015-01-01

    Summary Long-term consumption of fatty foods is associated with obesity, macrophage activation and inflammation, metabolic imbalance, and a reduced lifespan. We took advantage of Drosophila genetics to investigate the role of macrophages and the pathway(s) that govern their response to dietary stress. Flies fed a lipid-rich diet presented with increased fat storage, systemic activation of JAK-STAT signaling, reduced insulin sensitivity, hyperglycemia, and a shorter lifespan. Drosophila macrophages produced the JAK-STAT-activating cytokine upd3, in a scavenger-receptor (crq) and JNK-dependent manner. Genetic depletion of macrophages or macrophage-specific silencing of upd3 decreased JAK-STAT activation and rescued insulin sensitivity and the lifespan of Drosophila, but did not decrease fat storage. NF-κB signaling made no contribution to the phenotype observed. These results identify an evolutionarily conserved “scavenger receptor-JNK-type 1 cytokine” cassette in macrophages, which controls glucose metabolism and reduces lifespan in Drosophila maintained on a lipid-rich diet via activation of the JAK-STAT pathway. PMID:25601202

  6. TPL-2 Regulates Macrophage Lipid Metabolism and M2 Differentiation to Control TH2-Mediated Immunopathology

    PubMed Central

    Entwistle, Lewis J.; Khoury, Hania; Papoutsopoulou, Stamatia; Mahmood, Radma; Mansour, Nuha R.; Ching-Cheng Huang, Stanley; Pearce, Edward J.; Pedro S. de Carvalho, Luiz; Ley, Steven C.

    2016-01-01

    Persistent TH2 cytokine responses following chronic helminth infections can often lead to the development of tissue pathology and fibrotic scarring. Despite a good understanding of the cellular mechanisms involved in fibrogenesis, there are very few therapeutic options available, highlighting a significant medical need and gap in our understanding of the molecular mechanisms of TH2-mediated immunopathology. In this study, we found that the Map3 kinase, TPL-2 (Map3k8; Cot) regulated TH2-mediated intestinal, hepatic and pulmonary immunopathology following Schistosoma mansoni infection or S. mansoni egg injection. Elevated inflammation, TH2 cell responses and exacerbated fibrosis in Map3k8–/–mice was observed in mice with myeloid cell-specific (LysM) deletion of Map3k8, but not CD4 cell-specific deletion of Map3k8, indicating that TPL-2 regulated myeloid cell function to limit TH2-mediated immunopathology. Transcriptional and metabolic assays of Map3k8–/–M2 macrophages identified that TPL-2 was required for lipolysis, M2 macrophage activation and the expression of a variety of genes involved in immuno-regulatory and pro-fibrotic pathways. Taken together this study identified that TPL-2 regulated TH2-mediated inflammation by supporting lipolysis and M2 macrophage activation, preventing TH2 cell expansion and downstream immunopathology and fibrosis. PMID:27487182

  7. Mice spermatogonial stem cells transplantation induces macrophage migration into the seminiferous epithelium and lipid body formation: high-resolution light microscopy and ultrastructural studies.

    PubMed

    Dias, Felipe F; Chiarini-Garcia, Hélio; Parreira, Gleydes G; Melo, Rossana C N

    2011-12-01

    Transplantation of spermatogonial stem cells (SSCs), the male germline stem cells, in experimental animal models has been successfully used to study mechanisms involved in SSC self-renewal and to restore fertility. However, there are still many challenges associated with understanding the recipient immune response for SSCs use in clinical therapies. Here, we have undertaken a detailed structural study of macrophages elicited by SSCs transplantation in mice using both high-resolution light microscopy (HRLM) and transmission electron microscopy (TEM). We demonstrate that SSCs transplantation elicits a rapid and potent recruitment of macrophages into the seminiferous epithelium (SE). Infiltrating macrophages were derived from differentiation of peritubular monocyte-like cells into typical activated macrophages, which actively migrate through the SE, accumulate in the tubule lumen, and direct phagocytosis of differentiating germ cells and spermatozoa. Quantitative TEM analyses revealed increased formation of lipid bodies (LBs), organelles recognized as intracellular platforms for synthesis of inflammatory mediators and key markers of macrophage activation, within both infiltrating macrophages and Sertoli cells. LBs significantly increased in number and size in parallel to the augmented macrophage migration during different times post-transplantation. Our findings suggest that LBs may be involved with immunomodulatory mechanisms regulating the seminiferous tubule niche after SSC transplantation.

  8. Effects of dietary thia fatty acids on lipid composition, morphology and macrophage function of Atlantic salmon (Salmo salar L.) kidney.

    PubMed

    Gjøen, Tor; Kleveland, Ellen Johanne; Moya-Falcón, Corina; Frøystad, Marianne K; Vegusdal, Anne; Hvattum, Erlend; Berge, Rolf K; Ruyter, Bente

    2007-09-01

    High lipid levels are being used in modern salmonid diets to promote rapid growth; however there is a limiting supply of the traditional fish oils as the fish farming industry expands. One way to utilize the lipid sources better, could be to find ways to stimulate fatty acid (FA) oxidation so that Atlantic salmon use more energy for muscle growth and less for storage in perivisceral adipose tissue. We have previously shown that dietary inclusion of the thia FA tetradecylthioacetic acid (TTA) promoted hepatic beta-oxidation and reduced total body lipid levels. However, dietary TTA also had some negative effects, leading to accumulation of sulfone and sulfoxide metabolites of TTA in the kidney and increasing mortality rates, particularly at low water temperatures. Therefore we also wish to investigate the effects of TTA on kidney function at high and low temperatures, including some immune system parameters. The production of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) immunoreactive material from exogenously added arachidonic acid in isolated head kidney macrophages was affected by both diet and temperature. The phagocytic activity in these cells was reduced by DTA in the 12 degrees C group and there was significantly higher protein degradation in head kidney macrophages at 12 degrees C compared to 5 degrees C in all dietary groups. Interestingly, the incorporation of thia FAs in the kidney was higher at 5 degrees C (0.3% TTA and 0.6% DTA) than at 12 degrees C (0.1% TTA and 0.5% DTA). Additionally, there were lower levels of saturated FAs, while higher levels of polyunsaturated FAs (PUFAs) in the kidney of TTA fed fish at 5 degrees C. We also observed temperature-independent tubular dilatation and a reduction in the density of melanomacrophages of the kidney in salmon fed TTA. Nevertheless, the mRNA expression of some immune-relevant genes in head kidney tissue was not affected by TTA-inclusion in salmon diets. In conclusion, it is clear that 0.6% TTA

  9. Adipocyte Fatty Acid Binding Protein Potentiates Toxic Lipids-Induced Endoplasmic Reticulum Stress in Macrophages via Inhibition of Janus Kinase 2-dependent Autophagy

    PubMed Central

    Hoo, Ruby L. C.; Shu, Lingling; Cheng, Kenneth K. Y.; Wu, Xiaoping; Liao, Boya; Wu, Donghai; Zhou, Zhiguang; Xu, Aimin

    2017-01-01

    Lipotoxicity is implicated in the pathogenesis of obesity-related inflammatory complications by promoting macrophage infiltration and activation. Endoplasmic reticulum (ER) stress and adipocyte fatty acid binding protein (A-FABP) play key roles in obesity and mediate inflammatory activity through similar signaling pathways. However, little is known about their interplay in lipid-induced inflammatory responses. Here, we showed that prolonged treatment of palmitic acid (PA) increased ER stress and expression of A-FABP, which was accompanied by reduced autophagic flux in macrophages. Over-expression of A-FABP impaired PA-induced autophagy associating with enhanced ER stress and pro-inflammatory cytokine production, while genetic ablation or pharmacological inhibition of A-FABP reversed the conditions. PA-induced expression of autophagy-related protein (Atg)7 was attenuated in A-FABP over-expressed macrophages, but was elevated in A-FABP-deficient macrophages. Mechanistically, A-FABP potentiated the effects of PA by inhibition of Janus Kinase (JAK)2 activity, thus diminished PA-induced Atg7 expression contributing to impaired autophagy and further augmentation of ER stress. These findings suggest that A-FABP acts as autophagy inhibitor to instigate toxic lipids-induced ER stress through inhibition of JAK2-dependent autophagy, which in turn triggers inflammatory responses in macrophages. A-FABP-JAK2 axis may represent an important pathological pathway contributing to obesity-related inflammatory diseases. PMID:28094778

  10. Regulatory effects of curcumin on lipid accumulation in monocytes/macrophages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent evidence suggests potential benefits from phytochemicals and micronutrients in protecting against oxidative and lipid-mediated damage, but the molecular mechanisms of these actions are still unclear. Here we investigated whether the dietary polyphenol curcumin can modulate the accumulation of...

  11. Resveratrol prevents the impairment of advanced glycosylation end products (AGE) on macrophage lipid homeostasis by suppressing the receptor for AGE via peroxisome proliferator-activated receptor gamma activation.

    PubMed

    Zhang, Yihua; Luo, Zhidan; Ma, Liqun; Xu, Qiang; Yang, Qihong; Si, Liangyi

    2010-05-01

    Advanced glycosylation end products (AGE) and its receptor (RAGE) axis is involved in the regulation of lipid homeostasis and is critical in the pathogenesis of diabetic atherosclerosis. We investigated the protective role of resveratrol against the AGE-induced impairment on macrophage lipid homeostasis. In THP-1-derived macrophages, RAGE was dose-dependently induced by AGE and played a key role in the AGE-induced cholesterol accumulation. Resveratrol markedly reduced RAGE expression via peroxisome proliferator-activated receptor (PPAR) gamma but not PPARalpha or AMP-activated protein kinase. Importantly, pretreatment with resveratrol significantly ameliorated AGE-induced up-regulation of scavenger receptor-A (SR-A) and down-regulation of ATP-binding cassette (ABC) A1 and ABCG1 and thus effectively prevented the cholesterol accumulation in macrophages as shown by cellular cholesterol analysis and oil red O staining. Moreover, blockade of PPARgamma abolished all these effects of resveratrol. Collectively, our results indicate that resveratrol prevents the impairment of AGE on macrophage lipid homeostasis partially by suppressing RAGE via PPARgamma activation, which might provide new insight into the protective role of resveratrol against diabetic atherosclerosis.

  12. Iron Together with Lipid Downregulates Protein Levels of Ceruloplasmin in Macrophages Associated with Rapid Foam Cell Formation

    PubMed Central

    Hao, Shuangying; Zhang, Meng; Li, Kuanyu

    2016-01-01

    Aim: Iron accumulation in foam cells was previously shown to be involved in atherogenesis. However, the mechanism for iron accumulation was not clarified. Ceruloplasmin (Cp) is an important factor in cellular iron efflux and was found to be downregulated in atherosclerotic plaques in our previous study. The current study is to investigate the role of Cp in atherosclerosis. Methods: We used RAW264.7 cells, a well-accepted cell model of atherosclerosis, which were treated with lipopolysaccharides (LPS), ferric ammonium citrate (FAC) or deferoxamine, and oxidized low density lipoprotein (ox-LDL) to detect the regulation of Cp and its influence in iron efflux and lipid accumulation using biochemical and histological assays. Results: Our results showed that the Cp protein level increased after 200-µM FAC treatment in LPS-activated RAW264.7 cells. Ox-LDL treatment (50 µg/ml) moderately reduced both mRNA and protein levels and ferroxidase activity of Cp (p < 0.05). No significant difference was observed in the expression of ferritin and ferroportin, two important iron-related proteins for iron storage and efflux, respectively, after ox-LDL treatment. However, co-treatment with ox-LDL and FAC drastically reduced the expression of Cp. Accordingly, the ferroxidase activities simultaneously decreased, whereas the protein levels of Ft and Fpn1 significantly increased, indicating further iron accumulation. Moreover, co-treatment with FAC and ox-LDL enhanced the accumulation of cholesterol compared with ox-LDL-only treatment to trigger apoptosis. Conclusion: Our findings suggest that physiological interaction of iron and lipid obstructs iron efflux and accelerates the lipid accumulation in macrophages during foam cell formation, which implicates the role of iron in the pathology of atherosclerosis. PMID:27040361

  13. Nocardia brasiliensis Cell Wall Lipids Modulate Macrophage and Dendritic Responses That Favor Development of Experimental Actinomycetoma in BALB/c Mice

    PubMed Central

    Trevino-Villarreal, J. Humberto; Vera-Cabrera, Lucio; Valero-Guillén, Pedro L.

    2012-01-01

    Nocardia brasiliensis is a Gram-positive facultative intracellular bacterium frequently isolated from human actinomycetoma. However, the pathogenesis of this infection remains unknown. Here, we used a model of bacterial delipidation with benzine to investigate the role of N. brasiliensis cell wall-associated lipids in experimental actinomycetoma. Delipidation of N. brasiliensis with benzine resulted in complete abolition of actinomycetoma without affecting bacterial viability. Chemical analyses revealed that trehalose dimycolate and an unidentified hydrophobic compound were the principal compounds extracted from N. brasiliensis with benzine. By electron microscopy, the extracted lipids were found to be located in the outermost membrane layer of the N. brasiliensis cell wall. They also appeared to confer acid-fastness. In vitro, the extractable lipids from the N. brasiliensis cell wall induced the production of the proinflammatory cytokines interleukin-1β (IL-1β), IL-6, and CCL-2 in macrophages. The N. brasiliensis cell wall extractable lipids inhibited important macrophage microbicidal effects, such as tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) production, phagocytosis, bacterial killing, and major histocompatibility complex class II (MHC-II) expression in response to gamma interferon (IFN-γ). In dendritic cells (DCs), N. brasiliensis cell wall-associated extractable lipids suppressed MHC-II, CD80, and CD40 expression while inducing tumor growth factor β (TGF-β) production. Immunization with delipidated N. brasiliensis induced partial protection preventing actinomycetoma. These findings suggest that N. brasiliensis cell wall-associated lipids are important for actinomycetoma development by inducing inflammation and modulating the responses of macrophages and DCs to N. brasiliensis. PMID:22851755

  14. Drug Targeting to Macrophages With Solid Lipid Nanoparticles Harboring Paromomycin: an In Vitro Evaluation Against L. major and L. tropica.

    PubMed

    Kharaji, Maryam Heidari; Doroud, Delaram; Taheri, Tahereh; Rafati, Sima

    2016-10-01

    Leishmaniasis is a worldwide disease that leads to high mortality and morbidity in human populations. Today, leishmaniasis is managed via drug therapy. The drugs that are already in clinical use are limited to a number of toxic chemical compounds and their parasite drug resistance is increasing. It is therefore essential, in order to circumvent the current difficulties, to design a new anti-leishmanial drug treatment strategy. Besides producing new, active anti-leishmanial entities, another promising strategy could be developing novel delivery systems and formulations of the existing pharmaceutical ingredients to improve drug efficacy. In the present study, paromomycin sulfate (PM), as one of the promising anti-leishmanial drugs, was formulated in solid lipid nanoparticles (SLN), and its in vitro efficacy was investigated against different strains of Leishmania using a MTT test, Parasite-Rescue-Transformation-Assay, SYTO Green staining, and fluorescent microscope imaging. The results show that PM-loaded SLN is significantly more effective than PM in inhibiting parasite propagation (P < 0.05) and that cytotoxicity of PM-SLN formulations is size dependent. According to our results, delivery of the drugs to the macrophages via nanoparticle utilization seems to be an accessible and practical approach.

  15. Effects of copper sulfate-oxidized or myeloperoxidase-modified LDL on lipid loading and programmed cell death in macrophages under hypoxia

    PubMed Central

    Vlaminck, Benoit; Calay, Damien; Genin, Marie; Sauvage, Aude; Ninane, Noelle; Zouaoui Boudjeltia, Karim; Raes, Martine; Michiels, Carine

    2014-01-01

    Atheromatous plaques contain heavily lipid-loaded macrophages that die, hence generating the necrotic core of these plaques. Since plaque instability and rupture is often correlated with a large necrotic core, it is important to understand the mechanisms underlying foam cell death. Furthermore, macrophages within the plaque are associated with hypoxic areas but little is known about the effect of low oxygen partial pressure on macrophage death. The aim of this work was to unravel macrophage death mechanisms induced by oxidized low-density lipoproteins (LDL) both under normoxia and hypoxia. Differentiated macrophages were incubated in the presence of native, copper sulfate-oxidized, or myeloperoxidase-modified LDL. The unfolded protein response, apoptosis, and autophagy were then investigated. The unfolded protein response and autophagy were triggered by myeloperoxidase-modified LDL and, to a larger extent, by copper sulfate-oxidized LDL. Electron microscopy observations showed that oxidized LDL induced excessive autophagy and apoptosis under normoxia, which were less marked under hypoxia. Myeloperoxidase-modified LDL were more toxic and induced a higher level of apoptosis. Hypoxia markedly decreased apoptosis and cell death, as marked by caspase activation. In conclusion, the cell death pathways induced by copper sulfate-oxidized and myeloperoxidase-modified LDL are different and are differentially modulated by hypoxia. PMID:27774474

  16. Toll like receptor 2 and CC chemokine receptor 5 cluster in the lipid raft enhances the susceptibility of Leishmania donovani infection in macrophages.

    PubMed

    Majumdar, Suchandra Bhattacharyya; Bhattacharya, Parna; Bhattacharjee, Surajit; Majumder, Saikat; Banerjee, Sayantan; Majumdar, Subrata

    2014-01-01

    In experimental visceral leishmaniasis the causative obligate protozoan parasite, L. donovani invades and multiplies inside of macrophages, one of the sentries of the mammalian immune system. The initial host-parasite interaction between the Leishmania promastigote and the macrophage takes place at the plasma membrane interface. To trace any possible interaction between Toll-like receptor 2 (TLR2) and CC chemokine receptor 5 (CCR5) during early Leishmania-macrophage interactions, it was observed that the expression of both TLR2 and CCR5 were significantly increased, along with their recruitment to the lipid raft. TLR2 silencing attenuates CCR5 expression and restricts L. donovani infection, indicating a regulatory role of TLR2 and CCR5 during infection. Silencing of CCR5 and TLR2 markedly reduced the number of intracellular parasites in macrophages by host protective cytokine responses, while raft disruption using beta-MCD affected TLR2/CCR5 cross-talk and resulted in a significant reduction in parasite invasion. In vivo RNA interference of TLR2 and CCR5 using shRNA plasmids rendered protection in Leishmania donovani-infected mice. Thus, this study for the first time demonstrates the importance of TLR2/CCR5 crosstalk as a significant determinant of Leishmania donovani entry in host macrophages.

  17. Pulmonary surfactant protein A and surfactant lipids upregulate IRAK-M, a negative regulator of TLR-mediated inflammation in human macrophages

    PubMed Central

    Nguyen, Huy A.; Rajaram, Murugesan V. S.; Meyer, Douglas A.

    2012-01-01

    Alveolar macrophages (AMs) are exposed to frequent challenges from inhaled particulates and microbes and function as a first line of defense with a highly regulated immune response because of their unique biology as prototypic alternatively activated macrophages. Lung collectins, particularly surfactant protein A (SP-A), contribute to this activation state by fine-tuning the macrophage inflammatory response. During short-term (10 min–2 h) exposure, SP-A's regulation of human macrophage responses occurs through decreased activity of kinases required for proinflammatory cytokine production. However, AMs are continuously exposed to surfactant, and the biochemical pathways underlying long-term reduction of proinflammatory cytokine activity are not known. We investigated the molecular mechanism(s) underlying SP-A- and surfactant lipid-mediated suppression of proinflammatory cytokine production in response to Toll-like receptor (TLR) 4 (TLR4) activation over longer time periods. We found that exposure of human macrophages to SP-A for 6–24 h upregulates expression of IL-1 receptor-associated kinase M (IRAK-M), a negative regulator of TLR-mediated NF-κB activation. Exposure to Survanta, a natural bovine lung extract lacking SP-A, also enhances IRAK-M expression, but at lower magnitude and for a shorter duration than SP-A. Surfactant-mediated upregulation of IRAK-M in macrophages suppresses TLR4-mediated TNF-α and IL-6 production in response to LPS, and IRAK-M knockdown by small interfering RNA reverses this suppression. In contrast to TNF-α and IL-6, the surfactant components upregulate LPS-mediated immunoregulatory IL-10 production, an effect reversed by IRAK-M knockdown. In conclusion, these data identify an important signaling regulator in human macrophages that is used by surfactant to control the long-term alveolar inflammatory response, i.e., enhanced IRAK-M activity. PMID:22886503

  18. Soluble Glucan Is Internalized and Trafficked to the Golgi Apparatus in Macrophages via a Clathrin-Mediated, Lipid Raft-Regulated Mechanism

    PubMed Central

    Goldman, Matthew P.; Kalbfleisch, John H.; Williams, David L.

    2012-01-01

    Glucans are natural product carbohydrates that stimulate immunity. Glucans are internalized by the pattern recognition receptor, Dectin-1. Glucans were thought to be trafficked to phagolysosomes, but this is unproven. We examined the internalization and trafficking of soluble glucans in macrophages. Incubation of macrophages with glucan resulted in internalization of Dectin-1 and glucan. Inhibition of clathrin blocked internalization of the Dectin-1/glucan complex. Lipid raft depletion resulted in decreased Dectin levels and glucan uptake. Once internalized, glucans colocalized with early endosomes at 0 to 15 min, with the Golgi apparatus at 15 min to 24 h, and with Dectin-1 immediately (0 h) and again later (15 min-24 h). Glucans did not colocalize with lysosomes at any time interval examined. We conclude that the internalization of Dectin-1/glucan complexes in macrophages is mediated by clathrin and negatively regulated by lipid rafts and/or caveolin-1. Upon internalization, soluble glucans are trafficked via endosomes to the Golgi apparatus, not lysosomes. PMID:22700434

  19. p62-enriched inclusion bodies in macrophages protect against atherosclerosis

    PubMed Central

    Sergin, Ismail; Bhattacharya, Somashubhra; Emanuel, Roy; Esen, Emel; Stokes, Carl J.; Evans, Trent D.; Arif, Batool; Curci, John A.; Razani, Babak

    2016-01-01

    Autophagy is a catabolic cellular mechanism that degrades dysfunctional proteins and organelles. Atherosclerotic plaque formation is enhanced in mice with macrophages that cannot undergo autophagy because of a deficiency of an autophagy component such as ATG5. We showed that exposure of macrophages to atherogenic lipids led to an increase in the abundance of the autophagy chaperone p62, which colocalized with polyubiquitinated proteins in cytoplasmic inclusions. p62 accumulation was increased in ATG5-null macrophages, which had large cytoplasmic ubiquitin-positive p62 inclusions. Aortas from atherosclerotic mice and plaques from human endarterectomy samples showed increased abundance of p62 and polyubiquitinated proteins that co-localized with plaque macrophages, suggesting that p62-enriched protein aggregates were characteristic of atherosclerosis. The formation of the cytoplasmic inclusions depended on p62 because lipid-loaded p62-null macrophages accumulated polyubiquitinated proteins in a diffuse cytoplasmic pattern. The failure of these aggregates to form was associated with increased secretion of IL-1β and enhanced macrophage apoptosis, which depended on the p62 ubiquitin-binding domain and at least partly involved p62-mediated clearance of NLRP3 inflammasomes. Consistent with our in vitro observations, p62-deficient mice formed greater numbers of more complex atherosclerotic plaques, and p62 deficiency further increased atherosclerotic plaque burden in mice with a macrophage-specific ablation of ATG5. Together, these data suggested that sequestration of cytotoxic ubiquitinated proteins by p62 protects against atherogenesis, a condition in which the clearance of protein aggregates is disrupted. PMID:26732762

  20. Macrophage phenotypes in atherosclerosis.

    PubMed

    Colin, Sophie; Chinetti-Gbaguidi, Giulia; Staels, Bart

    2014-11-01

    Initiation and progression of atherosclerosis depend on local inflammation and accumulation of lipids in the vascular wall. Although many cells are involved in the development and progression of atherosclerosis, macrophages are fundamental contributors. For nearly a decade, the phenotypic heterogeneity and plasticity of macrophages has been studied. In atherosclerotic lesions, macrophages are submitted to a large variety of micro-environmental signals, such as oxidized lipids and cytokines, which influence the phenotypic polarization and activation of macrophages resulting in a dynamic plasticity. The macrophage phenotype spectrum is characterized, at the extremes, by the classical M1 macrophages induced by T-helper 1 (Th-1) cytokines and by the alternative M2 macrophages induced by Th-2 cytokines. M2 macrophages can be further classified into M2a, M2b, M2c, and M2d subtypes. More recently, additional plaque-specific macrophage phenotypes have been identified, termed as Mox, Mhem, and M4. Understanding the mechanisms and functional consequences of the phenotypic heterogeneity of macrophages will contribute to determine their potential role in lesion development and plaque stability. Furthermore, research on macrophage plasticity could lead to novel therapeutic approaches to counteract cardiovascular diseases such as atherosclerosis. The present review summarizes our current knowledge on macrophage subsets in atherosclerotic plaques and mechanism behind the modulation of the macrophage phenotype.

  1. Curcuma oil attenuates accelerated atherosclerosis and macrophage foam-cell formation by modulating genes involved in plaque stability, lipid homeostasis and inflammation.

    PubMed

    Singh, Vishal; Rana, Minakshi; Jain, Manish; Singh, Niharika; Naqvi, Arshi; Malasoni, Richa; Dwivedi, Anil Kumar; Dikshit, Madhu; Barthwal, Manoj Kumar

    2015-01-14

    In the present study, the anti-atherosclerotic effect and the underlying mechanism of curcuma oil (C. oil), a lipophilic fraction from turmeric (Curcuma longa L.), was evaluated in a hamster model of accelerated atherosclerosis and in THP-1 macrophages. Male golden Syrian hamsters were subjected to partial carotid ligation (PCL) or FeCl3-induced arterial oxidative injury (Ox-injury) after 1 week of treatment with a high-cholesterol (HC) diet or HC diet plus C. oil (100 and 300 mg/kg, orally). Hamsters fed with the HC diet were analysed at 1, 3 and 5 weeks following carotid injury. The HC diet plus C. oil-fed group was analysed at 5 weeks. In hyperlipidaemic hamsters with PCL or Ox-injury, C. oil (300 mg/kg) reduced elevated plasma and aortic lipid levels, arterial macrophage accumulation, and stenosis when compared with those subjected to arterial injury alone. Similarly, elevated mRNA transcripts of matrix metalloproteinase-2 (MMP-2), MMP-9, cluster of differentiation 45 (CD45), TNF-α, interferon-γ (IFN-γ), IL-1β and IL-6 were reduced in atherosclerotic arteries, while those of transforming growth factor-β (TGF-β) and IL-10 were increased after the C. oil treatment (300 mg/kg). The treatment with C. oil prevented HC diet- and oxidised LDL (OxLDL)-induced lipid accumulation, decreased the mRNA expression of CD68 and CD36, and increased the mRNA expression of PPARα, LXRα, ABCA1 and ABCG1 in both hyperlipidaemic hamster-derived peritoneal and THP-1 macrophages. The administration of C. oil suppressed the mRNA expression of TNF-α, IL-1β, IL-6 and IFN-γ and increased the expression of TGF-β in peritoneal macrophages. In THP-1 macrophages, C. oil supplementation prevented OxLDL-induced production of TNF-α and IL-1β and increased the levels of TGF-β. The present study shows that C. oil attenuates arterial injury-induced accelerated atherosclerosis, inflammation and macrophage foam-cell formation.

  2. LXR Agonism Upregulates the Macrophage ABCA1/Syntrophin Protein Complex That Can Bind ApoA-I and Stabilized ABCA1 Protein, but Complex Loss Does Not Inhibit Lipid Efflux.

    PubMed

    Tamehiro, Norimasa; Park, Min Hi; Hawxhurst, Victoria; Nagpal, Kamalpreet; Adams, Marv E; Zannis, Vassilis I; Golenbock, Douglas T; Fitzgerald, Michael L

    2015-11-24

    Macrophage ABCA1 effluxes lipid and has anti-inflammatory activity. The syntrophins, which are cytoplasmic PDZ protein scaffolding factors, can bind ABCA1 and modulate its activity. However, many of the data assessing the function of the ABCA1-syntrophin interaction are based on overexpression in nonmacrophage cells. To assess endogenous complex function in macrophages, we derived immortalized macrophages from Abca1(+/+) and Abca1(-/-) mice and show their phenotype recapitulates primary macrophages. Abca1(+/+) lines express the CD11B and F4/80 macrophage markers and markedly upregulate cholesterol efflux in response to LXR nuclear hormone agonists. In contrast, immortalized Abca1(-/-) macrophages show no efflux to apoA-I. In response to LPS, Abca1(-/-) macrophages display pro-inflammatory changes, including an increased level of expression of cell surface CD14, and 11-26-fold higher levels of IL-6 and IL-12 mRNA. Given recapitulation of phenotype, we show with these lines that the ABCA1-syntrophin protein complex is upregulated by LXR agonists and can bind apoA-I. Moreover, in immortalized macrophages, combined α1/β2-syntrophin loss modulated ABCA1 cell surface levels and induced pro-inflammatory gene expression. However, loss of all three syntrophin isoforms known to bind ABCA1 did not impair lipid efflux in immortalized or primary macrophages. Thus, the ABCA1-syntrophin protein complex is not essential for ABCA1 macrophage lipid efflux but does directly interact with apoA-I and can modulate the pool of cell surface ABCA1 stabilized by apoA-I.

  3. Atherogenic Lipids and Lipoproteins Trigger CD36-TLR2-Dependent Apoptosis in Macrophages Undergoing Endoplasmic Reticulum Stress

    PubMed Central

    Seimon, Tracie A.; Nadolski, Marissa J.; Liao, Xianghai; Magallon, Jorge; Nguyen, Matthew; Feric, Nicole T.; Koschinsky, Marlys L.; Harkewicz, Richard; Witztum, Joseph L.; Tsimikas, Sotirios; Golenbock, Douglas; Moore, Kathryn J.; Tabas, Ira

    2010-01-01

    SUMMARY Macrophage apoptosis in advanced atheromata, a key process in plaque necrosis, involves the combination of ER stress with other pro-apoptotic stimuli. We show here that oxidized phospholipids, oxidized LDL, saturated fatty acids (SFAs), and lipoprotein(a) trigger apoptosis in ER-stressed macrophages through a mechanism requiring both CD36 and toll-like receptor 2 (TLR2). In vivo, macrophage apoptosis was induced in SFA-fed, ER-stressed wild-type but not Cd36−/− or Tlr2−/− mice. For atherosclerosis, we combined TLR2 deficiency with that of TLR4, which can also promote apoptosis in ER-stressed macrophages. Advanced lesions of fat-fed Ldlr−/− mice transplanted with Tlr4−/−Tlr2−/− bone marrow were markedly protected from macrophage apoptosis and plaque necrosis compared with WT → Ldlr−/− lesions. These findings provide insight into how atherogenic lipoproteins trigger macrophage apoptosis in the setting of ER stress and how TLR activation might promote macrophage apoptosis and plaque necrosis in advanced atherosclerosis. PMID:21035758

  4. Involvement of caspase-3, lipid peroxidation and TNF-alpha in causing apoptosis of macrophages by coordinately expressed Salmonella phenotype under stress conditions.

    PubMed

    Chanana, Vishal; Majumdar, Siddharth; Rishi, Praveen

    2007-03-01

    Invasive Salmonella has been reported to induce apoptosis of macrophages as a part of its infection process, which may allow it to avoid detection by the innate immune system. However, the bacterial components capable of inducing apoptosis, particularly under the environments offered by the host have not been fully identified. Therefore, in the present study, attempts were made to evaluate the apoptotic potential of Salmonella enterica serovar Typhi (S. typhi) outer membrane protein expressed under stress conditions like iron, oxidative and anaerobic simulating the in vivo situations encountered by the pathogen. Analysis of data revealed that a coordinately expressed 69kDa outer membrane protein (OMP) expressed with enhanced intensity under iron, oxidative and anaerobic stress conditions caused apoptotic cell death in 51% of macrophages, whereas OMPs of S. typhi extracted under normal conditions accounted for apoptotic cell death in only 31% of macrophages. A significantly enhanced activity of caspase-3 was observed during macrophage-apoptosis induced by this protein. A significant increase in the extent of lipid peroxidation (levels of oxidant) and decrease in the activities of antioxidants was also observed which correlated with the increased generation of tumor necrosis factor-alpha, interleukine-1alpha and interleukine-6. These results suggest that caspase-3 and tumor necrosis factor-alpha in conjunction with other cytokines may induce apoptotic cell death through the up-regulation of oxidants and down-regulation of antioxidants. These findings may be relevant for the better understanding of the disease pathophysiology and for the future developments of diagnostic and preventive strategies during the host-pathogen interactions.

  5. Molecular chaperones and photoreceptor function

    PubMed Central

    Kosmaoglou, Maria; Schwarz, Nele; Bett, John S.; Cheetham, Michael E.

    2008-01-01

    Molecular chaperones facilitate and regulate protein conformational change within cells. This encompasses many fundamental cellular processes: including the correct folding of nascent chains; protein transport and translocation; signal transduction and protein quality control. Chaperones are, therefore, important in several forms of human disease, including neurodegeneration. Within the retina, the highly specialized photoreceptor cell presents a fascinating paradigm to investigate the specialization of molecular chaperone function and reveals unique chaperone requirements essential to photoreceptor function. Mutations in several photoreceptor proteins lead to protein misfolding mediated neurodegeneration. The best characterized of these are mutations in the molecular light sensor, rhodopsin, which cause autosomal dominant retinitis pigmentosa. Rhodopsin biogenesis is likely to require chaperones, while rhodopsin misfolding involves molecular chaperones in quality control and the cellular response to protein aggregation. Furthermore, the specialization of components of the chaperone machinery to photoreceptor specific roles has been revealed by the identification of mutations in molecular chaperones that cause inherited retinal dysfunction and degeneration. These chaperones are involved in several important cellular pathways and further illuminate the essential and diverse roles of molecular chaperones. PMID:18490186

  6. Inclusion bodies enriched for p62 and polyubiquitinated proteins in macrophages protect against atherosclerosis.

    PubMed

    Sergin, Ismail; Bhattacharya, Somashubhra; Emanuel, Roy; Esen, Emel; Stokes, Carl J; Evans, Trent D; Arif, Batool; Curci, John A; Razani, Babak

    2016-01-05

    Autophagy is a catabolic cellular mechanism that degrades dysfunctional proteins and organelles. Atherosclerotic plaque formation is enhanced in mice with macrophages deficient for the critical autophagy protein ATG5. We showed that exposure of macrophages to lipids that promote atherosclerosis increased the abundance of the autophagy chaperone p62 and that p62 colocalized with polyubiquitinated proteins in cytoplasmic inclusions, which are characterized by insoluble protein aggregates. ATG5-null macrophages developed further p62 accumulation at the sites of large cytoplasmic ubiquitin-positive inclusion bodies. Aortas from atherosclerotic mice and plaques from human endarterectomy samples showed increased abundance of p62 and polyubiquitinated proteins that colocalized with plaque macrophages, suggesting that p62-enriched protein aggregates were characteristic of atherosclerosis. The formation of the cytoplasmic inclusions depended on p62 because lipid-loaded p62-null macrophages accumulated polyubiquitinated proteins in a diffuse cytoplasmic pattern. Lipid-loaded p62-null macrophages also exhibited increased secretion of interleukin-1β (IL-1β) and had an increased tendency to undergo apoptosis, which depended on the p62 ubiquitin-binding domain and at least partly involved p62-mediated clearance of NLRP3 inflammasomes. Consistent with our in vitro observations, p62-deficient mice formed greater numbers of more complex atherosclerotic plaques, and p62 deficiency further increased atherosclerotic plaque burden in mice with a macrophage-specific ablation of ATG5. Together, these data suggested that sequestration of cytotoxic ubiquitinated proteins by p62 protects against atherogenesis, a condition in which the clearance of protein aggregates is disrupted.

  7. Experimental Models of Foamy Macrophages and Approaches for Dissecting the Mechanisms of Lipid Accumulation and Consumption during Dormancy and Reactivation of Tuberculosis

    PubMed Central

    Santucci, Pierre; Bouzid, Feriel; Smichi, Nabil; Poncin, Isabelle; Kremer, Laurent; De Chastellier, Chantal; Drancourt, Michel; Canaan, Stéphane

    2016-01-01

    Despite a slight decline since 2014, tuberculosis (TB) remains the major deadly infectious disease worldwide with about 1.5 million deaths each year and with about one-third of the population being latently infected with Mycobacterium tuberculosis, the etiologic agent of TB. During primo-infection, the recruitment of immune cells leads to the formation of highly organized granulomas. Among the different cells, one outstanding subpopulation is the foamy macrophage (FM), characterized by the abundance of triacylglycerol-rich lipid bodies (LB). M. tuberculosis can reside in FM, where it acquires, from host LB, the neutral lipids which are subsequently processed and stored by the bacilli in the form of intracytosolic lipid inclusions (ILI). Although host LB can be viewed as a reservoir of nutrients for the pathogen during latency, the molecular mechanisms whereby intraphagosomal mycobacteria interact with LB and assimilate the LB-derived lipids are only beginning to be understood. Past studies have emphasized that these physiological processes are critical to the M. tuberculosis infectious-life cycle, for propagation of the infection, establishment of the dormancy state and reactivation of the disease. In recent years, several animal and cellular models have been developed with the aim of dissecting these complex processes and of determining the nature and contribution of their key players. Herein, we review some of the in vitro and in vivo models which allowed to gain significant insight into lipid accumulation and consumption in M. tuberculosis, two important events that are directly linked to pathogenicity, granuloma formation/maintenance and survival of the tubercle bacillus under non-replicative conditions. We also discuss the advantages and limitations of each model, hoping that this will serve as a guide for future investigations dedicated to persistence and innovative therapeutic approaches against TB. PMID:27774438

  8. Molecular chaperones and neuronal proteostasis

    PubMed Central

    Smith, Heather L.; Li, Wenwen; Cheetham, Michael E.

    2015-01-01

    Protein homeostasis (proteostasis) is essential for maintaining the functionality of the proteome. The disruption of proteostasis, due to genetic mutations or an age-related decline, leads to aberrantly folded proteins that typically lose their function. The accumulation of misfolded and aggregated protein is also cytotoxic and has been implicated in the pathogenesis of neurodegenerative diseases. Neurons have developed an intrinsic protein quality control network, of which molecular chaperones are an essential component. Molecular chaperones function to promote efficient folding and target misfolded proteins for refolding or degradation. Increasing molecular chaperone expression can suppress protein aggregation and toxicity in numerous models of neurodegenerative disease; therefore, molecular chaperones are considered exciting therapeutic targets. Furthermore, mutations in several chaperones cause inherited neurodegenerative diseases. In this review, we focus on the importance of molecular chaperones in neurodegenerative diseases, and discuss the advances in understanding their protective mechanisms. PMID:25770416

  9. Chaperone activation by unfolding.

    PubMed

    Foit, Linda; George, Jenny S; Zhang, Bin W; Brooks, Charles L; Bardwell, James C A

    2013-04-02

    Conditionally disordered proteins can alternate between highly ordered and less ordered configurations under physiological conditions. Whereas protein function is often associated with the ordered conformation, for some of these conditionally unstructured proteins, the opposite applies: Their activation is associated with their unfolding. An example is the small periplasmic chaperone HdeA, which is critical for the ability of enteric bacterial pathogens like Escherichia coli to survive passage through extremely acidic environments, such as the human stomach. At neutral pH, HdeA is a chaperone-inactive dimer. On a shift to low pH, however, HdeA monomerizes, partially unfolds, and becomes rapidly active in preventing the aggregation of substrate proteins. By mutating two aspartic acid residues predicted to be responsible for the pH-dependent monomerization of HdeA, we have succeeded in isolating an HdeA mutant that is active at neutral pH. We find this HdeA mutant to be substantially destabilized, partially unfolded, and mainly monomeric at near-neutral pH at a concentration at which it prevents aggregation of a substrate protein. These results provide convincing evidence for direct activation of a protein by partial unfolding.

  10. Carboxypeptidase-M is regulated by lipids and CSFs in macrophages and dendritic cells and expressed selectively in tissue granulomas and foam cells

    PubMed Central

    Tsakiris, Ioannis; Torocsik, Daniel; Gyongyosi, Adrienn; Dozsa, Aniko; Szatmari, Istvan; Szanto, Attila; Soos, Gyorgyike; Nemes, Zoltan; Igali, Laszlo; Marton, Ildiko; Takats, Zoltan; Nagy, Laszlo; Dezso, Balazs

    2012-01-01

    Granulomatous inflammations, characterized by the presence of activated macrophages (MAs) forming epithelioid cell (EPC) clusters, are usually easy to recognize. However, in ambiguous cases the use of a MA marker that expresses selectively in EPCs may be needed. Here, we report that carboxypeptidase-M (CPM), a MA-differentiation marker, is preferentially induced in EPCs of all granuloma types studied, but not in resting MAs. As CPM is not expressed constitutively in MAs, this allows utilization of CPM-immunohistochemistry in diagnostics of minute granuloma detection when dense non-granulomatous MAs are also present. Despite this rule, hardly any detectable CPM was found in advanced/active tubercle caseous disease, albeit in early tuberculosis granuloma, MAs still expressed CPM. Indeed, in vitro both the CPM-protein and -mRNA became downregulated when MAs were infected with live mycobacteria. In vitro, MA-CPM transcript is neither induced remarkably by interferon-γ, known to cause classical MA activation, nor by IL-4, an alternative MA activator. Instead, CPM is selectively expressed in lipid-laden MAs, including the foam cells of atherosclerotic plaques, xanthomatous lesions and lipid pneumonias. By using serum, rich in lipids, and low-density lipoprotein (LDL) or VLDL, CPM upregulation could be reproduced in vitro in monocyte-derived MAs both at transcriptional and protein levels, and the increase is repressed under lipid-depleted conditions. The microarray analyses support the notion that CPM induction correlates with a robust progressive increase in CPM gene expression during monocyte to MA maturation and dendritic cell (DC) differentiation mediated by granulocyte–MA-colony-stimulating factor+IL-4. M-CSF alone also induced CPM. These results collectively indicate that CPM upregulation in MAs is preferentially associated with increased lipid uptake, and exposure to CSF, features of EPCs, also. Therefore, CPM-immunohistochemistry is useful for granuloma and

  11. Revisiting the Interaction between the Chaperone Skp and Lipopolysaccharide

    PubMed Central

    Burmann, Björn M.; Holdbrook, Daniel A.; Callon, Morgane; Bond, Peter J.; Hiller, Sebastian

    2015-01-01

    The bacterial outer membrane comprises two main classes of components, lipids and membrane proteins. These nonsoluble compounds are conveyed across the aqueous periplasm along specific molecular transport routes: the lipid lipopolysaccharide (LPS) is shuttled by the Lpt system, whereas outer membrane proteins (Omps) are transported by chaperones, including the periplasmic Skp. In this study, we revisit the specificity of the chaperone-lipid interaction of Skp and LPS. High-resolution NMR spectroscopy measurements indicate that LPS interacts with Skp nonspecifically, accompanied by destabilization of the Skp trimer and similar to denaturation by the nonnatural detergent lauryldimethylamine-N-oxide (LDAO). Bioinformatic analysis of amino acid conservation, structural analysis of LPS-binding proteins, and MD simulations further confirm the absence of a specific LPS binding site on Skp, making a biological relevance of the interaction unlikely. Instead, our analysis reveals a highly conserved salt-bridge network, which likely has a role for Skp function. PMID:25809264

  12. Glutamine Modulates Macrophage Lipotoxicity

    PubMed Central

    He, Li; Weber, Kassandra J.; Schilling, Joel D.

    2016-01-01

    Obesity and diabetes are associated with excessive inflammation and impaired wound healing. Increasing evidence suggests that macrophage dysfunction is responsible for these inflammatory defects. In the setting of excess nutrients, particularly dietary saturated fatty acids (SFAs), activated macrophages develop lysosome dysfunction, which triggers activation of the NLRP3 inflammasome and cell death. The molecular pathways that connect lipid stress to lysosome pathology are not well understood, but may represent a viable target for therapy. Glutamine uptake is increased in activated macrophages leading us to hypothesize that in the context of excess lipids glutamine metabolism could overwhelm the mitochondria and promote the accumulation of toxic metabolites. To investigate this question we assessed macrophage lipotoxicity in the absence of glutamine using LPS-activated peritoneal macrophages exposed to the SFA palmitate. We found that glutamine deficiency reduced lipid induced lysosome dysfunction, inflammasome activation, and cell death. Under glutamine deficient conditions mTOR activation was decreased and autophagy was enhanced; however, autophagy was dispensable for the rescue phenotype. Rather, glutamine deficiency prevented the suppressive effect of the SFA palmitate on mitochondrial respiration and this phenotype was associated with protection from macrophage cell death. Together, these findings reveal that crosstalk between activation-induced metabolic reprogramming and the nutrient microenvironment can dramatically alter macrophage responses to inflammatory stimuli. PMID:27077881

  13. Maresin-like lipid mediators are produced by leukocytes and platelets and rescue reparative function of diabetes-impaired macrophages.

    PubMed

    Hong, Song; Lu, Yan; Tian, Haibin; Alapure, Bhagwat V; Wang, Quansheng; Bunnell, Bruce A; Laborde, James Monroe

    2014-10-23

    Nonhealing diabetic wounds are associated with impaired macrophage (Mf) function. Leukocytes and platelets (PLT) play crucial roles in wound healing by poorly understood mechanisms. Here we report the identification and characterization of the maresin-like(L) mediators 14,22-dihydroxy-docosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acids, 14S,22-diHDHA (maresin-L1), and 14R,22-diHDHA (maresin-L2) that are produced by leukocytes and PLT and involved in wound healing. We show that 12-lipoxygenase-initiated 14S-hydroxylation or cytochrome P450 catalyzed 14R-hydroxylation and P450-initiated ω(22)-hydroxylation are required for maresin-L biosynthesis. Maresin-L treatment restores reparative functions of diabetic Mfs, suggesting that maresin-Ls act as autocrine/paracrine factors responsible for, at least in part, the reparative functions of leukocytes and PLT in wounds. Additionally, maresin-L ameliorates Mf inflammatory activation and has the potential to suppress the chronic inflammation in diabetic wounds caused by activation of Mfs. These findings provide initial insights into maresin-L biosynthesis and mechanism of action and potentially offer a therapeutic option for better treatment of diabetic wounds.

  14. Activation of GPR55 Receptors Exacerbates oxLDL-Induced Lipid Accumulation and Inflammatory Responses, while Reducing Cholesterol Efflux from Human Macrophages

    PubMed Central

    Lanuti, Mirko; Talamonti, Emanuela; Maccarrone, Mauro; Chiurchiù, Valerio

    2015-01-01

    The G protein-coupled receptor GPR55 has been proposed as a new cannabinoid receptor associated with bone remodelling, nervous system excitability, vascular homeostasis as well as in several pathophysiological conditions including obesity and cancer. However, its physiological role and underlying mechanism remain unclear. In the present work, we demonstrate for the first time its presence in human macrophages and its increased expression in ox-LDL-induced foam cells. In addition, pharmacological activation of GPR55 by its selective agonist O-1602 increased CD36- and SRB-I-mediated lipid accumulation and blocked cholesterol efflux by downregulating ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, as well as enhanced cytokine- and pro-metalloprotease-9 (pro-MMP-9)-induced proinflammatory responses in foam cells. Treatment with cannabidiol, a selective antagonist of GPR55, counteracted these pro-atherogenic and proinflammatory O-1602-mediated effects. Our data suggest that GPR55 could play deleterious role in ox-LDL-induced foam cells and could be a novel pharmacological target to manage atherosclerosis and other related cardiovascular diseases. PMID:25970609

  15. Human granulocyte-macrophage colony-stimulating factor DNA cationic-lipid complexed autologous tumour cell vaccination in the treatment of canine B-cell multicentric lymphoma.

    PubMed

    Turek, M M; Thamm, D H; Mitzey, A; Kurzman, I D; Huelsmeyer, M K; Dubielzig, R R; Vail, D M

    2007-12-01

    This study describes the development of an human granulocyte-macrophage colony-stimulating factor DNA cationic-lipid complexed autologous tumour cell vaccine (hGM-CSF CLDC ATCV) and its implementation, following a chemotherapy treatment protocol, in a randomized, placebo-controlled, double-blinded clinical trial in pet dogs with naturally occurring lymphoma. We hypothesized that the use of this vaccine would result in an antitumour immune response leading to improved first remission duration and overall survival in dogs with B-cell lymphoma when compared with chemotherapy alone. Immune stimulation generated by hGM-CSF CLDC ATCV was assessed by means of surrogate in vivo analysis (delayed-type hypersensitivity [DTH]) as well as an ex vivo cellular assay (lymphocyte proliferation assay). The vaccine approach considered in the current report did not result in clinically improved outcomes. A small measure of immunomodulation was documented by DTH and several modifications to the approach are suggested. This report illustrates the feasibility of clinical trials with vaccine strategies using companion animals with non-Hodgkin's lymphoma.

  16. An ssDNA aptamer against mannose-capped lipoarabinomannan enhances anti-tuberculosis activity of macrophages through downregulation of lipid-sensing nuclear receptor peroxisome proliferator-activated receptor γ expression.

    PubMed

    Pan, Qin; Yan, Jiamin; Liu, Qi; Yuan, Chunhui; Zhang, Xiao-Lian

    2017-02-16

    Mannose-capped lipoarabinomannan (ManLAM) is an immunomodulatory epitope of Mycobacterium tuberculosis (Mtb). We previously generated an aptamer (ZXL1) that specifically binds to ManLAM from the virulent Mtb H37Rv strain and reported that ZXL1 functioned as an antagonist, inhibiting the ManLAM-induced immunosuppression of dendritic cells (DCs). In the present study, we found that ZXL1 inhibited Mtb entry into murine macrophages. ZXL1 enhanced IL-1β and IL-12 mRNA expression and cytokine production in ManLAM-treated macrophages but decreased IL-10 production. Inducible nitric oxide synthase (iNOS) expression in the macrophages was upregulated in the presence of ZXL1 after stimulation with ManLAM. ZXL1 also inhibited the expression of the lipid-sensing nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ). These results suggest that ZXL1 promotes anti-tuberculosis activity through the downregulation of PPAR γ expression, which may contribute to M1 macrophage polarization and Mtb killing by macrophages.

  17. Histone chaperone networks shaping chromatin function.

    PubMed

    Hammond, Colin M; Strømme, Caroline B; Huang, Hongda; Patel, Dinshaw J; Groth, Anja

    2017-03-01

    The association of histones with specific chaperone complexes is important for their folding, oligomerization, post-translational modification, nuclear import, stability, assembly and genomic localization. In this way, the chaperoning of soluble histones is a key determinant of histone availability and fate, which affects all chromosomal processes, including gene expression, chromosome segregation and genome replication and repair. Here, we review the distinct structural and functional properties of the expanding network of histone chaperones. We emphasize how chaperones cooperate in the histone chaperone network and via co-chaperone complexes to match histone supply with demand, thereby promoting proper nucleosome assembly and maintaining epigenetic information by recycling modified histones evicted from chromatin.

  18. Gaucher iPSC-derived macrophages produce elevated levels of inflammatory mediators and serve as a new platform for therapeutic development

    PubMed Central

    Panicker, Leelamma M.; Miller, Diana; Awad, Ola; Bose, Vivek; Lun, Yu; Park, Tea Soon; Zambidis, Elias T.; Sgambato, Judi A.; Feldman, Ricardo A.

    2014-01-01

    Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid beta-glucocerebrosidase (GBA) gene. The hallmark of GD is the presence of lipid-laden Gaucher macrophages, which infiltrate bone marrow and other organs. These pathological macrophages are believed to be the source of elevated levels of inflammatory mediators present in the serum of GD patients. The alteration in the immune environment caused by GD is believed to play a role in the increased risk of developing multiple myeloma and other malignancies in GD patients. To determine directly whether Gaucher macrophages are abnormally activated and if their functional defects can be reversed by pharmacological intervention, we generated GD macrophages by directed differentiation of human iPS cells (hiPSC) derived from patients with types 1, 2, and 3 GD. GD hiPSC-derived macrophages expressed higher levels of TNF alpha, IL-6, and IL-1beta than control cells, and this phenotype was exacerbated by treatment with LPS. In addition, GD hiPSC macrophages exhibited a striking delay in clearance of phagocytosed red blood cells, recapitulating the presence of RBC remnants in Gaucher macrophages from bone marrow aspirates. Incubation of GD hiPSC macrophages with recombinant glucocerebrosidase, or with the chaperones isofagomine and ambroxol, corrected the abnormal phenotypes of GD macrophages to an extent that reflected their known clinical efficacies. We conclude that Gaucher macrophages are the likely source of the elevated levels of inflammatory mediators in the serum of GD patients, and that GD hiPSC are valuable new tools for studying disease mechanisms and drug discovery. PMID:24801745

  19. Involvement of TLR6 in the induction of COX-2, PGE2 and IL-10 in macrophages by lipids from virulent S2P and attenuated R1A Babesia bovis strains.

    PubMed

    Gimenez, G; Belaunzarán, M L; Magalhães, K G; Poncini, C V; Lammel, E M; González Cappa, S M; Bozza, P T; Isola, E L D

    2016-06-15

    Toll like receptors (TLRs) are involved in the modulation of diverse host genes expression through a complex network of signalling events that allow for an appropriate response to a microbial pathogen. In the present work we used TLR6KO mice in order to study the role of TLR6 in the immune discrimination of lipids from two Babesia bovis strains, attenuated R1A (LA) and virulent S2P (LV), and the consequent macrophage activation. We demonstrated that TLR6 is required for lipid body induction in murine peritoneal macrophages by both LA and LV. Interestingly, as regards IL-10 and COX-2/PGE2 pathway induction by LA and LV, we observed differences in the biological effects produced by these lipid extracts. Our results indicate a role of TLR6 in the down-modulation of these immunoregulators only in the case of LA, whereas this receptor was not implicated in pro-inflammatory TNFα, IL-6 and KC release induced by LA. Remarkably, LV did not exert the down-modulatory effect observed for LA, supporting the notion that LA and LV possess different lipid composition that could correlate with the polar pathogenic effect of both B. bovis strains.

  20. Macrophages in Synovial Inflammation

    PubMed Central

    Kennedy, Aisling; Fearon, Ursula; Veale, Douglas J.; Godson, Catherine

    2011-01-01

    Synovial macrophages are one of the resident cell types in synovial tissue and while they remain relatively quiescent in the healthy joint, they become activated in the inflamed joint and, along with infiltrating monocytes/macrophages, regulate secretion of pro-inflammatory cytokines and enzymes involved in driving the inflammatory response and joint destruction. Synovial macrophages are positioned throughout the sub-lining layer and lining layer at the cartilage–pannus junction and mediate articular destruction. Sub-lining macrophages are now also considered as the most reliable biomarker for disease severity and response to therapy in rheumatoid arthritis (RA). There is a growing understanding of the molecular drivers of inflammation and an appreciation that the resolution of inflammation is an active process rather than a passive return to homeostasis, and this has implications for our understanding of the role of macrophages in inflammation. Macrophage phenotype determines the cytokine secretion profile and tissue destruction capabilities of these cells. Whereas inflammatory synovial macrophages have not yet been classified into one phenotype or another it is widely known that TNFα and IL-l, characteristically released by M1 macrophages, are abundant in RA while IL-10 activity, characteristic of M2 macrophages, is somewhat diminished. Here we will briefly review our current understanding of macrophages and macrophage polarization in RA as well as the elements implicated in controlling polarization, such as cytokines and transcription factors like NFκB, IRFs and NR4A, and pro-resolving factors, such as LXA4 and other lipid mediators which may promote a non-inflammatory, pro-resolving phenotype, and may represent a novel therapeutic paradigm. PMID:22566842

  1. Lipid antigen presentation through CD1d pathway in mouse lung epithelial cells, macrophages and dendritic cells and its suppression by poly-dispersed single-walled carbon nanotubes.

    PubMed

    Rizvi, Zaigham Abbas; Puri, Niti; Saxena, Rajiv K

    2015-09-01

    Effect of poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) was examined on lipid antigen presentation through CD1d pathway on three cell lines, LA4, MHS, and JAWSII used as prototype antigen presenting cells (APCs). CD1d molecule was expressed on 80-90% MHS (prototype macrophages) and JAWSII (prototype dendritic cells) cells whereas <5% LA4 cells (lung epithelial cells, non-classical APCs) expressed CD1d. Treatment with AF-SWCNTs but not with pristine SWCNTs resulted in a significant decline in the level of CD1d mRNA as well as mRNA levels of some other intracellular proteins involved in lipid antigen presentation pathway (MTP, ApoE, prosaposin, SR-BI and LDLr). Lipid antigen presentation was assessed by first incubating the cells with a prototype lipid antigen (α-Glactosylceramide or αGC) and then staining with L363 monoclonal antibody that detects αGC bound to CD1d molecule. While 100% MHS and JAWSII cells presented αGC, only 20% LA4 cells presented the CD1d antigen. Treatment with AF-SWCNTs resulted in a 30-40% decrease in αGC antigen presentation in all three cell lines. These results show that AF-SWCNT treatment down regulated the lipid antigen presentation pathway in all three cell lines and significantly lowered the ability of these cell lines to present αGC antigen.

  2. Kaempferol suppresses lipid accumulation in macrophages through the downregulation of cluster of differentiation 36 and the upregulation of scavenger receptor class B type I and ATP-binding cassette transporters A1 and G1.

    PubMed

    Li, Xiu-Ying; Kong, Ling-Xi; Li, Juan; He, Hai-Xia; Zhou, Yuan-Da

    2013-02-01

    The accumulation of foam cells in atherosclerotic lesions is a hallmark of early-stage atherosclerosis. Kaempferol has been shown to inhibit oxidized low-density lipoprotein (oxLDL) uptake by macrophages; however, the underlying molecular mechanisms are not yet fully investigated. In this study, we shown that treatment with kaempferol markedly suppresses oxLDL-induced macrophage foam cell formation, which occurs due to a decrease in lipid accumulation and an increase in cholesterol efflux from THP-1-derived macrophages. Additionally, the kaempferol treatment of macrophages led to the downregulation of cluster of differentiation 36 (CD36) protein levels, the upregulation of ATP-binding cassette (ABC) transporter A1 (ABCA1), scavenger receptor class B type I (SR-BI) and ABCG1 protein levels, while no effects on scavenger receptor A (SR-A) expression were observed. Kaempferol had similar effects on the mRNA and protein expression of ABCA1, SR-BI, SR-A, CD36 and ABCG1. The reduced CD36 expression following kaempferol treatment involved the inhibition of c-Jun-activator protein-1 (AP-1) nuclear translocation. The inhibition of AP-1 using the inhibitor, SP600125, confirmed this involvement, as the AP-1 inhibition significantly augmented the kaempferol-induced reduction in CD36 expression. Accordingly, the kaempferol-mediated suppression of lipid accumulation in macrophages was also augmented by SP600125. The increased expression of ABCA1, SR-BI and ABCG1 following kaempferol treatment was accompanied by the enhanced protein expression of heme oxygenase-1 (HO-1). This increase was reversed following the knockdown of the HO-1 gene using small hairpin RNA (shRNA). Moreover, the kaempferol-mediated attenuation of lipid accumulation and the promotion of cholesterol efflux was also inhibited by HO-1 shRNA. In conclusion, the c-Jun-AP‑1-dependent downregulation of CD36 and the HO-1-dependent upregulation of ABCG1, SR-BI and ABCA1 may mediate the beneficial effects of

  3. Pro-oxidant activity of indicaxanthin from Opuntia ficus indica modulates arachidonate metabolism and prostaglandin synthesis through lipid peroxide production in LPS-stimulated RAW 264.7 macrophages

    PubMed Central

    Allegra, M.; D’Acquisto, F.; Tesoriere, L.; Attanzio, A.; Livrea, M.A.

    2014-01-01

    Macrophages come across active prostaglandin (PG) metabolism during inflammation, shunting early production of pro-inflammatory towards anti-inflammatory mediators terminating the process. This work for the first time provides evidence that a phytochemical may modulate the arachidonate (AA) metabolism in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, promoting the ultimate formation of anti-inflammatory cyclopentenone 15deoxy-PGJ2. Added 1 h before LPS, indicaxanthin from Opuntia Ficus Indica prevented activation of nuclear factor-κB (NF-κB) and over-expression of PGE2 synthase-1 (mPGES-1), but up-regulated cyclo-oxygenase-2 (COX-2) and PGD2 synthase (H-PGDS), with final production of the anti-inflammatory cyclopentenone. The effects were positively related with concentration between 50 and 100 µM. Indicaxanthin did not have any effect in the absence of LPS. A kinetic study investigating the redox status of LPS-stimulated macrophages between 0.5 and 12 h, either in the absence or in the presence of 50–100 µM indicaxanthin, revealed a differential control of ROS production, with early (0.5–3 h) modest inhibition, followed by a progressive (3–12 h) concentration-dependent enhancement over the level induced by LPS alone. In addition, indicaxanthin caused early (0.5–3 h) concentration-dependent elevation of conjugated diene lipid hydroperoxides, and production of hydroxynonenal-protein adducts, over the amount induced by LPS. In LPS-stimulated macrophages indicaxanthin did not affect PG metabolism when co-incubated with either an inhibitor of NADPH oxidase or vitamin E. It is concluded that LPS-induced pro-oxidant activity of indicaxanthin at the membrane level allows formation of signaling intermediates whose accumulation modulates PG biosynthetic pathway in inflamed macrophages. PMID:25180166

  4. Pro-oxidant activity of indicaxanthin from Opuntia ficus indica modulates arachidonate metabolism and prostaglandin synthesis through lipid peroxide production in LPS-stimulated RAW 264.7 macrophages.

    PubMed

    Allegra, M; D'Acquisto, F; Tesoriere, L; Attanzio, A; Livrea, M A

    2014-01-01

    Macrophages come across active prostaglandin (PG) metabolism during inflammation, shunting early production of pro-inflammatory towards anti-inflammatory mediators terminating the process. This work for the first time provides evidence that a phytochemical may modulate the arachidonate (AA) metabolism in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, promoting the ultimate formation of anti-inflammatory cyclopentenone 15deoxy-PGJ2. Added 1 h before LPS, indicaxanthin from Opuntia Ficus Indica prevented activation of nuclear factor-κB (NF-κB) and over-expression of PGE2 synthase-1 (mPGES-1), but up-regulated cyclo-oxygenase-2 (COX-2) and PGD2 synthase (H-PGDS), with final production of the anti-inflammatory cyclopentenone. The effects were positively related with concentration between 50 and 100 µM. Indicaxanthin did not have any effect in the absence of LPS. A kinetic study investigating the redox status of LPS-stimulated macrophages between 0.5 and 12 h, either in the absence or in the presence of 50-100 µM indicaxanthin, revealed a differential control of ROS production, with early (0.5-3 h) modest inhibition, followed by a progressive (3-12 h) concentration-dependent enhancement over the level induced by LPS alone. In addition, indicaxanthin caused early (0.5-3 h) concentration-dependent elevation of conjugated diene lipid hydroperoxides, and production of hydroxynonenal-protein adducts, over the amount induced by LPS. In LPS-stimulated macrophages indicaxanthin did not affect PG metabolism when co-incubated with either an inhibitor of NADPH oxidase or vitamin E. It is concluded that LPS-induced pro-oxidant activity of indicaxanthin at the membrane level allows formation of signaling intermediates whose accumulation modulates PG biosynthetic pathway in inflamed macrophages.

  5. Pharmacological Targeting of the Hsp70 Chaperone

    PubMed Central

    Patury, Srikanth; Miyata, Yoshinari; Gestwicki, Jason E.

    2009-01-01

    The molecular chaperone, heat shock protein 70 (Hsp70), acts at multiple steps in a protein’s life cycle, including during the processes of folding, trafficking, remodeling and degradation. To accomplish these various tasks, the activity of Hsp70 is shaped by a host of co-chaperones, which bind to the core chaperone and influence its functions. Genetic studies have strongly linked Hsp70 and its co-chaperones to numerous diseases, including cancer, neurodegeneration and microbial pathogenesis, yet the potential of this chaperone as a therapeutic target remains largely underexplored. Here, we review the current state of Hsp70 as a drug target, with a special emphasis on the important challenges and opportunities imposed by its co-chaperones, protein-protein interactions and allostery. PMID:19860737

  6. Systems biology of molecular chaperone networks.

    PubMed

    Csermely, Péter; Korcsmáros, Tamás; Kovács, István A; Szalay, Máté S; Soti, Csaba

    2008-01-01

    Molecular chaperones are not only fascinating molecular machines that help the folding, refolding, activation or assembly of other proteins, but also have a number of functions. These functions can be understood only by considering the emergent properties of cellular networks--and that of chaperones as special network constituents. As a notable example for the network-related roles of chaperones they may act as genetic buffers stabilizing the phenotype of various cells and organisms, and may serve as potential regulators of evolvability. Why are chaperones special in the context of cellular networks? Chaperones: (1) have weak links, i.e. low affinity, transient interactions with most of their partners; (2) connect hubs, i.e. act as 'masterminds' of the cell being close to several centre proteins with a lot of neighbours; and (3) are in the overlaps of network modules, which confers upon them a special regulatory role. Importantly, chaperones may uncouple or even quarantine modules of protein-protein interaction networks, signalling networks, genetic regulatory networks and membrane organelle networks during stress, which gives an additional chaperone-mediated protection for the cell at the network-level. Moreover, chaperones are essential to rebuild inter-modular contacts after stress by their low affinity, 'quasi-random' sampling of the potential interaction partners in different cellular modules. This opens the way to the chaperone-regulated modular evolution of cellular networks, and helps us to design novel therapeutic and anti-ageing strategies.

  7. Macrophages in atherosclerosis: a dynamic balance

    PubMed Central

    Moore, Kathryn; Sheedy, Frederick; Fisher, Edward

    2015-01-01

    Preface Atherosclerosis is a chronic inflammatory disease arising from an imbalance in lipid metabolism and a maladaptive immune response driven by the accumulation of cholesterol-laden macrophages in the artery wall. Through the analysis of animal models of atherosclerosis progression and regression, there is a growing understanding that the balance of macrophages in the plaque is dynamic, with both macrophage numbers and an inflammatory phenotype influencing plaque fate. Here we summarize recently identified pro- and anti-inflammatory pathways linking lipid and inflammation biology with the retention of macrophages in plaques, as well as factors with the potential to promote their egress from these sites. PMID:23995626

  8. Collagenase Production by Endotoxin-Activated Macrophages

    PubMed Central

    Wahl, Larry M.; Wahl, Sharon M.; Mergenhagen, Stephan E.; Martin, George R.

    1974-01-01

    Peritoneal exudate macrophages, when exposed to bacterial lipopolysaccharide in culture, were found to produce collagenase (EC 3.4.24.3). This enzyme was not detected in extracts of the macrophages or in media from nonstimulated macrophage cultures. Lipidcontaining fractions of the lipopolysaccharide, including a glycolipid from the rough mutant of Salmonella minnesota (R595) and lipid A, were potent stimulators of collagenase production. The lipid-free polysaccharide fraction had no effect. Cycloheximide prevented the production of collagenase by endotoxin-treated macrophages, suggesting that it was newly synthesized. Images PMID:4372628

  9. Lipid mediators in innate immunity against tuberculosis: opposing roles of PGE2 and LXA4 in the induction of macrophage death.

    PubMed

    Chen, Minjian; Divangahi, Maziar; Gan, Huixian; Shin, Daniel S J; Hong, Song; Lee, David M; Serhan, Charles N; Behar, Samuel M; Remold, Heinz G

    2008-11-24

    Virulent Mycobacterium tuberculosis (Mtb) induces a maladaptive cytolytic death modality, necrosis, which is advantageous for the pathogen. We report that necrosis of macrophages infected with the virulent Mtb strains H37Rv and Erdmann depends on predominant LXA(4) production that is part of the antiinflammatory and inflammation-resolving action induced by Mtb. Infection of macrophages with the avirulent H37Ra triggers production of high levels of the prostanoid PGE(2), which promotes protection against mitochondrial inner membrane perturbation and necrosis. In contrast to H37Ra infection, PGE(2) production is significantly reduced in H37Rv-infected macrophages. PGE(2) acts by engaging the PGE(2) receptor EP2, which induces cyclic AMP production and protein kinase A activation. To verify a role for PGE(2) in control of bacterial growth, we show that infection of prostaglandin E synthase (PGES)(-/-) macrophages in vitro with H37Rv resulted in significantly higher bacterial burden compared with wild-type macrophages. More importantly, PGES(-/-) mice harbor significantly higher Mtb lung burden 5 wk after low-dose aerosol infection with virulent Mtb. These in vitro and in vivo data indicate that PGE(2) plays a critical role in inhibition of Mtb replication.

  10. Macrophage Polarization.

    PubMed

    Murray, Peter J

    2017-02-10

    Macrophage polarization refers to how macrophages have been activated at a given point in space and time. Polarization is not fixed, as macrophages are sufficiently plastic to integrate multiple signals, such as those from microbes, damaged tissues, and the normal tissue environment. Three broad pathways control polarization: epigenetic and cell survival pathways that prolong or shorten macrophage development and viability, the tissue microenvironment, and extrinsic factors, such as microbial products and cytokines released in inflammation. A plethora of advances have provided a framework for rationally purifying, describing, and manipulating macrophage polarization. Here, I assess the current state of knowledge about macrophage polarization and enumerate the major questions about how activated macrophages regulate the physiology of normal and damaged tissues.

  11. RNA Chaperones Step Out of Hfq's Shadow.

    PubMed

    Attaiech, Laetitia; Glover, J N Mark; Charpentier, Xavier

    2017-04-01

    The stability and function of regulatory small RNAs (sRNAs) often require a specialized RNA-binding protein called an RNA chaperone. Recent findings show that proteins containing a ProQ/FinO domain constitute a new class of RNA chaperones that could play key roles in post-transcriptional gene regulation throughout bacterial species.

  12. Review: Beta-thalassemia and molecular chaperones.

    PubMed

    Sumera, Afshan; Radhakrishnan, Ammu; Baba, Abdul Aziz; George, Elizabeth

    2015-04-01

    Thalassemia is known as a diverse single gene disorder, which is prevalent worldwide. The molecular chaperones are set of proteins that help in two important processes while protein synthesis and degradation include folding or unfolding and assembly or disassembly, thereby helping in cell homeostasis. This review recaps current knowledge regarding the role of molecular chaperones in thalassemia, with a focus on beta thalassemia.

  13. Chaperone addiction of toxin–antitoxin systems

    PubMed Central

    Bordes, Patricia; Sala, Ambre Julie; Ayala, Sara; Texier, Pauline; Slama, Nawel; Cirinesi, Anne-Marie; Guillet, Valérie; Mourey, Lionel; Genevaux, Pierre

    2016-01-01

    Bacterial toxin–antitoxin (TA) systems, in which a labile antitoxin binds and inhibits the toxin, can promote adaptation and persistence by modulating bacterial growth in response to stress. Some atypical TA systems, known as tripartite toxin–antitoxin–chaperone (TAC) modules, include a molecular chaperone that facilitates folding and protects the antitoxin from degradation. Here we use a TAC module from Mycobacterium tuberculosis as a model to investigate the molecular mechanisms by which classical TAs can become ‘chaperone-addicted'. The chaperone specifically binds the antitoxin at a short carboxy-terminal sequence (chaperone addiction sequence, ChAD) that is not present in chaperone-independent antitoxins. In the absence of chaperone, the ChAD sequence destabilizes the antitoxin, thus preventing toxin inhibition. Chaperone–ChAD pairs can be transferred to classical TA systems or to unrelated proteins and render them chaperone-dependent. This mechanism might be used to optimize the expression and folding of heterologous proteins in bacterial hosts for biotechnological or medical purposes. PMID:27827369

  14. Artemin as an efficient molecular chaperone.

    PubMed

    Shahangian, S Shirin; Rasti, Behnam; Sajedi, Reza H; Khodarahmi, Reza; Taghdir, Majid; Ranjbar, Bijan

    2011-12-01

    Artemin is an abundant thermostable protein in Artemia encysted embryos under stress. It is considered as a stress protein, as its highly regulated expression is associated with stress resistance in this crustacea. In the present study, artemin has been shown to be a potent molecular chaperone with high efficacy. Artemin is capable of inhibiting the chemical aggregation of proteins such as carbonic anhydrase (CA) and horseradish peroxidase (HRP) at unique molar ratios of chaperone to substrates (1:40 and 1:26 for CA and HRP, respectively). Furthermore, it can also enhance refolding yield of these substrates by nearly 50%. The refolding promotion of CA is checked and verified through a sensitive fluorimetric technique. Based on these experiments, artemin showed higher chaperone activity than other chaperones. The evaluation of artemin surface using ANS showed it to be highly hydrophobic, probably resulting in its high efficacy. These results suggest that artemin can be considered a novel low molecular weight chaperone.

  15. Gene expression in macrophage-rich human atherosclerotic lesions. 15-lipoxygenase and acetyl low density lipoprotein receptor messenger RNA colocalize with oxidation specific lipid-protein adducts.

    PubMed Central

    Ylä-Herttuala, S; Rosenfeld, M E; Parthasarathy, S; Sigal, E; Särkioja, T; Witztum, J L; Steinberg, D

    1991-01-01

    Oxidatively modified low density lipoprotein (LDL) exhibits several potentially atherogenic properties, and inhibition of LDL oxidation in rabbits decreases the rate of the development of atherosclerotic lesions. In vitro studies have suggested that cellular lipoxygenases may be involved in LDL oxidation, and we have shown previously that 15-lipoxygenase and oxidized LDL are present in rabbit atherosclerotic lesions. We now report that epitopes of oxidized LDL are also found in macrophage-rich areas of human fatty streaks as well as in more advanced human atherosclerotic lesions. Using in situ hybridization and immunostaining techniques, we also report that 15-lipoxygenase mRNA and protein colocalize to the same macrophage-rich areas. Moreover, these same lesions express abundant mRNA for the acetyl LDL receptor but no detectable mRNA for the LDL receptor. We suggest that atherogenesis in human arteries may be linked to macrophage-induced oxidative modification of LDL mediated by 15-lipoxygenase, leading to subsequent enhanced macrophage uptake, partly by way of the acetyl LDL receptor. Images PMID:2010531

  16. Azasugar inhibitors as pharmacological chaperones for Krabbe disease

    SciTech Connect

    Hill, Chris H.; Viuff, Agnete H.; Spratley, Samantha J.; Salamone, Stéphane; Christensen, Stig H.; Read, Randy J.; Moriarty, Nigel W.; Jensen, Henrik H.; Deane, Janet E.

    2015-03-23

    Krabbe disease is a devastating neurodegenerative disorder characterized by rapid demyelination of nerve fibers. This disease is caused by defects in the lysosomal enzyme β-galactocerebrosidase (GALC), which hydrolyzes the terminal galactose from glycosphingolipids. These lipids are essential components of eukaryotic cell membranes: substrates of GALC include galactocerebroside, the primary lipid component of myelin, and psychosine, a cytotoxic metabolite. Mutations of GALC that cause misfolding of the protein may be responsive to pharmacological chaperone therapy (PCT), whereby small molecules are used to stabilize these mutant proteins, thus correcting trafficking defects and increasing residual catabolic activity in cells. Here we describe a new approach for the synthesis of galacto-configured azasugars and the characterization of their interaction with GALC using biophysical, biochemical and crystallographic methods. We identify that the global stabilization of GALC conferred by azasugar derivatives, measured by fluorescence-based thermal shift assays, is directly related to their binding affinity, measured by enzyme inhibition. X-ray crystal structures of these molecules bound in the GALC active site reveal which residues participate in stabilizing interactions, show how potency is achieved and illustrate the penalties of aza/iminosugar ring distortion. The structure–activity relationships described here identify the key physical properties required of pharmacological chaperones for Krabbe disease and highlight the potential of azasugars as stabilizing agents for future enzyme replacement therapies. This work lays the foundation for new drug-based treatments of Krabbe disease.

  17. Azasugar inhibitors as pharmacological chaperones for Krabbe disease

    DOE PAGES

    Hill, Chris H.; Viuff, Agnete H.; Spratley, Samantha J.; ...

    2015-03-23

    Krabbe disease is a devastating neurodegenerative disorder characterized by rapid demyelination of nerve fibers. This disease is caused by defects in the lysosomal enzyme β-galactocerebrosidase (GALC), which hydrolyzes the terminal galactose from glycosphingolipids. These lipids are essential components of eukaryotic cell membranes: substrates of GALC include galactocerebroside, the primary lipid component of myelin, and psychosine, a cytotoxic metabolite. Mutations of GALC that cause misfolding of the protein may be responsive to pharmacological chaperone therapy (PCT), whereby small molecules are used to stabilize these mutant proteins, thus correcting trafficking defects and increasing residual catabolic activity in cells. Here we describe amore » new approach for the synthesis of galacto-configured azasugars and the characterization of their interaction with GALC using biophysical, biochemical and crystallographic methods. We identify that the global stabilization of GALC conferred by azasugar derivatives, measured by fluorescence-based thermal shift assays, is directly related to their binding affinity, measured by enzyme inhibition. X-ray crystal structures of these molecules bound in the GALC active site reveal which residues participate in stabilizing interactions, show how potency is achieved and illustrate the penalties of aza/iminosugar ring distortion. The structure–activity relationships described here identify the key physical properties required of pharmacological chaperones for Krabbe disease and highlight the potential of azasugars as stabilizing agents for future enzyme replacement therapies. This work lays the foundation for new drug-based treatments of Krabbe disease.« less

  18. Ethyl 2,4,6-trihydroxybenzoate is an agonistic ligand for liver X receptor that induces cholesterol efflux from macrophages without affecting lipid accumulation in HepG2 cells.

    PubMed

    Hoang, Minh-Hien; Jia, Yaoyao; Jun, Hee-jin; Lee, Ji-Hae; Lee, Dong-Ho; Hwang, Bang-Yeon; Kim, Woo-Jin; Lee, Hak-Ju; Lee, Sung-Joon

    2012-06-15

    The present study reports a novel liver X receptor (LXR) activator, ethyl 2,4,6-trihydroxybenzoate (ETB), isolated from Celtis biondii. Using a reporter gene assay, time-resolved fluorescence resonance energy transfer (TR-FRET), and surface plasmon resonance (SPR) analysis, we showed that ETB directly bound to and stimulated the transcriptional activity of LXR-α and LXR-β. In macrophages, hepatocytes, and intestinal cells, ETB suppressed cellular cholesterol accumulation in a dose-dependent manner and induced the transcriptional activation of LXR-α/-β-responsive genes. Notably, ETB did not induce lipogenic gene expression or cellular triglyceride accumulation in hepatocytes. These results suggest that ETB is a dual-LXR modulator that regulates the expression of key genes in cholesterol homeostasis in multiple cells without inducing lipid accumulation in HepG2 cells.

  19. Chaperone receptors: guiding proteins to intracellular compartments.

    PubMed

    Kriechbaumer, Verena; von Löffelholz, Ottilie; Abell, Ben M

    2012-01-01

    Despite mitochondria and chloroplasts having their own genome, 99% of mitochondrial proteins (Rehling et al., Nat Rev Mol Cell Biol 5:519-530, 2004) and more than 95% of chloroplast proteins (Soll, Curr Opin Plant Biol 5:529-535, 2002) are encoded by nuclear DNA, synthesised in the cytosol and imported post-translationally. Protein targeting to these organelles depends on cytosolic targeting factors, which bind to the precursor, and then interact with membrane receptors to deliver the precursor into a translocase. The molecular chaperones Hsp70 and Hsp90 have been widely implicated in protein targeting to mitochondria and chloroplasts, and receptors capable of recognising these chaperones have been identified at the surface of both these organelles (Schlegel et al., Mol Biol Evol 24:2763-2774, 2007). The role of these chaperone receptors is not fully understood, but they have been shown to increase the efficiency of protein targeting (Young et al., Cell 112:41-50, 2003; Qbadou et al., EMBO J 25:1836-1847, 2006). Whether these receptors contribute to the specificity of targeting is less clear. A class of chaperone receptors bearing tetratricopeptide repeat domains is able to specifically bind the highly conserved C terminus of Hsp70 and/or Hsp90. Interestingly, at least of one these chaperone receptors can be found on each organelle (Schlegel et al., Mol Biol Evol 24:2763-2774, 2007), which suggests a universal role in protein targeting for these chaperone receptors. This review will investigate the role that chaperone receptors play in targeting efficiency and specificity, as well as examining recent in silico approaches to find novel chaperone receptors.

  20. Molecular chaperones: functional mechanisms and nanotechnological applications

    NASA Astrophysics Data System (ADS)

    Rosario Fernández-Fernández, M.; Sot, Begoña; María Valpuesta, José

    2016-08-01

    Molecular chaperones are a group of proteins that assist in protein homeostasis. They not only prevent protein misfolding and aggregation, but also target misfolded proteins for degradation. Despite differences in structure, all types of chaperones share a common general feature, a surface that recognizes and interacts with the misfolded protein. This and other, more specialized properties can be adapted for various nanotechnological purposes, by modification of the original biomolecules or by de novo design based on artificial structures.

  1. Supercharging Chaperones: A Meeting Toolkit for Maximizing Learning for Youth and Chaperones

    ERIC Educational Resources Information Center

    Brandt, Brian

    2016-01-01

    Trip and conference chaperones are a wonderful resource in youth development programs. These well-intended volunteers, many parents of youth participating in the event, want the best experience for the youth but are not necessarily trained in positive youth development. A consequence of this circumstance is that not all chaperones provide the best…

  2. Visualizing chaperone-assisted protein folding

    PubMed Central

    Horowitz, Scott; Salmon, Loïc; Koldewey, Philipp; Ahlstrom, Logan S.; Martin, Raoul; Quan, Shu; Afonine, Pavel V.; van den Bedem, Henry; Wang, Lili; Xu, Qingping; Trievel, Raymond C.; Brooks, Charles L.; Bardwell, James CA

    2016-01-01

    Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding, where obtaining structural ensembles of chaperone:substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a novel structural biology approach based on X-ray crystallography, termed Residual Electron and Anomalous Density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy. This study resulted in a series of snapshots depicting the various folding states of Im7 while bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone. PMID:27239796

  3. Chaperones in hepatitis C virus infection

    PubMed Central

    Khachatoorian, Ronik; French, Samuel W

    2016-01-01

    The hepatitis C virus (HCV) infects approximately 3% of the world population or more than 185 million people worldwide. Each year, an estimated 350000-500000 deaths occur worldwide due to HCV-associated diseases including cirrhosis and hepatocellular carcinoma. HCV is the most common indication for liver transplantation in patients with cirrhosis worldwide. HCV is an enveloped RNA virus classified in the genus Hepacivirus in the Flaviviridae family. The HCV viral life cycle in a cell can be divided into six phases: (1) binding and internalization; (2) cytoplasmic release and uncoating; (3) viral polyprotein translation and processing; (4) RNA genome replication; (5) encapsidation (packaging) and assembly; and (6) virus morphogenesis (maturation) and secretion. Many host factors are involved in the HCV life cycle. Chaperones are an important group of host cytoprotective molecules that coordinate numerous cellular processes including protein folding, multimeric protein assembly, protein trafficking, and protein degradation. All phases of the viral life cycle require chaperone activity and the interaction of viral proteins with chaperones. This review will present our current knowledge and understanding of the role of chaperones in the HCV life cycle. Analysis of chaperones in HCV infection will provide further insights into viral/host interactions and potential therapeutic targets for both HCV and other viruses. PMID:26783419

  4. The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding

    PubMed Central

    Woodford, Mark R.; Dunn, Diana M.; Blanden, Adam R.; Capriotti, Dante; Loiselle, David; Prodromou, Chrisostomos; Panaretou, Barry; Hughes, Philip F.; Smith, Aaron; Ackerman, Wendi; Haystead, Timothy A.; Loh, Stewart N.; Bourboulia, Dimitra; Schmidt, Laura S.; Marston Linehan, W.; Bratslavsky, Gennady; Mollapour, Mehdi

    2016-01-01

    Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors. PMID:27353360

  5. Chaperones get in touch: the Hip-Hop connection.

    PubMed

    Frydman, J; Höhfeld, J

    1997-03-01

    Recent findings emphasize that different molecular chaperones cooperate during intracellular protein biogenesis. Mechanistic aspects of chaperone cooperation are now emerging from studies on the regulation of certain signal transduction pathways mediated by Hsc70 and Hsp90 in the eukaryotic cytosol. Efficient cooperation appears to be achieved through a defined regulation of Hsc70 activity by the chaperone cofactors Hip and Hop.

  6. Histone chaperone-mediated nucleosome assembly process.

    PubMed

    Fan, Hsiu-Fang; Liu, Zi-Ning; Chow, Sih-Yao; Lu, Yi-Han; Li, Hsin

    2015-01-01

    A huge amount of information is stored in genomic DNA and this stored information resides inside the nucleus with the aid of chromosomal condensation factors. It has been reported that the repeat nucleosome core particle (NCP) consists of 147-bp of DNA and two copies of H2A, H2B, H3 and H4. Regulation of chromosomal structure is important to many processes inside the cell. In vivo, a group of histone chaperones facilitate and regulate nucleosome assembly. How NCPs are constructed with the aid of histone chaperones remains unclear. In this study, the histone chaperone-mediated nucleosome assembly process was investigated using single-molecule tethered particle motion (TPM) experiments. It was found that Asf1 is able to exert more influence than Nap1 and poly glutamate acid (PGA) on the nucleosome formation process, which highlights Asf1's specific role in tetrasome formation. Thermodynamic parameters supported a model whereby energetically favored nucleosomal complexes compete with non-nucleosomal complexes. In addition, our kinetic findings propose the model that histone chaperones mediate nucleosome assembly along a path that leads to enthalpy-favored products with free histones as reaction substrates.

  7. Phenylalanine hydroxylase misfolding and pharmacological chaperones.

    PubMed

    Underhaug, Jarl; Aubi, Oscar; Martinez, Aurora

    2012-01-01

    Phenylketonuria (PKU) is a loss-of-function inborn error of metabolism. As many other inherited diseases the main pathologic mechanism in PKU is an enhanced tendency of the mutant phenylalanine hydroxylase (PAH) to misfold and undergo ubiquitin-dependent degradation. Recent alternative approaches with therapeutic potential for PKU aim at correcting the PAH misfolding, and in this respect pharmacological chaperones are the focus of increasing interest. These compounds, which often resemble the natural ligands and show mild competitive inhibition, can rescue the misfolded proteins by stimulating their renaturation in vivo. For PKU, a few studies have proven the stabilization of PKU-mutants in vitro, in cells, and in mice by pharmacological chaperones, which have been found either by using the tetrahydrobiopterin (BH(4)) cofactor as query structure for shape-focused virtual screening or by high-throughput screening of small compound libraries. Both approaches have revealed a number of compounds, most of which bind at the iron-binding site, competitively with respect to BH(4). Furthermore, PAH shares a number of ligands, such as BH(4), amino acid substrates and inhibitors, with the other aromatic amino acid hydroxylases: the neuronal/neuroendocrine enzymes tyrosine hydroxylase (TH) and the tryptophan hydroxylases (TPHs). Recent results indicate that the PAH-targeted pharmacological chaperones should also be tested on TH and the TPHs, and eventually be derivatized to avoid unwanted interactions with these other enzymes. After derivatization and validation in animal models, the PAH-chaperoning compounds represent novel possibilities in the treatment of PKU.

  8. Changes in transcriptome of macrophages in atherosclerosis

    PubMed Central

    Chistiakov, Dimitry A; Bobryshev, Yuri V; Orekhov, Alexander N

    2015-01-01

    Macrophages display significant phenotypic heterogeneity. Two growth factors, macrophage colony-stimulating factor and chemokine (C-X-C motif) ligand 4, drive terminal differentiation of monocytes to M0 and M4 macrophages respectively. Compared to M0 macrophages, M4 cells have a unique transcriptome, with expression of surface markers such as S100A8, mannose receptor CD206 and matrix metalloproteinase 7. M4 macrophages did not express CD163, a scavenger receptor for haemoglobin/haptoglobin complex. Depending on the stimuli, M0 macrophages could polarize towards the proinflammatory M1 subset by treatment with lipopolysaccharide or interferon-γ. These macrophages produce a range of proinflammatory cytokines, nitric oxide, reactive oxygen species and exhibit high chemotactic and phagocytic activity. The alternative M2 type could be induced from M0 macrophage by stimulation with interleukin (IL)-4. M2 macrophages express high levels of CD206 and produce anti-inflammatory cytokines IL-10 and transforming growth factor-β. M1, M2 and M4 macrophages could be found in atherosclerotic plaques. In the plaque, macrophages are subjected to the intensive influence not only by cytokines and chemokines but also with bioactive lipids such as cholesterol and oxidized phospholipids. Oxidized phospholipids induce a distinct Mox phenotype in murine macrophages that express a unique panel of antioxidant enzymes under control of the redox-regulated transcription factor Klf2, resistant to lipid accumulation. In unstable human lesions, atheroprotective M(Hb) and HA-mac macrophage subsets could be found. These two subsets are induced by the haemoglobin/haptoglobin complex, highly express haeme oxygenase 1 and CD163, and are implicated in clearance of haemoglobin and erythrocyte remnants. In atherogenesis, the macrophage phenotype is plastic and could therefore be switched to proinflammatory (i.e. proatherogenic) and anti-inflammatory (i.e. atheroprotective). The aim of this review was to

  9. Study on the chaperone properties of conserved GTPases.

    PubMed

    Wang, Xiang; Xue, Jiaying; Sun, Zhe; Qin, Yan; Gong, Weimin

    2012-01-01

    As a large family of hydrolases, GTPases are widespread in cells and play the very important biological function of hydrolyzing GTP into GDP and inorganic phosphate through binding with it. GTPases are involved in cell cycle regulation, protein synthesis, and protein transportation. Chaperones can facilitate the folding or refolding of nascent peptides and denatured proteins to their native states. However, chaperones do not occur in the native structures in which they can perform their normal biological functions. In the current study, the chaperone activity of the conserved GTPases of Escherichia coli is tested by the chemical denaturation and chaperone-assisted renaturation of citrate synthase and α-glucosidase. The effects of ribosomes and nucleotides on the chaperone activity are also examined. Our data indicate that these conserved GTPases have chaperone properties, and may be ancestral protein folding factors that have appeared before dedicated chaperones.

  10. Emerging novel concept of chaperone therapies for protein misfolding diseases

    PubMed Central

    SUZUKI, Yoshiyuki

    2014-01-01

    Chaperone therapy is a newly developed molecular therapeutic approach to protein misfolding diseases. Among them we found unstable mutant enzyme proteins in a few lysosomal diseases, resulting in rapid intracellular degradation and loss of function. Active-site binding low molecular competitive inhibitors (chemical chaperones) paradoxically stabilized and enhanced the enzyme activity in somatic cells by correction of the misfolding of enzyme protein. They reached the brain through the blood-brain barrier after oral administration, and corrected pathophysiology of the disease. In addition to these inhibitory chaperones, non-competitive chaperones without inhibitory bioactivity are being developed. Furthermore molecular chaperone therapy utilizing the heat shock protein and other chaperone proteins induced by small molecules has been experimentally tried to handle abnormally accumulated proteins as a new approach particularly to neurodegenerative diseases. These three types of chaperones are promising candidates for various types of diseases, genetic or non-genetic, and neurological or non-neurological, in addition to lysosomal diseases. PMID:24814990

  11. Adipocyte fetuin-A contributes to macrophage migration into adipose tissue and polarization of macrophages.

    PubMed

    Chatterjee, Priyajit; Seal, Soma; Mukherjee, Sandip; Kundu, Rakesh; Mukherjee, Sutapa; Ray, Sukanta; Mukhopadhyay, Satinath; Majumdar, Subeer S; Bhattacharya, Samir

    2013-09-27

    Macrophage infiltration into adipose tissue during obesity and their phenotypic conversion from anti-inflammatory M2 to proinflammatory M1 subtype significantly contributes to develop a link between inflammation and insulin resistance; signaling molecule(s) for these events, however, remains poorly understood. We demonstrate here that excess lipid in the adipose tissue environment may trigger one such signal. Adipose tissue from obese diabetic db/db mice, high fat diet-fed mice, and obese diabetic patients showed significantly elevated fetuin-A (FetA) levels in respect to their controls; partially hepatectomized high fat diet mice did not show noticeable alteration, indicating adipose tissue to be the source of this alteration. In adipocytes, fatty acid induces FetA gene and protein expressions, resulting in its copious release. We found that FetA could act as a chemoattractant for macrophages. To simulate lipid-induced inflammatory conditions when proinflammatory adipose tissue and macrophages create a niche of an altered microenvironment, we set up a transculture system of macrophages and adipocytes; the addition of fatty acid to adipocytes released FetA into the medium, which polarized M2 macrophages to M1. This was further confirmed by direct FetA addition to macrophages. Taken together, lipid-induced FetA from adipocytes is an efficient chemokine for macrophage migration and polarization. These findings open a new dimension for understanding obesity-induced inflammation.

  12. Adipocyte Fetuin-A Contributes to Macrophage Migration into Adipose Tissue and Polarization of Macrophages*

    PubMed Central

    Chatterjee, Priyajit; Seal, Soma; Mukherjee, Sandip; Kundu, Rakesh; Mukherjee, Sutapa; Ray, Sukanta; Mukhopadhyay, Satinath; Majumdar, Subeer S.; Bhattacharya, Samir

    2013-01-01

    Macrophage infiltration into adipose tissue during obesity and their phenotypic conversion from anti-inflammatory M2 to proinflammatory M1 subtype significantly contributes to develop a link between inflammation and insulin resistance; signaling molecule(s) for these events, however, remains poorly understood. We demonstrate here that excess lipid in the adipose tissue environment may trigger one such signal. Adipose tissue from obese diabetic db/db mice, high fat diet-fed mice, and obese diabetic patients showed significantly elevated fetuin-A (FetA) levels in respect to their controls; partially hepatectomized high fat diet mice did not show noticeable alteration, indicating adipose tissue to be the source of this alteration. In adipocytes, fatty acid induces FetA gene and protein expressions, resulting in its copious release. We found that FetA could act as a chemoattractant for macrophages. To simulate lipid-induced inflammatory conditions when proinflammatory adipose tissue and macrophages create a niche of an altered microenvironment, we set up a transculture system of macrophages and adipocytes; the addition of fatty acid to adipocytes released FetA into the medium, which polarized M2 macrophages to M1. This was further confirmed by direct FetA addition to macrophages. Taken together, lipid-induced FetA from adipocytes is an efficient chemokine for macrophage migration and polarization. These findings open a new dimension for understanding obesity-induced inflammation. PMID:23943623

  13. Impaired macrophage autophagy increases the immune response in obese mice by promoting proinflammatory macrophage polarization

    PubMed Central

    Liu, Kun; Zhao, Enpeng; Ilyas, Ghulam; Lalazar, Gadi; Lin, Yu; Haseeb, Muhammad; Tanaka, Kathryn E; Czaja, Mark J

    2015-01-01

    Recent evidence that excessive lipid accumulation can decrease cellular levels of autophagy and that autophagy regulates immune responsiveness suggested that impaired macrophage autophagy may promote the increased innate immune activation that underlies obesity. Primary bone marrow-derived macrophages (BMDM) and peritoneal macrophages from high-fat diet (HFD)-fed mice had decreased levels of autophagic flux indicating a generalized impairment of macrophage autophagy in obese mice. To assess the effects of decreased macrophage autophagy on inflammation, mice with a Lyz2-Cre-mediated knockout of Atg5 in macrophages were fed a HFD and treated with low-dose lipopolysaccharide (LPS). Knockout mice developed systemic and hepatic inflammation with HFD feeding and LPS. This effect was liver specific as knockout mice did not have increased adipose tissue inflammation. The mechanism by which the loss of autophagy promoted inflammation was through the regulation of macrophage polarization. BMDM and Kupffer cells from knockout mice exhibited abnormalities in polarization with both increased proinflammatory M1 and decreased anti-inflammatory M2 polarization as determined by measures of genes and proteins. The heightened hepatic inflammatory response in HFD-fed, LPS-treated knockout mice led to liver injury without affecting steatosis. These findings demonstrate that autophagy has a critical regulatory function in macrophage polarization that downregulates inflammation. Defects in macrophage autophagy may underlie inflammatory disease states such as the decrease in macrophage autophagy with obesity that leads to hepatic inflammation and the progression to liver injury. PMID:25650776

  14. Impaired macrophage autophagy increases the immune response in obese mice by promoting proinflammatory macrophage polarization.

    PubMed

    Liu, Kun; Zhao, Enpeng; Ilyas, Ghulam; Lalazar, Gadi; Lin, Yu; Haseeb, Muhammad; Tanaka, Kathryn E; Czaja, Mark J

    2015-01-01

    Recent evidence that excessive lipid accumulation can decrease cellular levels of autophagy and that autophagy regulates immune responsiveness suggested that impaired macrophage autophagy may promote the increased innate immune activation that underlies obesity. Primary bone marrow-derived macrophages (BMDM) and peritoneal macrophages from high-fat diet (HFD)-fed mice had decreased levels of autophagic flux indicating a generalized impairment of macrophage autophagy in obese mice. To assess the effects of decreased macrophage autophagy on inflammation, mice with a Lyz2-Cre-mediated knockout of Atg5 in macrophages were fed a HFD and treated with low-dose lipopolysaccharide (LPS). Knockout mice developed systemic and hepatic inflammation with HFD feeding and LPS. This effect was liver specific as knockout mice did not have increased adipose tissue inflammation. The mechanism by which the loss of autophagy promoted inflammation was through the regulation of macrophage polarization. BMDM and Kupffer cells from knockout mice exhibited abnormalities in polarization with both increased proinflammatory M1 and decreased anti-inflammatory M2 polarization as determined by measures of genes and proteins. The heightened hepatic inflammatory response in HFD-fed, LPS-treated knockout mice led to liver injury without affecting steatosis. These findings demonstrate that autophagy has a critical regulatory function in macrophage polarization that downregulates inflammation. Defects in macrophage autophagy may underlie inflammatory disease states such as the decrease in macrophage autophagy with obesity that leads to hepatic inflammation and the progression to liver injury.

  15. Histone chaperone specificity in Rtt109 activation

    PubMed Central

    Park, Young-Jun; Sudhoff, Keely B; Andrews, Andrew J; Stargell, Laurie A; Luger, Karolin

    2008-01-01

    Rtt109 is a histone acetyltransferase that requires a histone chaperone for the acetylation of histone 3 at lysine 56 (H3K56). Rtt109 forms a complex with the chaperone Vps75 in vivo and is implicated in DNA replication and repair. Here we show that both Rtt109 and Vps75 bind histones with high affinity, but only the complex is efficient for catalysis. The C-terminal acidic domain of Vps75 contributes to activation of Rtt109 and is necessary for in vivo functionality of Vps75, but it is not required for interaction with either Rtt109 or histones. We demonstrate that Vps75 is a structural homolog of yeast Nap1 by solving its crystal structure. Nap1 and Vps75 interact with histones and Rtt109 with comparable affinities. However, only Vps75 stimulates Rtt109 enzymatic activity. Our data highlight the functional specificity of Vps75 in Rtt109 activation. PMID:19172749

  16. Molecular chaperones and hypoxic-ischemic encephalopathy

    PubMed Central

    Hua, Cong; Ju, Wei-na; Jin, Hang; Sun, Xin; Zhao, Gang

    2017-01-01

    Hypoxic-ischemic encephalopathy (HIE) is a disease that occurs when the brain is subjected to hypoxia, resulting in neuronal death and neurological deficits, with a poor prognosis. The mechanisms underlying hypoxic-ischemic brain injury include excitatory amino acid release, cellular proteolysis, reactive oxygen species generation, nitric oxide synthesis, and inflammation. The molecular and cellular changes in HIE include protein misfolding, aggregation, and destruction of organelles. The apoptotic pathways activated by ischemia and hypoxia include the mitochondrial pathway, the extrinsic Fas receptor pathway, and the endoplasmic reticulum stress-induced pathway. Numerous treatments for hypoxic-ischemic brain injury caused by HIE have been developed over the last half century. Hypothermia, xenon gas treatment, the use of melatonin and erythropoietin, and hypoxic-ischemic preconditioning have proven effective in HIE patients. Molecular chaperones are proteins ubiquitously present in both prokaryotes and eukaryotes. A large number of molecular chaperones are induced after brain ischemia and hypoxia, among which the heat shock proteins are the most important. Heat shock proteins not only maintain protein homeostasis; they also exert anti-apoptotic effects. Heat shock proteins maintain protein homeostasis by helping to transport proteins to their target destinations, assisting in the proper folding of newly synthesized polypeptides, regulating the degradation of misfolded proteins, inhibiting the aggregation of proteins, and by controlling the refolding of misfolded proteins. In addition, heat shock proteins exert anti-apoptotic effects by interacting with various signaling pathways to block the activation of downstream effectors in numerous apoptotic pathways, including the intrinsic pathway, the endoplasmic reticulum-stress mediated pathway and the extrinsic Fas receptor pathway. Molecular chaperones play a key role in neuroprotection in HIE. In this review, we

  17. Recombination of ozone via the chaperon mechanism

    NASA Astrophysics Data System (ADS)

    Ivanov, Mikhail V.; Schinke, Reinhard

    2006-03-01

    The recombination of ozone via the chaperon mechanism, i.e., ArO +O2→Ar+O3 and ArO2+O→Ar+O3, is studied by means of classical trajectories and a pairwise additive Ar -O3 potential energy surface. The recombination rate coefficient has a strong temperature dependence, which approximately can be described by T-n with n ≈3. It is negligible for temperatures above 700 K or so, but it becomes important for low temperatures. The calculations unambiguously affirm the conclusions of Hippler et al. [J. Chem. Phys. 93, 6560 (1990)] and Luther et al. [Phys. Chem. Chem. Phys. 7, 2764 (2005)] that the chaperon mechanism makes a sizable contribution to the recombination of O3 at room temperature and below. The dependence of the chaperon recombination rate coefficient on the isotopomer, studied for two different isotope combinations, is only in rough qualitative agreement with the experimental data. The oxygen atom isotope exchange reaction involving ArO and ArO2 van der Waals complexes is also investigated; the weak binding of O or O2 to Ar has only a small effect.

  18. A molecular mechanism of chaperone-client recognition

    PubMed Central

    He, Lichun; Sharpe, Timothy; Mazur, Adam; Hiller, Sebastian

    2016-01-01

    Molecular chaperones are essential in aiding client proteins to fold into their native structure and in maintaining cellular protein homeostasis. However, mechanistic aspects of chaperone function are still not well understood at the atomic level. We use nuclear magnetic resonance spectroscopy to elucidate the mechanism underlying client recognition by the adenosine triphosphate-independent chaperone Spy at the atomic level and derive a structural model for the chaperone-client complex. Spy interacts with its partially folded client Im7 by selective recognition of flexible, locally frustrated regions in a dynamic fashion. The interaction with Spy destabilizes a partially folded client but spatially compacts an unfolded client conformational ensemble. By increasing client backbone dynamics, the chaperone facilitates the search for the native structure. A comparison of the interaction of Im7 with two other chaperones suggests that the underlying principle of recognizing frustrated segments is of a fundamental nature. PMID:28138538

  19. Stress and molecular chaperones in disease.

    PubMed

    Macario, A J; Conway de Macario, E

    2000-01-01

    Stress, a common phenomenon in today's society, is suspected of playing a role in the development of disease. Stressors of various types, psychological, physical, and biological, abound. They occur in the working and social environments, in air, soil, water, food, and medicines. Stressors impact on cells directly or indirectly, cause protein denaturation, and elicit a stress response. This is mediated by stress (heat-shock) genes and proteins, among which are those named molecular chaperones because they assist other proteins to achieve and maintain a functional shape (the native configuration), and to recover it when partially lost due to stress. Denatured proteins tend to aggregate and precipitate. The same occurs with abnormal proteins due to mutations, or to failure of post-transcriptional or post-translational mechanisms. These abnormal proteins need the help of molecular chaperones as much as denatured molecules do, especially during stress. A cell with normal antistress mechanisms, including a complete and functional set of chaperones, may be able to withstand stress if its intensity is not beyond that which will cause irreversible protein damage. There is a certain threshold that normal cells have above which they cannot cope with stress. A cell with an abnormal protein that has an intrinsic tendency to misfold and aggregate is more vulnerable to stress than normal counterparts. Furthermore, these abnormal proteins may precipitate even in the absence of stress and cause diseases named proteinopathies. It is possible that stress contributes to the pathogenesis of proteinopathies by promoting protein aggregation, even in cells that possess a normal chaperoning system. Examples of proteinopathies are age-related degenerative disorders with protein deposits in various tissues, most importantly in the brain where the deposits are associated with neuronal degeneration. It is conceivable that stress enhances the progression of these diseases by facilitating

  20. New insights into the roles of molecular chaperones in Chlamydomonas and Volvox.

    PubMed

    Nordhues, André; Miller, Stephen M; Mühlhaus, Timo; Schroda, Michael

    2010-01-01

    The unicellular green alga Chlamydomonas reinhardtii has been used as a model organism for many decades, mainly to study photosynthesis and flagella/cilia. Only recently, Chlamydomonas has received much attention because of its ability to produce hydrogen and nonpolar lipids that have promise as biofuels. The best-studied multicellular cousin of Chlamydomonas reinhardtii is Volvox carteri, whose life cycle comprises events that have clear parallels in higher plants and/or animals, making it an excellent system in which to study fundamental developmental processes. Molecular chaperones are proteins that guide other cellular proteins through their life cycle. They assist in de novo folding of nascent chains, mediate assembly and disassembly of protein complexes, facilitate protein transport across membranes, disassemble protein aggregates, fold denatured proteins back to the native state, and transfer unfoldable proteins to proteolytic degradation. Hence, molecular chaperones regulate protein function under all growth conditions and play important roles in many basic cellular and developmental processes. The aim of this chapter is to describe recent advances toward understanding molecular chaperone biology in Chlamydomonas and Volvox.

  1. Histone chaperones link histone nuclear import and chromatin assembly.

    PubMed

    Keck, Kristin M; Pemberton, Lucy F

    2013-01-01

    Histone chaperones are proteins that shield histones from nonspecific interactions until they are assembled into chromatin. After their synthesis in the cytoplasm, histones are bound by different histone chaperones, subjected to a series of posttranslational modifications and imported into the nucleus. These evolutionarily conserved modifications, including acetylation and methylation, can occur in the cytoplasm, but their role in regulating import is not well understood. As part of histone import complexes, histone chaperones may serve to protect the histones during transport, or they may be using histones to promote their own nuclear localization. In addition, there is evidence that histone chaperones can play an active role in the import of histones. Histone chaperones have also been shown to regulate the localization of important chromatin modifying enzymes. This review is focused on the role histone chaperones play in the early biogenesis of histones, the distinct cytoplasmic subcomplexes in which histone chaperones have been found in both yeast and mammalian cells and the importins/karyopherins and nuclear localization signals that mediate the nuclear import of histones. We also address the role that histone chaperone localization plays in human disease. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.

  2. Macrophage-mediated cholesterol handling in atherosclerosis.

    PubMed

    Chistiakov, Dimitry A; Bobryshev, Yuri V; Orekhov, Alexander N

    2016-01-01

    Formation of foam cells is a hallmark at the initial stages of atherosclerosis. Monocytes attracted by pro-inflammatory stimuli attach to the inflamed vascular endothelium and penetrate to the arterial intima where they differentiate to macrophages. Intimal macrophages phagocytize oxidized low-density lipoproteins (oxLDL). Several scavenger receptors (SR), including CD36, SR-A1 and lectin-like oxLDL receptor-1 (LOX-1), mediate oxLDL uptake. In late endosomes/lysosomes of macrophages, oxLDL are catabolysed. Lysosomal acid lipase (LAL) hydrolyses cholesterol esters that are enriched in LDL to free cholesterol and free fatty acids. In the endoplasmic reticulum (ER), acyl coenzyme A: cholesterol acyltransferase-1 (ACAT1) in turn catalyses esterification of cholesterol to store cholesterol esters as lipid droplets in the ER of macrophages. Neutral cholesteryl ester hydrolases nCEH and NCEH1 are involved in a secondary hydrolysis of cholesterol esters to liberate free cholesterol that could be then out-flowed from macrophages by cholesterol ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 and SR-BI. In atherosclerosis, disruption of lipid homoeostasis in macrophages leads to cholesterol accumulation and formation of foam cells.

  3. A Mouse Macrophage Lipidome*♦

    PubMed Central

    Dennis, Edward A.; Deems, Raymond A.; Harkewicz, Richard; Quehenberger, Oswald; Brown, H. Alex; Milne, Stephen B.; Myers, David S.; Glass, Christopher K.; Hardiman, Gary; Reichart, Donna; Merrill, Alfred H.; Sullards, M. Cameron; Wang, Elaine; Murphy, Robert C.; Raetz, Christian R. H.; Garrett, Teresa A.; Guan, Ziqiang; Ryan, Andrea C.; Russell, David W.; McDonald, Jeffrey G.; Thompson, Bonne M.; Shaw, Walter A.; Sud, Manish; Zhao, Yihua; Gupta, Shakti; Maurya, Mano R.; Fahy, Eoin; Subramaniam, Shankar

    2010-01-01

    We report the lipidomic response of the murine macrophage RAW cell line to Kdo2-lipid A, the active component of an inflammatory lipopolysaccharide functioning as a selective TLR4 agonist and compactin, a statin inhibitor of cholesterol biosynthesis. Analyses of lipid molecular species by dynamic quantitative mass spectrometry and concomitant transcriptomic measurements define the lipidome and demonstrate immediate responses in fatty acid metabolism represented by increases in eicosanoid synthesis and delayed responses characterized by sphingolipid and sterol biosynthesis. Lipid remodeling of glycerolipids, glycerophospholipids, and prenols also take place, indicating that activation of the innate immune system by inflammatory mediators leads to alterations in a majority of mammalian lipid categories, including unanticipated effects of a statin drug. Our studies provide a systems-level view of lipid metabolism and reveal significant connections between lipid and cell signaling and biochemical pathways that contribute to innate immune responses and to pharmacological perturbations. PMID:20923771

  4. Bacterial proteostasis balances energy and chaperone utilization efficiently.

    PubMed

    Santra, Mantu; Farrell, Daniel W; Dill, Ken A

    2017-03-28

    Chaperones are protein complexes that help to fold and disaggregate a cell's proteins. It is not understood how four major chaperone systems of Escherichia coli work together in proteostasis: the recognition, sorting, folding, and disaggregating of the cell's many different proteins. Here, we model this machine. We combine extensive data on chaperoning, folding, and aggregation rates with expression levels of proteins and chaperones measured at different growth rates. We find that the proteostasis machine recognizes and sorts a client protein based on two biophysical properties of the client's misfolded state (M state): its stability and its kinetic accessibility from its unfolded state (U state). The machine is energy-efficient (the sickest proteins use the most ATP-expensive chaperones), comprehensive (it can handle any type of protein), and economical (the chaperone concentrations are just high enough to keep the whole proteome folded and disaggregated but no higher). The cell needs higher chaperone levels in two situations: fast growth (when protein production rates are high) and very slow growth (to mitigate the effects of protein degradation). This type of model complements experimental knowledge by showing how the various chaperones work together to achieve the broad folding and disaggregation needs of the cell.

  5. Chaperones rescue luciferase folding by separating its domains.

    PubMed

    Scholl, Zackary N; Yang, Weitao; Marszalek, Piotr E

    2014-10-10

    Over the last 50 years, significant progress has been made toward understanding how small single-domain proteins fold. However, very little is known about folding mechanisms of medium and large multidomain proteins that predominate the proteomes of all forms of life. Large proteins frequently fold cotranslationally and/or require chaperones. Firefly (Photinus pyralis) luciferase (Luciferase, 550 residues) has been a model of a cotranslationally folding protein whose extremely slow refolding (approximately days) is catalyzed by chaperones. However, the mechanism by which Luciferase misfolds and how chaperones assist Luciferase refolding remains unknown. Here we combine single-molecule force spectroscopy (atomic force microscopy (AFM)/single-molecule force spectroscopy) with steered molecular dynamic computer simulations to unravel the mechanism of chaperone-assisted Luciferase refolding. Our AFM and steered molecular dynamic results show that partially unfolded Luciferase, with the N-terminal domain remaining folded, can refold robustly without chaperones. Complete unfolding causes Luciferase to get trapped in very stable non-native configurations involving interactions between N- and C-terminal residues. However, chaperones allow the completely unfolded Luciferase to refold quickly in AFM experiments, strongly suggesting that chaperones are able to sequester non-natively contacting residues. More generally, we suggest that many chaperones, rather than actively promoting the folding, mimic the ribosomal exit tunnel and physically separate protein domains, allowing them to fold in a cotranslational-like sequential process.

  6. Chaperones Rescue Luciferase Folding by Separating Its Domains*

    PubMed Central

    Scholl, Zackary N.; Yang, Weitao; Marszalek, Piotr E.

    2014-01-01

    Over the last 50 years, significant progress has been made toward understanding how small single-domain proteins fold. However, very little is known about folding mechanisms of medium and large multidomain proteins that predominate the proteomes of all forms of life. Large proteins frequently fold cotranslationally and/or require chaperones. Firefly (Photinus pyralis) luciferase (Luciferase, 550 residues) has been a model of a cotranslationally folding protein whose extremely slow refolding (approximately days) is catalyzed by chaperones. However, the mechanism by which Luciferase misfolds and how chaperones assist Luciferase refolding remains unknown. Here we combine single-molecule force spectroscopy (atomic force microscopy (AFM)/single-molecule force spectroscopy) with steered molecular dynamic computer simulations to unravel the mechanism of chaperone-assisted Luciferase refolding. Our AFM and steered molecular dynamic results show that partially unfolded Luciferase, with the N-terminal domain remaining folded, can refold robustly without chaperones. Complete unfolding causes Luciferase to get trapped in very stable non-native configurations involving interactions between N- and C-terminal residues. However, chaperones allow the completely unfolded Luciferase to refold quickly in AFM experiments, strongly suggesting that chaperones are able to sequester non-natively contacting residues. More generally, we suggest that many chaperones, rather than actively promoting the folding, mimic the ribosomal exit tunnel and physically separate protein domains, allowing them to fold in a cotranslational-like sequential process. PMID:25160632

  7. Bacterial proteostasis balances energy and chaperone utilization efficiently

    PubMed Central

    Santra, Mantu; Farrell, Daniel W.; Dill, Ken A.

    2017-01-01

    Chaperones are protein complexes that help to fold and disaggregate a cell’s proteins. It is not understood how four major chaperone systems of Escherichia coli work together in proteostasis: the recognition, sorting, folding, and disaggregating of the cell’s many different proteins. Here, we model this machine. We combine extensive data on chaperoning, folding, and aggregation rates with expression levels of proteins and chaperones measured at different growth rates. We find that the proteostasis machine recognizes and sorts a client protein based on two biophysical properties of the client’s misfolded state (M state): its stability and its kinetic accessibility from its unfolded state (U state). The machine is energy-efficient (the sickest proteins use the most ATP-expensive chaperones), comprehensive (it can handle any type of protein), and economical (the chaperone concentrations are just high enough to keep the whole proteome folded and disaggregated but no higher). The cell needs higher chaperone levels in two situations: fast growth (when protein production rates are high) and very slow growth (to mitigate the effects of protein degradation). This type of model complements experimental knowledge by showing how the various chaperones work together to achieve the broad folding and disaggregation needs of the cell. PMID:28292901

  8. Toward Instituting a Chaperone Policy in Outpatient Pediatric Clinics

    ERIC Educational Resources Information Center

    Feldman, Kenneth W.; Jenkins, Carol; Laney, Tyler; Seidel, Kristy

    2009-01-01

    Objectives: We sought to evaluate child, parent and medical provider preferences for chaperones for outpatient encounters and to evaluate the acceptability and frequency of utilization following institution of a chaperone policy. Secondarily, we sought to understand what medical history and examinations teens consider "sensitive." Design: We…

  9. Molecular chaperones as rational drug targets for Parkinson's disease therapeutics.

    PubMed

    Kalia, S K; Kalia, L V; McLean, P J

    2010-12-01

    Parkinson's disease is a neurodegenerative movement disorder that is caused, in part, by the loss of dopaminergic neurons within the substantia nigra pars compacta of the basal ganglia. The presence of intracellular protein aggregates, known as Lewy bodies and Lewy neurites, within the surviving nigral neurons is the defining neuropathological feature of the disease. Accordingly, the identification of specific genes mutated in families with Parkinson's disease and of genetic susceptibility variants for idiopathic Parkinson's disease has implicated abnormalities in proteostasis, or the handling and elimination of misfolded proteins, in the pathogenesis of this neurodegenerative disorder. Protein folding and the refolding of misfolded proteins are regulated by a network of interactive molecules, known as the chaperone system, which is composed of molecular chaperones and co-chaperones. The chaperone system is intimately associated with the ubiquitin-proteasome system and the autophagy-lysosomal pathway which are responsible for elimination of misfolded proteins and protein quality control. In addition to their role in proteostasis, some chaperone molecules are involved in the regulation of cell death pathways. Here we review the role of the molecular chaperones Hsp70 and Hsp90, and the cochaperones Hsp40, BAG family members such as BAG5, CHIP and Hip in modulating neuronal death with a focus on dopaminergic neurodegeneration in Parkinson's disease. We also review current progress in preclinical studies aimed at targetting the chaperone system to prevent neurodegeneration. Finally, we discuss potential future chaperone-based therapeutics for the symptomatic treatment and possible disease modification of Parkinson's disease.

  10. Mitochondrial chaperones may be targets for anti-cancer drugs

    Cancer.gov

    Scientists at NCI have found that a mitochondrial chaperone protein, TRAP1, may act indirectly as a tumor suppressor as well as a novel target for developing anti-cancer drugs. Chaperone proteins, such as TRAP1, help other proteins adapt to stress, but sc

  11. Chaperones as potential therapeutics for Krabbe disease.

    PubMed

    Graziano, Adriana Carol Eleonora; Pannuzzo, Giovanna; Avola, Rosanna; Cardile, Venera

    2016-11-01

    Krabbe's disease (KD) is an autosomal recessive, neurodegenerative disorder. It is classified among the lysosomal storage diseases (LSDs). It was first described in , but the genetic defect for the galactocerebrosidase (GALC) gene was not discovered until the beginning of the 1970s, 20 years before the GALC cloning. Recently, in 2011, the crystal structures of the GALC enzyme and the GALC-product complex were obtained. For this, compared with other LSDs, the research on possible therapeutic interventions is much more recent. Thus, it is not surprising that some treatment options are still under preclinical investigation, whereas their relevance for other pathologies of the same group has already been tested in clinical studies. This is specifically the case for pharmacological chaperone therapy (PCT), a promising strategy for selectively correcting defective protein folding and trafficking and for enhancing enzyme activity by small molecules. These compounds bind directly to a partially folded biosynthetic intermediate, stabilize the protein, and allow completion of the folding process to yield a functional protein. Here, we review the chaperones that have demonstrated potential therapeutics during preclinical studies for KD, underscoring the requirement to invigorate research for KD-addressed PCT that will benefit from recent insights into the molecular understanding of GALC structure, drug design, and development in cellular models. © 2016 Wiley Periodicals, Inc.

  12. Nanomedicine engulfed by macrophages for targeted tumor therapy

    PubMed Central

    Li, Siwen; Feng, Song; Ding, Li; Liu, Yuxi; Zhu, Qiuyun; Qian, Zhiyu; Gu, Yueqing

    2016-01-01

    Macrophages, exhibiting high intrinsic accumulation and infiltration into tumor tissues, are a novel drug vehicle for directional drug delivery. However, the low drug-loading (DL) capacity and the drug cytotoxicity to the cell vehicle have limited the application of macrophages in tumor therapy. In this study, different drugs involving small molecular and nanoparticle drugs were loaded into intrinsic macrophages to find a better way to overcome these limitations. Their DL capacity and cytotoxicity to the macrophages were first compared. Furthermore, their phagocytic ratio, dynamic distributions, and tumoricidal effects were also investigated. Results indicated that more lipid-soluble molecules and DL particles can be phagocytized by macrophages than hydrophilic ones. In addition, the N-succinyl-N′-octyl chitosan (SOC) DL particles showed low cytotoxicity to the macrophage itself, while the dynamic biodistribution of macrophages engulfed with different particles/small molecules showed similar profiles, mainly excreted from liver to intestine pathway. Furthermore, macrophages loaded with SOC–paclitaxel (PTX) particles exhibited greater therapeutic efficacies than those of macrophages directly carrying small molecular drugs such as doxorubicin and PTX. Interestingly, macrophages displayed stronger targeting ability to the tumor site hypersecreting chemokine in immunocompetent mice in comparison to the tumor site secreting low levels of chemokine in immunodeficiency mice. Finally, results demonstrated that macrophages carrying SOC–PTX are a promising pharmaceutical preparation for tumor-targeted therapy. PMID:27601898

  13. Nanomedicine engulfed by macrophages for targeted tumor therapy.

    PubMed

    Li, Siwen; Feng, Song; Ding, Li; Liu, Yuxi; Zhu, Qiuyun; Qian, Zhiyu; Gu, Yueqing

    Macrophages, exhibiting high intrinsic accumulation and infiltration into tumor tissues, are a novel drug vehicle for directional drug delivery. However, the low drug-loading (DL) capacity and the drug cytotoxicity to the cell vehicle have limited the application of macrophages in tumor therapy. In this study, different drugs involving small molecular and nanoparticle drugs were loaded into intrinsic macrophages to find a better way to overcome these limitations. Their DL capacity and cytotoxicity to the macrophages were first compared. Furthermore, their phagocytic ratio, dynamic distributions, and tumoricidal effects were also investigated. Results indicated that more lipid-soluble molecules and DL particles can be phagocytized by macrophages than hydrophilic ones. In addition, the N-succinyl-N'-octyl chitosan (SOC) DL particles showed low cytotoxicity to the macrophage itself, while the dynamic biodistribution of macrophages engulfed with different particles/small molecules showed similar profiles, mainly excreted from liver to intestine pathway. Furthermore, macrophages loaded with SOC-paclitaxel (PTX) particles exhibited greater therapeutic efficacies than those of macrophages directly carrying small molecular drugs such as doxorubicin and PTX. Interestingly, macrophages displayed stronger targeting ability to the tumor site hypersecreting chemokine in immunocompetent mice in comparison to the tumor site secreting low levels of chemokine in immunodeficiency mice. Finally, results demonstrated that macrophages carrying SOC-PTX are a promising pharmaceutical preparation for tumor-targeted therapy.

  14. Goniothalamin enhances the ATPase activity of the molecular chaperone Hsp90 but inhibits its chaperone activity.

    PubMed

    Yokoyama, Yuhei; Ohtaki, Aguru; Jantan, Ibrahim; Yohda, Masafumi; Nakamoto, Hitoshi

    2015-03-01

    Hsp90 is an ATP-dependent molecular chaperone that is involved in important cellular pathways such as signal transduction pathways. It is a potential cancer drug target because it plays a critical role for stabilization and activation of oncoproteins. Thus, small molecule compounds that control the Hsp90 function are useful to elucidate potential lead compounds against cancer. We studied effect of a naturally occurring styryl-lactone goniothalamin on the activity of Hsp90. Although many drugs targeting Hsp90 inhibit the ATPase activity of Hsp90, goniothalamin enhanced rather than inhibited the ATPase activity of a cyanobacterial Hsp90 (HtpG) and a yeast Hsp90. It increased both K(m) and k(cat) of the Hsp90s. Domain competition assays and tryptophan fluorescence measurements with various truncated derivatives of HtpG indicated that goniothalamin binds to the N-terminal domain of HtpG. Goniothalamin did not influence on the interaction of HtpG with a non-native protein or the anti-aggregation activity of HtpG significantly. However, it inhibited the activity of HtpG that assists refolding of a non-native protein in cooperation with the Hsp70 chaperone system. This is the first report to show that a small molecule that binds to the N-terminal domain of Hsp90 activates its ATPase activity, while inhibiting the chaperone function of Hsp90.

  15. Monocyte and Macrophage Dynamics during Atherogenesis

    PubMed Central

    Ley, Klaus; Miller, Yury I.; Hedrick, Catherine C.

    2011-01-01

    Vascular inflammation is associated with and in large part driven by changes in the leukocyte compartment of the vessel wall. Here, we focus on monocyte influx during atherosclerosis, the most common form of vascular inflammation. Although the arterial wall contains a large number of resident macrophages and some resident dendritic cells, atherosclerosis drives a rapid influx of inflammatory monocytes (Ly-6C+ in mice) and other monocytes (Ly-6C− in mice, also known as patrolling monocytes). Once in the vessel wall, Ly-6C+ monocytes differentiate to a phenotype consistent with inflammatory macrophages and inflammatory dendritic cells. The phenotype of these cells is modulated by lipid uptake, Toll-like receptor ligands, hematopoietic growth factors, cytokines and chemokines. In addition to newly recruited macrophages, it is likely that resident macrophages also change their phenotype. Monocyte-derived inflammatory macrophages have a short half-life. After undergoing apoptosis, they may be taken up by surrounding macrophages or, if the phagocytic capacity is overwhelmed, can undergo secondary necrosis, a key event in forming the necrotic core of atherosclerotic lesions. In this review, we discuss these and other processes associated with monocytic cell dynamics in the vascular wall and their role in the initiation and progression of atherosclerosis. PMID:21677293

  16. Metabolism Supports Macrophage Activation

    PubMed Central

    Langston, P. Kent; Shibata, Munehiko; Horng, Tiffany

    2017-01-01

    Macrophages are found in most tissues of the body, where they have tissue- and context-dependent roles in maintaining homeostasis as well as coordinating adaptive responses to various stresses. Their capacity for specialized functions is controlled by polarizing signals, which activate macrophages by upregulating transcriptional programs that encode distinct effector functions. An important conceptual advance in the field of macrophage biology, emerging from recent studies, is that macrophage activation is critically supported by metabolic shifts. Metabolic shifts fuel multiple aspects of macrophage activation, and preventing these shifts impairs appropriate activation. These findings raise the exciting possibility that macrophage functions in various contexts could be regulated by manipulating their metabolism. Here, we review the rapidly evolving field of macrophage metabolism, discussing how polarizing signals trigger metabolic shifts and how these shifts enable appropriate activation and sustain effector activities. We also discuss recent studies indicating that the mitochondria are central hubs in inflammatory macrophage activation. PMID:28197151

  17. Unfolded protein response (UPR) signaling regulates arsenic trioxide-mediated macrophage innate immune function disruption

    SciTech Connect

    Srivastava, Ritesh K.; Li, Changzhao; Chaudhary, Sandeep C.; Ballestas, Mary E.; Elmets, Craig A.; Robbins, David J.; Matalon, Sadis; Deshane, Jessy S.; Afaq, Farrukh; Bickers, David R.; Athar, Mohammad

    2013-11-01

    Arsenic exposure is known to disrupt innate immune functions in humans and in experimental animals. In this study, we provide a mechanism by which arsenic trioxide (ATO) disrupts macrophage functions. ATO treatment of murine macrophage cells diminished internalization of FITC-labeled latex beads, impaired clearance of phagocytosed fluorescent bacteria and reduced secretion of pro-inflammatory cytokines. These impairments in macrophage functions are associated with ATO-induced unfolded protein response (UPR) signaling pathway characterized by the enhancement in proteins such as GRP78, p-PERK, p-eIF2α, ATF4 and CHOP. The expression of these proteins is altered both at transcriptional and translational levels. Pretreatment with chemical chaperon, 4-phenylbutyric acid (PBA) attenuated the ATO-induced activation in UPR signaling and afforded protection against ATO-induced disruption of macrophage functions. This treatment also reduced ATO-mediated reactive oxygen species (ROS) generation. Interestingly, treatment with antioxidant N-acetylcysteine (NAC) prior to ATO exposure, not only reduced ROS production and UPR signaling but also improved macrophage functions. These data demonstrate that UPR signaling and ROS generation are interdependent and are involved in the arsenic-induced pathobiology of macrophage. These data also provide a novel strategy to block the ATO-dependent impairment in innate immune responses. - Highlights: • Inorganic arsenic to humans and experimental animals disrupt innate immune responses. • The mechanism underlying arsenic impaired macrophage functions involves UPR signaling. • Chemical chaperon attenuates arsenic-mediated macrophage function impairment. • Antioxidant, NAC blocks impairment in arsenic-treated macrophage functions.

  18. Macrophage-derived foam cells freshly isolated from rabbit atherosclerotic lesions degrade modified lipoproteins, promote oxidation of low-density lipoproteins, and contain oxidation-specific lipid-protein adducts.

    PubMed Central

    Rosenfeld, M E; Khoo, J C; Miller, E; Parthasarathy, S; Palinski, W; Witztum, J L

    1991-01-01

    Pure macrophage-derived foam cells (MFC) were isolated from the aortas of rabbits made atherosclerotic by balloon deendothelialization followed by diet-induced hypercholesterolemia. The MFC were isolated under sterile conditions using an enzymatic digestion procedure and discontinuous density gradient centrifugation. The purity of the MFC preparations was verified immunocytochemically with the macrophage specific monoclonal antibody RAM-11. MFC plated in medium containing 0.5% FCS for 24 h contained approximately 600 micrograms cholesterol per mg cell protein, 80% of which was esterified cholesterol. The MFC specifically degraded low density lipoprotein (LDL), acetyl-LDL, copper oxidized LDL, and beta-very low density lipoprotein (beta-VLDL) at rates comparable to mouse peritoneal macrophages (MPM) in 5-h assays. MFC within sections of the atherosclerotic lesions from the ballooned rabbits as well as the MFC isolated from the same lesions in the presence of antioxidants, exhibited positive immunoreactivity with polyclonal guinea pig antisera and mouse monoclonal antibodies directed against malondialdehyde-LDL, and 4-hydroxynonal-LDL. The MFC also exhibited the capacity to induce the oxidation of LDL at rates comparable to those exhibited by MPM and rabbit aortic endothelial cells. These data provide direct evidence that arterial wall macrophages express modified LDL receptors in vivo, contain epitopes found in oxidized-LDL and are capable of oxidizing LDL even when maximally loaded with cholesterol. Images PMID:1985115

  19. Histone chaperones: assisting histone traffic and nucleosome dynamics.

    PubMed

    Gurard-Levin, Zachary A; Quivy, Jean-Pierre; Almouzni, Geneviève

    2014-01-01

    The functional organization of eukaryotic DNA into chromatin uses histones as components of its building block, the nucleosome. Histone chaperones, which are proteins that escort histones throughout their cellular life, are key actors in all facets of histone metabolism; they regulate the supply and dynamics of histones at chromatin for its assembly and disassembly. Histone chaperones can also participate in the distribution of histone variants, thereby defining distinct chromatin landscapes of importance for genome function, stability, and cell identity. Here, we discuss our current knowledge of the known histone chaperones and their histone partners, focusing on histone H3 and its variants. We then place them into an escort network that distributes these histones in various deposition pathways. Through their distinct interfaces, we show how they affect dynamics during DNA replication, DNA damage, and transcription, and how they maintain genome integrity. Finally, we discuss the importance of histone chaperones during development and describe how misregulation of the histone flow can link to disease.

  20. Chaperone-assisted refolding of Escherichia coli maltodextrin glucosidase.

    PubMed

    Paul, Subhankar; Punam, Shashikala; Chaudhuri, Tapan K

    2007-11-01

    In vitro refolding of maltodextrin glucosidase, a 69 kDa monomeric Escherichia coli protein, was studied in the presence of glycerol, dimethylsulfoxide, trimethylamine-N-oxide, ethylene glycol, trehalose, proline and chaperonins GroEL and GroES. Different osmolytes, namely proline, glycerol, trimethylamine-N-oxide and dimethylsulfoxide, also known as chemical chaperones, assist in protein folding through effective inhibition of the aggregation process. In the present study, it was observed that a few chemical chaperones effectively reduced the aggregation process of maltodextrin glucosidase and hence the in vitro refolding was substantially enhanced, with ethylene glycol being the exception. Although, the highest recovery of active maltodextrin glucosidase was achieved through the ATP-mediated GroEL/GroES-assisted refolding of denatured protein, the yield of correctly folded protein from glycerol- or proline-assisted spontaneous refolding process was closer to the chaperonin-assisted refolding. It was also observed that the combined application of chemical chaperones and molecular chaperone was more productive than their individual contribution towards the in vitro refolding of maltodextrin glucosidase. The chemical chaperones, except ethylene glycol, were found to provide different degrees of protection to maltodextrin glucosidase from thermal denaturation, whereas proline caused the highest protection. The observations from the present studies conclusively demonstrate that chemical or molecular chaperones, or the combination of both chaperones, could be used in the efficient refolding of recombinant E. coli maltodextrin glucosidase, which enhances the possibility of identifying or designing suitable small molecules that can act as chemical chaperones in the efficient refolding of various aggregate-prone proteins of commercial and medical importance.

  1. Liposomes as chaperone mimics with controllable affinity toward heat-denatured formate dehydrogenase from Candida boidinii.

    PubMed

    Yoshimoto, Makoto; Kozono, Ryohei; Tsubomura, Naoki

    2015-01-20

    Chaperone machinery in living systems can catch denatured enzymes and induce their reactivation. Chaperone mimics are beneficial for applying enzymatic reactions in vitro. In this work, the affinity between liposomes and thermally denatured enzymes was controlled to stabilize the enzyme activity. The model enzyme is formate dehydrogenase from Candida boidinii (CbFDH) which is a homodimer and negatively charged in the phosphate buffer solution (pH 7.2) used. The activity of free CbFDH readily decreased at 58 °C following the first-order kinetics with the half-life t1/2 of 27 min. The turbidity measurements showed that the denatured enzyme molecules formed aggregates. The liposomes composed of zwitterionic phosphatidylcholines (PCs) stabilized the CbFDH activity at 58 °C, as revealed with six different PCs. The PC liposomes were indicated to bind to the aggregate-prone enzyme molecules, allowing reactivation at 25 °C. The cofactor β-reduced nicotinamide adenine dinucleotide (NADH) also stabilized the enzyme activity. The affinity between liposomes and denatured CbFDH could be modulated by incorporating cationic 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP) in PC membranes. The t1/2 values significantly increased in the presence of liposomes ([lipid] = 1.5 mM) composed of PC and DOTAP at the mole fraction f(D) of 0.1. On the other hand, the DOTAP-rich liposomes (f(D) ≥ 0.7) showed strong affinity toward denatured CbFDH, accelerating its deactivation. The liposomes with low charge density function as chaperone mimics that can efficiently catch the denatured enzymes without interfering with their intramolecular interaction for reactivation.

  2. Structural basis for the antifolding activity of a molecular chaperone

    PubMed Central

    Huang, Chengdong; Rossi, Paolo; Saio, Tomohide; Kalodimos, Charalampos G.

    2016-01-01

    Molecular chaperones act on non-native proteins in the cell to prevent their aggregation, premature folding or misfolding. Different chaperones often exert distinct effects, such as acceleration or delay of folding, on client proteins via mechanisms that are poorly understood. Here we report the solution structure of SecB, a chaperone that exhibits strong antifolding activity, in complex with alkaline phosphatase (PhoA) and maltose binding protein (MBP) captured in their unfolded states. SecB uses long hydrophobic grooves that run around its disk-like shape to recognize and bind to multiple hydrophobic segments across the length of the non-native proteins. The multivalent binding mode results in proteins wrapping around SecB. This unique complex architecture alters the kinetics of protein binding to SecB and confers strong antifolding activity on the chaperone. The data show how the different architectures of chaperones result in distinct binding modes with non-native proteins that ultimately define the activity of the chaperone. PMID:27501151

  3. Structural basis for the antifolding activity of a molecular chaperone

    NASA Astrophysics Data System (ADS)

    Huang, Chengdong; Rossi, Paolo; Saio, Tomohide; Kalodimos, Charalampos G.

    2016-09-01

    Molecular chaperones act on non-native proteins in the cell to prevent their aggregation, premature folding or misfolding. Different chaperones often exert distinct effects, such as acceleration or delay of folding, on client proteins via mechanisms that are poorly understood. Here we report the solution structure of SecB, a chaperone that exhibits strong antifolding activity, in complex with alkaline phosphatase and maltose-binding protein captured in their unfolded states. SecB uses long hydrophobic grooves that run around its disk-like shape to recognize and bind to multiple hydrophobic segments across the length of non-native proteins. The multivalent binding mode results in proteins wrapping around SecB. This unique complex architecture alters the kinetics of protein binding to SecB and confers strong antifolding activity on the chaperone. The data show how the different architectures of chaperones result in distinct binding modes with non-native proteins that ultimately define the activity of the chaperone.

  4. Specificity of Intramembrane Protein–Lipid Interactions

    PubMed Central

    Contreras, Francesc-Xabier; Ernst, Andreas Max; Wieland, Felix; Brügger, Britta

    2011-01-01

    Our concept of biological membranes has markedly changed, from the fluid mosaic model to the current model that lipids and proteins have the ability to separate into microdomains, differing in their protein and lipid compositions. Since the breakthrough in crystallizing membrane proteins, the most powerful method to define lipid-binding sites on proteins has been X-ray and electron crystallography. More recently, chemical biology approaches have been developed to analyze protein–lipid interactions. Such methods have the advantage of providing highly specific cellular probes. With the advent of novel tools to study functions of individual lipid species in membranes together with structural analysis and simulations at the atomistic resolution, a growing number of specific protein–lipid complexes are defined and their functions explored. In the present article, we discuss the various modes of intramembrane protein–lipid interactions in cellular membranes, including examples for both annular and nonannular bound lipids. Furthermore, we will discuss possible functional roles of such specific protein–lipid interactions as well as roles of lipids as chaperones in protein folding and transport. PMID:21536707

  5. Plasma membrane appearance of phosphatidylethanolamine in stimulated macrophages

    SciTech Connect

    Sandra, A.; Cai, J. )

    1991-07-01

    Mouse peritoneal macrophages were labeled with (1-3H)ethanolamine, and the presence of radioactive (3H)phosphatidylethanolamine (PE) at the plasma membrane was monitored by reacting the cells with trinitrobenzene sulfonic acid (TNBS) under nonpenetrating conditions. Macrophages stimulated with either the calcium ionophore A23187 or zymosan demonstrated a larger proportion of radiolabeled PE in the plasma membrane than control, nonstimulated cells. In experiments in which macrophages were labeled with ethanolamine for increasing times, appearance of membrane 3(H)PE was stimulated as early as after 2 hr of labeling. Macrophages labeled for 24 hr, then stimulated and returned to fresh medium still reflected a higher amount of membrane 3(H)PE at 2 hr after the stimulation, suggesting stimulation results in long-term alterations in plasma membrane lipids. Protease-peptone-elicited macrophages, which are not stimulated by zymosan or ionophore, did not exhibit an increase in membrane 3(H)PE upon stimulation. The size of the TNBS-accessible radiolabeled PE pool increased proportionately with a second stimulation; however, a subsequent labeling of the cells with TNBS after brief warming increased the TNBS-accessible pool in control cells only. As shown in previous studies, macrophage stimulation resulted in an increased incorporation of lipid precursors into phospholipid. The mass of plasma membrane Tnp-PE relative to mass of PE was not increased in ionophore-treated macrophages in contrast to a small (approximately 22%) increase in zymosan-treated cells. These results are suggestive of alterations in lipid synthesis in stimulated macrophages and possible long-term changes in the structure and function of the plasma membrane of macrophages following stimulation.

  6. Molecular chaperone-mediated nuclear protein dynamics.

    PubMed

    Echtenkamp, Frank J; Freeman, Brian C

    2014-05-01

    Homeostasis requires effective action of numerous biological pathways including those working along a genome. The variety of processes functioning in the nucleus is considerable, yet the number of employed factors eclipses this total. Ideally, individual components assemble into distinct complexes and serially operate along a pathway to perform work. Adding to the complexity is a multitude of fluctuating internal and external signals that must be monitored to initiate, continue or halt individual activities. While cooperative interactions between proteins of the same process provide a mechanism for rapid and precise assembly, the inherent stability of such organized structures interferes with the proper timing of biological events. Further prolonging the longevity of biological complexes are crowding effects resulting from the high concentration of intracellular macromolecules. Hence, accessory proteins are required to destabilize the various assemblies to efficiently transition between structures, avoid off-pathway competitive interactions, and to terminate pathway activity. We suggest that molecular chaperones have evolved, in part, to manage these challenges by fostering a general and continuous dynamic protein environment within the nucleus.

  7. The Role of Macrophage Lipophagy in Reverse Cholesterol Transport

    PubMed Central

    2017-01-01

    Macrophage cholesterol efflux is a central step in reverse cholesterol transport, which helps to maintain cholesterol homeostasis and to reduce atherosclerosis. Lipophagy has recently been identified as a new step in cholesterol ester hydrolysis that regulates cholesterol efflux, since it mobilizes cholesterol from lipid droplets of macrophages via autophagy and lysosomes. In this review, we briefly discuss recent advances regarding the mechanisms of the cholesterol efflux pathway in macrophage foam cells, and present lipophagy as a therapeutic target in the treatment of atherosclerosis. PMID:28345315

  8. Macrophage elastase kills bacteria within murine macrophages.

    PubMed

    Houghton, A McGarry; Hartzell, William O; Robbins, Clinton S; Gomis-Rüth, F Xavier; Shapiro, Steven D

    2009-07-30

    Macrophages are aptly positioned to function as the primary line of defence against invading pathogens in many organs, including the lung and peritoneum. Their ability to phagocytose and clear microorganisms has been well documented. Macrophages possess several substances with which they can kill bacteria, including reactive oxygen species, nitric oxide, and antimicrobial proteins. We proposed that macrophage-derived proteinases may contribute to the antimicrobial properties of macrophages. Macrophage elastase (also known as matrix metalloproteinase 12 or MMP12) is an enzyme predominantly expressed in mature tissue macrophages and is implicated in several disease processes, including emphysema. Physiological functions for MMP12 have not been described. Here we show that Mmp12(-/-) mice exhibit impaired bacterial clearance and increased mortality when challenged with both gram-negative and gram-positive bacteria at macrophage-rich portals of entry, such as the peritoneum and lung. Intracellular stores of MMP12 are mobilized to macrophage phagolysosomes after the ingestion of bacterial pathogens. Once inside phagolysosomes, MMP12 adheres to bacterial cell walls where it disrupts cellular membranes resulting in bacterial death. The antimicrobial properties of MMP12 do not reside within its catalytic domain, but rather within the carboxy-terminal domain. This domain contains a unique four amino acid sequence on an exposed beta loop of the protein that is required for the observed antimicrobial activity. The present study represents, to our knowledge, the first report of direct antimicrobial activity by a matrix metallopeptidase, and describes a new antimicrobial peptide that is sequentially and structurally unique in nature.

  9. Effects of pH and Iminosugar Pharmacological Chaperones on Lysosomal Glycosidase Structure and Stability

    SciTech Connect

    Lieberman, Raquel L.; D’aquino, J. Alejandro; Ringe, Dagmar; Petsko, Gregory A.

    2009-06-05

    Human lysosomal enzymes acid-{beta}-glucosidase (GCase) and acid-{alpha}-galactosidase ({alpha}-Gal A) hydrolyze the sphingolipids glucosyl- and globotriaosylceramide, respectively, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease, respectively. We have investigated the structure and stability of GCase and {alpha}-Gal A in a neutral-pH environment reflective of the endoplasmic reticulum and an acidic-pH environment reflective of the lysosome. These details are important for the development of pharmacological chaperone therapy for Gaucher and Fabry disease, in which small molecules bind mutant enzymes in the ER to enable the mutant enzyme to meet quality control requirements for lysosomal trafficking. We report crystal structures of apo GCase at pH 4.5, at pH 5.5, and in complex with the pharmacological chaperone isofagomine (IFG) at pH 7.5. We also present thermostability analysis of GCase at pH 7.4 and 5.2 using differential scanning calorimetry. We compare our results with analogous experiments using {alpha}-Gal A and the chaperone 1-deoxygalactonijirimycin (DGJ), including the first structure of {alpha}-Gal A with DGJ. Both GCase and {alpha}-Gal A are more stable at lysosomal pH with and without their respective iminosugars bound, and notably, the stability of the GCase-IFG complex is pH sensitive. We show that the conformations of the active site loops in GCase are sensitive to ligand binding but not pH, whereas analogous galactose- or DGJ-dependent conformational changes in {alpha}-Gal A are not seen. Thermodynamic parameters obtained from {alpha}-Gal A unfolding indicate two-state, van't Hoff unfolding in the absence of the iminosugar at neutral and lysosomal pH, and non-two-state unfolding in the presence of DGJ. Taken together, these results provide insight into how GCase and {alpha}-Gal A are thermodynamically stabilized by iminosugars and suggest strategies for the development of new pharmacological

  10. Molecular chaperones encoded by a reduced nucleus: the cryptomonad nucleomorph.

    PubMed

    Archibald, J M; Cavalier-Smith, T; Maier, U; Douglas, S

    2001-06-01

    Molecular chaperones mediate the correct folding of nascent or denatured proteins and are found in both the organelles and cytoplasm of eukaryotic cells. Cryptomonad algae are unusual in possessing an extra cytoplasmic compartment (the periplastid space), the result of having engulfed and retained a photosynthetic eukaryote. Within the periplastid space is a diminutive nucleus (the nucleomorph) that encodes mostly genes for its own expression as well as a few needed by the plastid. Two plastid-encoded chaperones (GroEL and DnaK) and a nucleomorph-encoded chaperone (Cpn60) have been reported from the cryptomonad, Guillardia theta. Here we analyse G. theta nucleomorph genes for members of the cytosolic HSP70 and HSP90 families of molecular chaperones, a heat shock transcription factor (HSF), and all eight subunits of the group II chaperonin, CCT. These are presumably all active in the periplastid space, assisting in the maturation of polypeptides required by the cell; we propose a central role for them also in the structure and assembly of a putative relict mitotic apparatus. Curiously, none of the genes for co-chaperones of HSP70, HSP90, or CCT have been detected in the nucleomorph genome; they are either not needed or are encoded in the host nuclear genome and targeted back into the periplastid space. Endoplasmic reticulum (ER) homologs of HSP70 and HSP90 are also not present. Striking differences in the degree of conservation of the various nucleomorph-encoded molecular chaperones were observed. While the G. theta HSP70 and HSP90 homologs are well conserved, each of the eight CCT subunits (alpha, beta, gamma, delta, epsilon, eta, theta, and zeta) is remarkably divergent. Such differences are likely evidence for reduced/different functional constraints on the various molecular chaperones functioning in the periplastid space.

  11. Increased cellular free cholesterol in macrophage-specific Abca1 knock-out mice enhances pro-inflammatory response of macrophages.

    PubMed

    Zhu, Xuewei; Lee, Ji-Young; Timmins, Jenelle M; Brown, J Mark; Boudyguina, Elena; Mulya, Anny; Gebre, Abraham K; Willingham, Mark C; Hiltbold, Elizabeth M; Mishra, Nilamadhab; Maeda, Nobuyo; Parks, John S

    2008-08-22

    Macrophage-specific Abca1 knock-out (Abca1(-)(M)(/-)(M)) mice were generated to determine the role of macrophage ABCA1 expression in plasma lipoprotein concentrations and the innate immune response of macrophages. Plasma lipid and lipoprotein concentrations in chow-fed Abca1(-)(M)(/-)(M) and wild-type (WT) mice were indistinguishable. Compared with WT macrophages, Abca1(-)(M)(/-)(M) macrophages had a >95% reduction in ABCA1 protein, failed to efflux lipid to apoA-I, and had a significant increase in free cholesterol (FC) and membrane lipid rafts without induction of endoplasmic reticulum stress. Lipopolysaccharide (LPS)-treated Abca1(-)(M)(/-)(M) macrophages exhibited enhanced expression of pro-inflammatory cytokines and increased activation of the NF-kappaB and MAPK pathways, which could be diminished by silencing MyD88 or by chemical inhibition of NF-kappaB or MAPK. In vivo LPS injection also resulted in a higher pro-inflammatory response in Abca1(-)(M)(/-)(M) mice compared with WT mice. Furthermore, cholesterol depletion of macrophages with methyl-beta-cyclodextrin normalized FC content between the two genotypes and their response to LPS; cholesterol repletion of macrophages resulted in increased cellular FC accumulation and enhanced cellular response to LPS. Our results suggest that macrophage ABCA1 expression may protect against atherosclerosis by facilitating the net removal of excess lipid from macrophages and dampening pro-inflammatory MyD88-dependent signaling pathways by reduction of cell membrane FC and lipid raft content.

  12. A New Glucocerebrosidase Chaperone Reduces α-Synuclein and Glycolipid Levels in iPSC-Derived Dopaminergic Neurons from Patients with Gaucher Disease and Parkinsonism

    PubMed Central

    Aflaki, Elma; Borger, Daniel K.; Moaven, Nima; Stubblefield, Barbara K.; Rogers, Steven A.; Patnaik, Samarjit; Schoenen, Frank J.; Westbroek, Wendy; Zheng, Wei; Sullivan, Patricia; Fujiwara, Hideji; Sidhu, Rohini; Khaliq, Zayd M; Lopez, Grisel J.; Goldstein, David S.; Ory, Daniel S.; Marugan, Juan

    2016-01-01

    Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT Because GBA1 mutations are the most common

  13. Lipoxygenase products mediate the attachment of rat macrophages to glomeruli in vitro

    SciTech Connect

    Baud, L.; Sraer, J.; Delarue, F.; Bens, M.; Balavoine, F.; Schlondorff, D.; Ardaillou, R.; Sraer, J.D.

    1985-06-01

    Because there is an accumulation of macrophages in the Bowman's space during human and experimental glomerulonephritis, the authors have studied the binding of (/sup 3/H)-uridine labeled macrophages to isolated glomeruli. Binding was related to the glomerular protein and macrophage concentrations, temperature, time of incubation, and was a saturable process. Macrophage adherence depended on glomerular lipoxygenase activity but not on glomerular cyclooxygenase activity since preincubation of glomeruli with nordihydroguaiaretic acid (NDGA) inhibited this phenomenon whereas preincubation with indomethacin was ineffective. Glomeruli interacted with macrophages in converting arachidonic acid (C20:4) to prostaglandins (PG) since productions of 6 keto-PGF1 alpha, TXB2, and PGD2 by glomeruli and macrophages incubated in combination were much greater than the sums of their respective productions by glomeruli and macrophages incubated separately. Macrophages were the source of the supplementary synthesis of PG which was abolished when these cells were pretreated with aspirin. Stimulation of macrophages by glomeruli was blunted by pretreatment of glomeruli with NDGA. Production of PG and of 12-HETE by macrophages was stimulated by a lipid extract of glomeruli containing the oxygenated metabolites of C20:4. Direct addition of 12-HPETE also stimulated macrophage functions. These data suggest that macrophage attachment to glomeruli and macrophage stimulation in the presence of glomeruli depend on glomerular lipoxygenase activity.

  14. Inhibitors of the AAA+ Chaperone p97

    PubMed Central

    Chapman, Eli; Maksim, Nick; de la Cruz, Fabian; La Clair, James J.

    2015-01-01

    It is remarkable that a pathway as ubiquitous as protein quality control can be targeted to treat cancer. Bortezomib, an inhibitor of the proteasome, was first approved by the US Food and Drug Administration (FDA) more than 10 years ago to treat refractory myeloma and later extended to lymphoma. Its use has increased the survival rate of myeloma patients by as much as three years. This success was followed with the recent accelerated approval of the natural product derived proteasome inhibitor carfilzomib (Kyprolis®), which is used to treat patients with bortezomib-resistant multiple myeloma. The success of these two drugs has validated protein quality control as a viable target to fight select cancers, but begs the question why are proteasome inhibitors limited to lymphoma and myeloma? More recently, these limitations have encouraged the search for additional targets within the protein quality control system that might offer heightened cancer cell specificity, enhanced clinical utility, a lower rate of resistance, reduced toxicity, and mitigated side effects. One promising target is p97, an ATPase associated with various cellular activities (AAA+) chaperone. p97 figures prominently in protein quality control as well as serving a variety of other cellular functions associated with cancer. More than a decade ago, it was determined that up-regulation of p97 in many forms of cancer correlates with a poor clinical outcome. Since these initial discoveries, a mechanistic explanation for this observation has been partially illuminated, but details are lacking. Understandably, given this clinical correlation, myriad roles within the cell, and its importance in protein quality control, p97 has emerged as a potential therapeutic target. This review provides an overview of efforts towards the discovery of small molecule inhibitors of p97, offering a synopsis of efforts that parallel the excellent reviews that currently exist on p97 structure, function, and physiology. PMID

  15. A Novel Method for Assessing the Chaperone Activity of Proteins

    PubMed Central

    Hristozova, Nevena; Tompa, Peter; Kovacs, Denes

    2016-01-01

    Protein chaperones are molecular machines which function both during homeostasis and stress conditions in all living organisms. Depending on their specific function, molecular chaperones are involved in a plethora of cellular processes by playing key roles in nascent protein chain folding, transport and quality control. Among stress protein families–molecules expressed during adverse conditions, infection, and diseases–chaperones are highly abundant. Their molecular functions range from stabilizing stress-susceptible molecules and membranes to assisting the refolding of stress-damaged proteins, thereby acting as protective barriers against cellular damage. Here we propose a novel technique to test and measure the capability for protective activity of known and putative chaperones in a semi-high throughput manner on a plate reader. The current state of the art does not allow the in vitro measurements of chaperone activity in a highly parallel manner with high accuracy or high reproducibility, thus we believe that the method we report will be of significant benefit in this direction. The use of this method may lead to a considerable increase in the number of experimentally verified proteins with such functions, and may also allow the dissection of their molecular mechanism for a better understanding of their function. PMID:27564234

  16. Dissecting the Escherichia coli periplasmic chaperone network using differential proteomics

    PubMed Central

    Vertommen, Didier; Silhavy, Thomas J.; Collet, Jean-Francois

    2013-01-01

    β-barrel proteins, or outer membrane proteins (OMPs), perform many essential functions in Gram-negative bacteria, but questions remain about the mechanism by which they are assembled into the outer membrane (OM). In Escherichia coli, β-barrels are escorted across the periplasm by chaperones, most notably SurA and Skp. However, the contributions of these two chaperones to the assembly of the OM proteome remained unclear. We used differential proteomics to determine how the elimination of Skp and SurA affects the assembly of many OMPs. We have shown that removal of Skp has no impact on the levels of the 63 identified OM proteins. However, depletion of SurA in the skp strain has a marked impact on the OM proteome, diminishing the levels of almost all β-barrel proteins. Our results are consistent with a model in which SurA plays a primary chaperone role in E. coli. Furthermore, they suggest that while no OMPs prefer the Skp chaperone pathway in wild-type cells, most can use Skp efficiently when SurA is absent. Our data, which provide a unique glimpse into the protein content of the non-viable surA skp mutant, clarify the roles of the periplasmic chaperones in E. coli. PMID:22589188

  17. Enhancement of Anti-Inflammatory Activity of Curcumin Using Phosphatidylserine-Containing Nanoparticles in Cultured Macrophages.

    PubMed

    Wang, Ji; Kang, Yu-Xia; Pan, Wen; Lei, Wan; Feng, Bin; Wang, Xiao-Juan

    2016-06-20

    Macrophages are one kind of innate immune cells, and produce a variety of inflammatory cytokines in response to various stimuli, such as oxidized low density lipoprotein found in the pathogenesis of atherosclerosis. In this study, the effect of phosphatidylserine on anti-inflammatory activity of curcumin-loaded nanostructured lipid carriers was investigated using macrophage cultures. Different amounts of phosphatidylserine were used in the preparation of curcumin nanoparticles, their physicochemical properties and biocompatibilities were then compared. Cellular uptake of the nanoparticles was investigated using a confocal laser scanning microscope and flow cytometry analysis in order to determine the optimal phosphatidylserine concentration. In vitro anti-inflammatory activities were evaluated in macrophages to test whether curcumin and phosphatidylserine have interactive effects on macrophage lipid uptake behavior and anti-inflammatory responses. Here, we showed that macrophage uptake of phosphatidylserine-containing nanostructured lipid carriers increased with increasing amount of phosphatidylserine in the range of 0%-8%, and decreased when the phosphatidylserine molar ratio reached over 12%. curcumin-loaded nanostructured lipid carriers significantly inhibited lipid accumulation and pro-inflammatory factor production in cultured macrophages, and evidently promoted release of anti-inflammatory cytokines, when compared with curcumin or phosphatidylserine alone. These results suggest that the delivery system using PS-based nanoparticles has great potential for efficient delivery of drugs such as curcumin, specifically targeting macrophages and modulation of their anti-inflammatory functions.

  18. Deep Transcriptomic Profiling of M1 Macrophages Lacking Trpc3

    PubMed Central

    Kumarasamy, Sivarajan; Solanki, Sumeet; Atolagbe, Oluwatomisin T.; Joe, Bina; Birnbaumer, Lutz; Vazquez, Guillermo

    2017-01-01

    In previous studies using mice with macrophage-specific loss of TRPC3 we found a significant, selective effect of TRPC3 on the biology of M1, or inflammatory macrophages. Whereas activation of some components of the unfolded protein response and the pro-apoptotic mediators CamkII and Stat1 was impaired in Trpc3-deficient M1 cells, gathering insight about other molecular signatures within macrophages that might be affected by Trpc3 expression requires an alternative approach. In the present study we conducted RNA-seq analysis to interrogate the transcriptome of M1 macrophages derived from mice with macrophage-specific loss of TRPC3 and their littermate controls. We identified 160 significantly differentially expressed genes between the two groups, of which 62 were upregulated and 98 downregulated in control vs. Trpc3-deficient M1 macrophages. Gene ontology analysis revealed enrichment in processes associated to cellular movement and lipid signaling, whereas the enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways included networks for calcium signaling and cell adhesion molecules, among others. This is the first deep transcriptomic analysis of macrophages in the context of Trpc3 deficiency and the data presented constitutes a unique resource to further explore functions of TRPC3 in macrophage biology. PMID:28051144

  19. Control of cell cycle and cell growth by molecular chaperones.

    PubMed

    Aldea, Martí; Garí, Eloi; Colomina, Neus

    2007-11-01

    Cells adapt their size to both intrinsic and extrinsic demands and, among them, those that stem from growth and proliferation rates are crucial for cell size homeostasis. Here we revisit mechanisms that regulate cell cycle and cell growth in budding yeast. Cyclin Cln3, the most upstream activator of Start, is retained at the endoplasmic reticulum in early G(1) and released by specific chaperones in late G(1) to initiate the cell cycle. On one hand, these chaperones are rate-limiting for release of Cln3 and cell cycle entry and, on the other hand, they are required for key biosynthetic processes. We propose a model whereby the competition for specialized chaperones between growth and cycle machineries could gauge biosynthetic rates and set a critical size threshold at Start.

  20. ER chaperones in neurodegenerative disease: Folding and beyond.

    PubMed

    Garcia-Huerta, Paula; Bargsted, Leslie; Rivas, Alexis; Matus, Soledad; Vidal, Rene L

    2016-10-01

    Proteins along the secretory pathway are co-translationally translocated into the lumen of the endoplasmic reticulum (ER) as unfolded polypeptide chains. Afterwards, they are usually modified with N-linked glycans, correctly folded and stabilized by disulfide bonds. ER chaperones and folding enzymes control these processes. The accumulation of unfolded proteins in the ER activates a signaling response, termed the unfolded protein response (UPR). The hallmark of this response is the coordinated transcriptional up-regulation of ER chaperones and folding enzymes. In order to discuss the importance of the proper folding of certain substrates we will address the role of ER chaperones in normal physiological conditions and examine different aspects of its contribution in neurodegenerative disease. This article is part of a Special Issue entitled SI:ER stress.

  1. The conformational dynamics of the mitochondrial Hsp70 chaperone.

    PubMed

    Mapa, Koyeli; Sikor, Martin; Kudryavtsev, Volodymyr; Waegemann, Karin; Kalinin, Stanislav; Seidel, Claus A M; Neupert, Walter; Lamb, Don C; Mokranjac, Dejana

    2010-04-09

    Heat shock proteins 70 (Hsp70) represent a ubiquitous and conserved family of molecular chaperones involved in a plethora of cellular processes. The dynamics of their ATP hydrolysis-driven and cochaperone-regulated conformational cycle are poorly understood. We used fluorescence spectroscopy to analyze, in real time and at single-molecule resolution, the effects of nucleotides and cochaperones on the conformation of Ssc1, a mitochondrial member of the family. We report that the conformation of its ADP state is unexpectedly heterogeneous, in contrast to a uniform ATP state. Substrates are actively involved in determining the conformation of Ssc1. The J protein Mdj1 does not interact transiently with the chaperone, as generally believed, but rather is released slowly upon ATP hydrolysis. Analysis of the major bacterial Hsp70 revealed important differences between highly homologous members of the family, possibly explaining tuning of Hsp70 chaperones to meet specific functions in different organisms and cellular compartments.

  2. Chaperone-rich tumor cell lysate-mediated activation of antigen-presenting cells resists regulatory T cell suppression.

    PubMed

    Larmonier, Nicolas; Cantrell, Jessica; Lacasse, Collin; Li, Gang; Janikashvili, Nona; Situ, Elaine; Sepassi, Marjan; Andreansky, Samita; Katsanis, Emmanuel

    2008-04-01

    CD4(+)CD25(+) regulatory T lymphocytes (Tregs) critically contribute to the mechanisms of cancer-induced tolerance. These cells suppress anti-tumoral CD8(+) and CD4(+) T lymphocytes and can also restrain the function of APCs. We have previously documented the immunostimulatory effects of a chaperone-rich cell lysate (CRCL) anti-cancer vaccine. Tumor-derived CRCL induces tumor immunity in vivo, partly by promoting dendritic cell (DC) and macrophage activation. In the current study, we evaluated the effects of CD4(+)CD25(+)forkhead box P3(+) Tregs isolated from mice bearing 12B1 bcr-abl(+) leukemia on DC and macrophages that had been activated by 12B1-derived CRCL. CRCL-activated DC and macrophages resisted Treg suppression, as the production of proinflammatory cytokines, the activation of transcription factor NF-kappaB, and their immunostimulatory potential was unaffected by Tregs. Our results thus highlight CRCL as a powerful adjuvant endowed with the capacity to overcome tumor-induced Treg-inhibitory effects on APCs.

  3. Macrophage Pro-Resolving Mediators—the When and Where

    PubMed Central

    DALLI, JESMOND; SERHAN, CHARLES

    2016-01-01

    Macrophages and neutrophils orchestrate acute inflammation and host defense as well as the resolution phase and return to homeostasis. In this article, we review the contribution of macrophages to local lipid mediator (LM) levels and the regulation of macrophage LM profiles by neutrophils and neutrophil-derived microparticles. We carried out LM metabololipidomics profiling distinct phagocytes: neutrophils (PMN), apoptotic PMN, and macrophages. Efferocytosis increased specialized proresolving mediator (SPM) biosynthesis, including Resolvin D1 (RvD1), RvD2, and RvE2, which were further elevated by PMN microparticles. Using deuterium-labeled precursors (d8-arachidonic acid, d5-eicosapentaenoic acid, and d5-docosahexaenoic acid), apoptotic PMN and microparticles contributed to SPM biosynthesis during efferocytosis. Assessment of macrophage LM profiles in M2 macrophages demonstrated higher SPM levels in this macrophage subset, including maresin 1 (MaR1), and lower amounts of leukotriene B4 and prostaglandins than M1. Apoptotic PMN uptake by both macrophage subtypes led to modulation of their LM profiles. Leukotriene B4 was down-regulated in M2 whereas SPM including lipoxin A4 were increased. Conversely, uptake of apoptotic PMN by M2 macrophages reduced (~ 25%) overall LM. MaR1 displays potent tissue regenerative and anti-nociceptive actions in addition to its pro-resolving and anti-inflammatory actions. In addition the MaR1 biosynthetic intermediate 13S,14S-epoxy-Maresin is also bioactive, inhibiting LTB4 biosynthesis and switching macrophage phenotypes from M1 to M2. Together, these results establish LM signature profiles of human phagocytes and related subpopulations. They demonstrate microparticle regulation of specific macrophage endogenous LM during defined stages of acute inflammation and their dynamic changes in human primary phagocytes. PMID:27337457

  4. Heterogeneous expression of apolipoprotein-E by human macrophages

    PubMed Central

    Tedla, Nicodemus; Glaros, Elias N; Brunk, Ulf T; Jessup, Wendy; Garner, Brett

    2004-01-01

    Apolipoprotein-E (apoE) is expressed at high levels by macrophages. In addition to its role in lipid transport, macrophage-derived apoE plays an important role in immunoregulation. Previous studies have identified macrophage subpopulations that differ substantially in their ability to synthesize specific cytokines and enzymes, however, potential heterogeneous macrophage apoE expression has not been studied. Here we examined apoE expression in human THP-1 macrophages and monocyte-derived macrophages (MDM). Using immunocytochemistry and flow cytometry methods we reveal a striking heterogeneity in macrophage apoE expression in both cell types. In phorbol-ester-differentiated THP-1 macrophages, 5% of the cells over-expressed apoE at levels more than 50-fold higher than the rest of the population. ApoE over-expressing THP-1 macrophages contained condensed/fragmented nuclei and increased levels of activated caspase-3 indicating induction of apoptosis. In MDM, 3–5% of the cells also highly over-expressed apoE, up to 50-fold higher than the rest of the population; however, this was not associated with obvious nuclear alterations. The apoE over-expressing MDM were larger, more granular, and more autofluorescent than the majority of cells and they contained numerous vesicle-like structures that appeared to be coated by apoE. Flow cytometry experiments indicated that the apoE over-expressing subpopulation of MDM were positive for CD14, CD11b/Mac-1 and CD68. These observations suggest that specific macrophage subpopulations may be important for apoE-mediated immunoregulation and clearly indicate that subpopulation heterogeneity should be taken into account when investigating macrophage apoE expression. PMID:15500620

  5. Conformational dynamics of the molecular chaperone Hsp90

    PubMed Central

    Krukenberg, Kristin A.; Street, Timothy O.; Lavery, Laura A.; Agard, David A.

    2016-01-01

    The molecular chaperone Hsp90 is an essential eukaryotic protein that makes up 1–2% of all cytosolic proteins. Hsp90 is vital for the maturation and maintenance of a wide variety of substrate proteins largely involved in signaling and regulatory processes. Many of these substrates have also been implicated in cancer and other diseases making Hsp90 an attractive target for therapeutics. Hsp90 is a highly dynamic and flexible molecule that can adapt its conformation to the wide variety of substrate proteins with which it acts. Large conformational rearrangements are also required for the activation of these client proteins. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis shifts the equilibrium between a pre-existing set of conformational states in an organism-dependent manner. In vivo Hsp90 functions as part of larger heterocomplexes. The binding partners of Hsp90, co-chaperones, assist in the recruitment and activation of substrates, and many co-chaperones further regulate the conformational dynamics of Hsp90 by shifting the conformational equilibrium towards a particular state. Studies have also suggested alternative mechanisms for the regulation of Hsp90’s conformation. In this review, we discuss the structural and biochemical studies leading to our current understanding of the conformational dynamics of Hsp90 and the role that nucleotide, co-chaperones, post-translational modification and clients play in regulating Hsp90’s conformation. We also discuss the effects of current Hsp90 inhibitors on conformation and the potential for developing small molecules that inhibit Hsp90 by disrupting the conformational dynamics. PMID:21414251

  6. Orchestration of secretory protein folding by ER chaperones

    PubMed Central

    Gidalevitz, Tali; Stevens, Fred; Argon, Yair

    2013-01-01

    The endoplasmic reticulum is a major compartment of protein biogenesis in the cell, dedicated to production of secretory, membrane and organelle proteins. The secretome has distinct structural and post-translational characteristics, since folding in the ER occurs in an environment that is distinct in terms of its ionic composition, dynamics and requirements for quality contol. The folding machinery in the ER therefore includes chaperones and folding enzymes that introduce, monitor and react to disulfide bonds, glycans, and fluctuations of luminal calcium. We describe the major chaperone networks in the lumen and discuss how they have distinct modes of operation that enable cells to accomplish highly efficient production of the secretome. PMID:23507200

  7. Specific Chaperones and Regulatory Domains in Control of Amyloid Formation*

    PubMed Central

    Landreh, Michael; Rising, Anna; Presto, Jenny; Jörnvall, Hans; Johansson, Jan

    2015-01-01

    Many proteins can form amyloid-like fibrils in vitro, but only about 30 amyloids are linked to disease, whereas some proteins form physiological amyloid-like assemblies. This raises questions of how the formation of toxic protein species during amyloidogenesis is prevented or contained in vivo. Intrinsic chaperoning or regulatory factors can control the aggregation in different protein systems, thereby preventing unwanted aggregation and enabling the biological use of amyloidogenic proteins. The molecular actions of these chaperones and regulators provide clues to the prevention of amyloid disease, as well as to the harnessing of amyloidogenic proteins in medicine and biotechnology. PMID:26354437

  8. LPS-Induced Macrophage Activation and Plasma Membrane Fluidity Changes are Inhibited Under Oxidative Stress.

    PubMed

    de la Haba, Carlos; Morros, Antoni; Martínez, Paz; Palacio, José R

    2016-12-01

    Macrophage activation is essential for a correct and efficient response of innate immunity. During oxidative stress membrane receptors and/or membrane lipid dynamics can be altered, leading to dysfunctional cell responses. Our aim is to analyze membrane fluidity modifications and cell function under oxidative stress in LPS-activated macrophages. Membrane fluidity of individual living THP-1 macrophages was evaluated by the technique two-photon microscopy. LPS-activated macrophage function was determined by TNFα secretion. It was shown that LPS activation causes fluidification of macrophage plasma membrane and production of TNFα. However, oxidative stress induces rigidification of macrophage plasma membrane and inhibition of cell activation, which is evidenced by a decrease of TNFα secretion. Thus, under oxidative conditions macrophage proinflammatory response might develop in an inefficient manner.

  9. CXCL4 induces a unique transcriptome in monocyte-derived macrophages

    PubMed Central

    Gleissner, Christian A.; Shaked, Iftach; Little, Kristina M.; Ley, Klaus

    2012-01-01

    In atherosclerotic arteries, blood monocytes differentiate to macrophages in the presence of growth factors like macrophage colony-stimulation factor (MCSF) and chemokines like platelet factor 4 (CXCL4). To compare the gene expression signature of CXCL4-induced macrophages with MCSF-induced macrophages or macrophages polarized with IFN-γ/LPS (M1) or IL-4 (M2), we cultured primary human peripheral blood monocytes for six days. mRNA expression was measured by Affymetrix gene chips and differences were analyzed by Local Pooled Error test, Profile of Complex Functionality and Gene Set Enrichment Analysis. 375 genes were differentially expressed between MCSF- and CXCL4-induced macrophages, 206 of them overexpressed in CXCL4 macrophages coding for genes implicated in the inflammatory/immune response, antigen processing/presentation, and lipid metabolism. CXCL4-induced macrophages overexpressed some M1 and M2 genes and the corresponding cytokines at the protein level, however, their transcriptome clustered with neither M1 nor M2 transcriptomes. They almost completely lost the ability to phagocytose zymosan beads. Genes linked to atherosclerosis were not consistently up- or downregulated. Scavenger receptors showed lower and cholesterol efflux transporters higher expression in CXCL4- than MCSF-induced macrophages, resulting in lower LDL content. We conclude that CXCL4 induces a unique macrophage transcriptome distinct from known macrophage types, defining a new macrophage differentiation that we propose to call M4. PMID:20335529

  10. A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways

    PubMed Central

    Taipale, Mikko; Tucker, George; Peng, Jian; Krykbaeva, Irina; Lin, Zhen-Yuan; Larsen, Brett; Choi, Hyungwon; Berger, Bonnie; Gingras, Anne-Claude; Lindquist, Susan

    2014-01-01

    Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of co-factors (co-chaperones) that regulate their specificity and function. However, how these co-chaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We have combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone/co-chaperone/client interaction network in human cells. We uncover hundreds of novel chaperone clients, delineate their participation in specific co-chaperone complexes, and establish a surprisingly distinct network of protein/protein interactions for co-chaperones. As a salient example of the power of such analysis, we establish that NUDC family co-chaperones specifically associate with structurally related but evolutionarily distinct β-propeller folds. We provide a framework for deciphering the proteostasis network, its regulation in development and disease, and expand the use of chaperones as sensors for drug/target engagement. PMID:25036637

  11. Rhodopseudomonas sphaeroides lipid A derivatives block in vitro induction of tumor necrosis factor and endotoxin tolerance by smooth lipopolysaccharide and monophosphoryl lipid A.

    PubMed Central

    Henricson, B E; Perera, P Y; Qureshi, N; Takayama, K; Vogel, S N

    1992-01-01

    Rhodopseudomonas (Rhodobacter) sphaeroides diphosphoryl lipid A is a relatively inert species of lipid A but has been shown to antagonize the effects of toxic lipopolysaccharide (LPS) both in vivo and in vitro. The antagonist and its monophosphoryl derivative were examined for the ability to block tumor necrosis factor synthesis and reverse tolerance induction in vitro in macrophage cultures stimulated with bioactive preparations of smooth LPS, rough LPS, diphosphoryl lipid A, and monophosphoryl lipid A. Inhibition of agonist activity and reversal of tolerance by these novel penta-acylated lipid A antagonists provides new insight into macrophage-LPS interactions. PMID:1398939

  12. Amelioration of glucolipotoxicity-induced endoplasmic reticulum stress by a "chemical chaperone" in human THP-1 monocytes.

    PubMed

    Lenin, Raji; Maria, Mariawilliam Sneha; Agrawal, Madhur; Balasubramanyam, Jayashree; Mohan, Viswanathan; Balasubramanyam, Muthuswamy

    2012-01-01

    Chronic ER stress is emerging as a trigger that imbalances a number of systemic and arterial-wall factors and promote atherosclerosis. Macrophage apoptosis within advanced atherosclerotic lesions is also known to increase the risk of atherothrombotic disease. We hypothesize that glucolipotoxicity might mediate monocyte activation and apoptosis through ER stress. Therefore, the aims of this study are (a) to investigate whether glucolipotoxicity could impose ER stress and apoptosis in THP-1 human monocytes and (b) to investigate whether 4-Phenyl butyric acid (PBA), a chemical chaperone could resist the glucolipotoxicity-induced ER stress and apoptosis. Cells subjected to either glucolipotoxicity or tunicamycin exhibited increased ROS generation, gene and protein (PERK, GRP-78, IRE1α, and CHOP) expression of ER stress markers. In addition, these cells showed increased TRPC-6 channel expression and apoptosis as revealed by DNA damage and increased caspase-3 activity. While glucolipotoxicity/tunicamycin increased oxidative stress, ER stress, mRNA expression of TRPC-6, and programmed the THP-1 monocytes towards apoptosis, all these molecular perturbations were resisted by PBA. Since ER stress is one of the underlying causes of monocyte dysfunction in diabetes and atherosclerosis, our study emphasize that chemical chaperones such as PBA could alleviate ER stress and have potential to become novel therapeutics.

  13. RNA chaperones buffer deleterious mutations in E. coli

    PubMed Central

    Rudan, Marina; Schneider, Dominique; Warnecke, Tobias; Krisko, Anita

    2015-01-01

    Both proteins and RNAs can misfold into non-functional conformations. Protein chaperones promote native folding of nascent polypeptides and refolding of misfolded species, thereby buffering mutations that compromise protein structure and function. Here, we show that RNA chaperones can also act as mutation buffers that enhance organismal fitness. Using competition assays, we demonstrate that overexpression of select RNA chaperones, including three DEAD box RNA helicases (DBRHs) (CsdA, SrmB, RhlB) and the cold shock protein CspA, improves fitness of two independently evolved Escherichia coli mutator strains that have accumulated deleterious mutations during short- and long-term laboratory evolution. We identify strain-specific mutations that are deleterious and subject to buffering when introduced individually into the ancestral genotype. For DBRHs, we show that buffering requires helicase activity, implicating RNA structural remodelling in the buffering process. Our results suggest that RNA chaperones might play a fundamental role in RNA evolution and evolvability. DOI: http://dx.doi.org/10.7554/eLife.04745.001 PMID:25806682

  14. Reconfiguration of the proteasome during chaperone-mediated assembly.

    PubMed

    Park, Soyeon; Li, Xueming; Kim, Ho Min; Singh, Chingakham Ranjit; Tian, Geng; Hoyt, Martin A; Lovell, Scott; Battaile, Kevin P; Zolkiewski, Michal; Coffino, Philip; Roelofs, Jeroen; Cheng, Yifan; Finley, Daniel

    2013-05-23

    The proteasomal ATPase ring, comprising Rpt1-Rpt6, associates with the heptameric α-ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt carboxy-terminal tails inserting into pockets of the α-ring. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit. Here we report that the base subassembly of the Saccharomyces cerevisiae proteasome, which includes the Rpt ring, forms a high-affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6 and Rpn14. Chaperone-mediated dissociation was abrogated by a non-hydrolysable ATP analogue, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound α-pockets with poor specificity, except for Rpt6, which uniquely bound the α2/α3-pocket. Although the Rpt6 tail is not visualized within an α-pocket in mature proteasomes, it inserts into the α2/α3-pocket in the base-CP complex and is important for complex formation. Thus, the Rpt-CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme.

  15. Pharmacological chaperones for human α-N-acetylgalactosaminidase

    PubMed Central

    Clark, Nathaniel E.; Metcalf, Matthew C.; Best, Daniel; Fleet, George W. J.; Garman, Scott C.

    2012-01-01

    Schindler/Kanzaki disease is an inherited metabolic disease with no current treatment options. This neurologic disease results from a defect in the lysosomal α-N-acetylgalactosaminidase (α-NAGAL) enzyme. In this report, we show evidence that the iminosugar DGJNAc can inhibit, stabilize, and chaperone human α-NAGAL both in vitro and in vivo. We demonstrate that a related iminosugar DGJ (currently in phase III clinical trials for another metabolic disorder, Fabry disease) can also chaperone human α-NAGAL in Schindler/Kanzaki disease. The 1.4- and 1.5-Å crystal structures of human α-NAGAL complexes reveal the different binding modes of iminosugars compared with glycosides. We show how differences in two functional groups result in >9 kcal/mol of additional binding energy and explain the molecular interactions responsible for the unexpectedly high affinity of the pharmacological chaperones. These results open two avenues for treatment of Schindler/Kanzaki disease and elucidate the atomic basis for pharmacological chaperoning in the entire family of lysosomal storage diseases. PMID:23045655

  16. Hsp100/ClpB Chaperone Function and Mechanism

    SciTech Connect

    Vierling, Elizabeth

    2015-01-27

    The supported research investigated the mechanism of action of a unique class of molecular chaperones in higher plants, the Hsp100/ClpB proteins, with the ultimate goal of defining how these chaperones influence plant growth, development, stress tolerance and productivity. Molecular chaperones are essential effectors of cellular “protein quality control”, which comprises processes that ensure the proper folding, localization, activation and turnover of proteins. Hsp100/ClpB proteins are required for temperature acclimation in plants, optimal seed yield, and proper chloroplast development. The model plant Arabidopsis thaliana and genetic and molecular approaches were used to investigate two of the three members of the Hsp100/ClpB proteins in plants, cytosolic AtHsp101 and chloroplast-localized AtClpB-p. Investigating the chaperone activity of the Hsp100/ClpB proteins addresses DOE goals in that this activity impacts how “plants generate and assemble components” as well as “allowing for their self repair”. Additionally, Hsp100/ClpB protein function in plants is directly required for optimal “utilization of biological energy” and is involved in “mechanisms that control the architecture of energy transduction systems”.

  17. Super Spy variants implicate flexibility in chaperone action

    PubMed Central

    Quan, Shu; Wang, Lili; Petrotchenko, Evgeniy V; Makepeace, Karl AT; Horowitz, Scott; Yang, Jianyi; Zhang, Yang; Borchers, Christoph H; Bardwell, James CA

    2014-01-01

    Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These “Super Spy” variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function. DOI: http://dx.doi.org/10.7554/eLife.01584.001 PMID:24497545

  18. Macrophage activation and polarization.

    PubMed

    Martinez, Fernando Oneissi; Sica, Antonio; Mantovani, Alberto; Locati, Massimo

    2008-01-01

    Macrophages are widely distributed immune system cells that play an indispensable role in homeostasis and defense. They can be phenotypically polarized by the microenvironment to mount specific functional programs. Polarized macrophages can be broadly classified in two main groups: classically activated macrophages (or M1), whose prototypical activating stimuli are IFNgamma and LPS, and alternatively activated macrophages (or M2), further subdivided in M2a (after exposure to IL-4 or IL-13), M2b (immune complexes in combination with IL-1beta or LPS) and M2c (IL-10, TGFbeta or glucocorticoids). M1 exhibit potent microbicidal properties and promote strong IL-12-mediated Th1 responses, whilst M2 support Th2-associated effector functions. Beyond infection M2 polarized macrophages play a role in resolution of inflammation through high endocytic clearance capacities and trophic factor synthesis, accompanied by reduced pro-inflammatory cytokine secretion. Similar functions are also exerted by tumor-associated macrophages (TAM), which also display an alternative-like activation phenotype and play a detrimental pro-tumoral role. Here we review the main functions of polarized macrophages and discuss the perspectives of this field.

  19. Proatherogenic macrophage activities are targeted by the flavonoid quercetin.

    PubMed

    Lara-Guzman, Oscar J; Tabares-Guevara, Jorge H; Leon-Varela, Yudy M; Álvarez, Rafael M; Roldan, Miguel; Sierra, Jelver A; Londoño-Londoño, Julian A; Ramirez-Pineda, Jose R

    2012-11-01

    Many studies have demonstrated that the flavonoid quercetin protects against cardiovascular disease (CVD) and related risk factors. Atherosclerosis, the underlying cause of CVD, is also attenuated by oral quercetin administration in animal models. Although macrophages are key players during fatty streak formation and plaque progression and aggravation, little is known about the effects of quercetin on atherogenic macrophages. Here, we report that primary bone marrow-derived macrophages internalized less oxidized low-density lipoprotein (oxLDL) and accumulated less intracellular cholesterol in the presence of quercetin. This reduction of foam cell formation correlated with reduced surface expression of the oxLDL receptor CD36. Quercetin also targeted the lipopolysaccharide-dependent, oxLDL-independent pathway of lipid droplet formation in macrophages. In oxLDL-stimulated macrophages, quercetin inhibited reactive oxygen species production and interleukin (IL)-6 secretion. In a system that evaluated cholesterol crystal-induced IL-1β secretion via nucleotide-binding domain and leucine-rich repeat containing protein 3 inflammasome activation, quercetin also exhibited an inhibitory effect. Dyslipidemic apolipoprotein E-deficient mice chronically treated with intraperitoneal quercetin injections had smaller atheromatous lesions, reduced lipid deposition, and less macrophage and T cell inflammatory infiltrate in the aortic roots than vehicle-treated animals. Serum levels of total cholesterol and the lipid peroxidation product malondialdehyde were also reduced in these mice. Our results demonstrate that quercetin interferes with both key proatherogenic activities of macrophages, namely foam cell formation and pro-oxidant/proinflammatory responses, and these effects may explain the atheroprotective properties of this common flavonoid.

  20. miRNA in Macrophage Development and Function

    PubMed Central

    2016-01-01

    Abstract Significance: MicroRNAs (miRNAs) control cellular gene expression via primarily binding to 3′ or 5′ untranslated region of the target transcript leading to translational repression or mRNA degradation. In most cases, miRNAs have been observed to fine-tune the cellular responses and, therefore, act as a rheostat rather than an on/off switch. Transcription factor PU.1 is a master switch that controls monocyte/macrophage development from hematopoietic stem cells. Recent Advances: PU.1 induces a specific set of miRNAs while suppressing the miR17-92 cluster to regulate monocyte/macrophage development. In addition to development, miRNAs tightly control the macrophage polarization continuum from proinflammatory M1 or proreparative M2 by regulating expression of key transcription factors involved in the process of polarization. Critical Issues: miRNAs are intricately involved with fine-tuning fundamental macrophage functions such as phagocytosis, efferocytosis, inflammation, tissue repair, and tumor promotion. Macrophages are secretory cells that participate in intercellular communication by releasing regulatory molecules and microvesicles (MVs). MVs are bilayered lipid membranes packaging a hydrophilic cargo, including proteins and nucleic acids. Macrophage-derived MVs carry functionally active miRNAs that suppress gene expression in target cells via post-transcriptional gene silencing, thus regulating cell function. In summary, miRNAs fine-tune several major facets of macrophage development and function. Such fine-tuning is critical in preventing exaggerated macrophage response to endogenous or exogenous stimuli. Future Directions: A critical role of miRNAs in the regulation of innate immune response and macrophage biology, including development, differentiation, and activation, has emerged. A clear understanding of such regulation on macrophage function remains to be elucidated. Antioxid. Redox Signal. 25, 795–804. PMID:27353423

  1. Macrophages heterogeneity in atherosclerosis – implications for therapy

    PubMed Central

    Wilson, Heather M

    2010-01-01

    Abstract Atherosclerosis is a chronic inflammatory disease occurring within the artery wall and is an underlying cause of cardiovascular complications, including myocardial infarction, stroke and peripheral vascular disease. Its pathogenesis involves many immune cell types with a well accepted role for monocyte/macrophages. Cholesterol-loaded macrophages are a characteristic feature of plaques and are major players in all stages of plaque development. As well as modulating lipid metabolism, macrophages secrete inflammatory cytokines, chemokines and reactive oxygen and nitrogen species that drive pathogenesis. They also produce proteases and tissue factor that contribute to plaque rupture and thrombosis. Macrophages are however heterogeneous cells and when appropriately activated, they phagocytose cytotoxic lipoproteins, clear apoptotic bodies, secrete anti-inflammatory cytokines and synthesize matrix repair proteins that stabilize vulnerable plaques. Pharmacological modulation of macrophage activity therefore represents a potential therapeutic strategy for atherosclerosis. The aim of this review is to provide an overview of the current understanding of the different macrophage subsets and their monocyte precursors, and, the implications of these subsets for atherosclerosis. This will present a foundation for highlighting novel opportunities to exploit the heterogeneity of macrophages as important diagnostic and therapeutic targets for atherosclerosis and its associated diseases. PMID:20629993

  2. Chaperonopathies of senescence and the scrambling of interactions between the chaperoning and the immune systems.

    PubMed

    Macario, Alberto J L; Cappello, Francesco; Zummo, Giovanni; Conway de Macario, Everly

    2010-06-01

    Aging entails progressive deterioration of molecules and supramolecular structures, including Hsp chaperones and their complexes, paralleled by functional decline. Recent research has changed our views on Hsp chaperones. They work inside and outside cells in many locations, alone or forming teams, interacting with cells, receptors, and molecules that are not chaperones, in roles that are not typically attributed to chaperones, such as protein folding. Hsp chaperones form a physiological system with a variety of functions and interactions with other systems, for example, the immune system. We propose that chaperone malfunctioning due to structural damage or gene dysregulation during aging has an impact on the immune system, creating the conditions for an overall malfunction of both systems. Pathological chaperones cannot interact with the immune system as normal ones do, and this leads to an overall readjustment of the interactions that is apparent during senescence and is likely to cause many of its manifestations.

  3. Lipotoxicity in macrophages: evidence from diseases associated with the metabolic syndrome.

    PubMed

    Prieur, Xavier; Roszer, Tamás; Ricote, Mercedes

    2010-03-01

    Accumulation of lipid metabolites within non-adipose tissues can induce chronic inflammation by promoting macrophage infiltration and activation. Oxidized and glycated lipoproteins, free fatty acids, free cholesterol, triacylglycerols, diacylglycerols and ceramides have long been known to induce cellular dysfunction through their pro-inflammatory and pro-apoptotic properties. Emerging evidence suggests that macrophage activation by lipid metabolites and further modulation by lipid signaling represents a common pathogenic mechanism underlying lipotoxicity in atherosclerosis, obesity-associated insulin resistance and inflammatory diseases related to metabolic syndrome such as liver steatosis and chronic kidney disease. In this review, we discuss the latest discoveries that support the role of lipids in modulating the macrophage phenotype in different metabolic diseases. We describe the common mechanisms by which lipid derivatives, through modulation of macrophage function, promote plaque instability in the arterial wall, impair insulin responsiveness and contribute to inflammatory liver, muscle and kidney disease. We discuss the molecular mechanism of lipid activation of pro-inflammatory pathways (JNK, NFkappaB) and the key roles played by the PPAR and LXR nuclear receptors-lipid sensors that link lipid metabolism and inflammation.

  4. The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium

    PubMed Central

    Sittka, Alexandra; Pfeiffer, Verena; Tedin, Karsten; Vogel, Jörg

    2007-01-01

    The RNA chaperone, Hfq, plays a diverse role in bacterial physiology beyond its original role as a host factor required for replication of Qβ RNA bacteriophage. In this study, we show that Hfq is involved in the expression and secretion of virulence factors in the facultative intracellular pathogen, Salmonella typhimurium. A Salmonella hfq deletion strain is highly attenuated in mice after both oral and intraperitoneal infection, and shows a severe defect in invasion of epithelial cells and a growth defect in both epithelial cells and macrophages in vitro. Surprisingly, we find that these phenotypes are largely independent of the previously reported requirement of Hfq for expression of the stationary phase sigma factor, RpoS. Our results implicate Hfq as a key regulator of multiple aspects of virulence including regulation of motility and outer membrane protein (OmpD) expression in addition to invasion and intracellular growth. These pleiotropic effects are suggested to involve a network of regulatory small non-coding RNAs, placing Hfq at the centre of post-transcriptional regulation of virulence gene expression in Salmonella. In addition, the hfq mutation appears to cause a chronic activation of the RpoE-mediated envelope stress response which is likely due to a misregulation of membrane protein expression. PMID:17163975

  5. Measuring autophagy in macrophages.

    PubMed

    Harris, James; Hanrahan, Orla; De Haro, Sergio A

    2009-11-01

    Macroautophagy is a conserved intracellular homeostatic mechanism for the degradation of cytosolic constituents. Autophagy can promote cell survival by providing essential amino acids from the breakdown of macromolecules during periods of nutrient deprivation, and can remove damaged or excess organelles, such as mitochondria and peroxisomes. More recently, autophagy has been shown to play an important role in innate and adaptive immune responses to pathogenic bacteria in macrophages and dendritic cells. This unit presents protocols for the measurement of autophagy in macrophages.

  6. Influence of trehalose 6,6'-dimycolate (TDM) during mycobacterial infection of bone marrow macrophages.

    PubMed

    Indrigo, Jessica; Hunter, Robert L; Actor, Jeffrey K

    2002-07-01

    The relative role of surface lipids in the innate macrophage response to infection with mycobacteria remains unknown. Trehalose 6,6'-dimycolate (TDM), a major component of the mycobacterial cell wall, can elicit hypersensitive as well as T-cell-independent foreign body responses. The T-cell-independent contribution of TDM to the primary macrophage response to mycobacterial infection was investigated. Bone-marrow-derived macrophages isolated from C57BL/6 mice were infected with native Mycobacterium tuberculosis (MTB) or with MTB delipidated using petroleum ether extraction methods. The removal of surface lipids caused decreased bacterial survival in macrophages, but there was no loss of bacterial growth in broth culture. Bacterial survival within macrophages was restored upon reconstitution of the bacteria with purified TDM. The cytokine and chemokine parameters of the macrophage responses were also investigated. The amounts of IL-1beta, TNF-alpha, IL-6 and MIP-1alpha produced were significantly reduced following delipidation, but were restored upon reconstitution with TDM. The amount of IL-12 produced, but not the amount of IL-10 produced, was also significantly reduced upon macrophage infection with delipidated MTB. Furthermore, nitric oxide responses were not impaired upon infection with delipidated MTB, suggesting that intracellular survival and macrophage secretion of cytokines and chemokines are differentially controlled. These studies indicate that TDM is a major component contributing to the innate macrophage responses to MTB infection.

  7. Macrophagic CD146 promotes foam cell formation and retention during atherosclerosis.

    PubMed

    Luo, Yongting; Duan, Hongxia; Qian, Yining; Feng, Liqun; Wu, Zhenzhen; Wang, Fei; Feng, Jing; Yang, Dongling; Qin, Zhihai; Yan, Xiyun

    2017-03-01

    The persistence of cholesterol-engorged macrophages (foam cells) in the artery wall fuels the development of atherosclerosis. However, the mechanism that regulates the formation of macrophage foam cells and impedes their emigration out of inflamed plaques is still elusive. Here, we report that adhesion receptor CD146 controls the formation of macrophage foam cells and their retention within the plaque during atherosclerosis exacerbation. CD146 is expressed on the macrophages in human and mouse atheroma and can be upregulated by oxidized low-density lipoprotein (oxLDL). CD146 triggers macrophage activation by driving the internalization of scavenger receptor CD36 during lipid uptake. In response to oxLDL, macrophages show reduced migratory capacity toward chemokines CCL19 and CCL21; this capacity can be restored by blocking CD146. Genetic deletion of macrophagic CD146 or targeting of CD146 with an antibody result in much less complex plaques in high-fat diet-fed ApoE(-/-) mice by causing lipid-loaded macrophages to leave plaques. Collectively, our findings identify CD146 as a novel retention signal that traps macrophages within the artery wall, and a promising therapeutic target in atherosclerosis treatment.

  8. Macrophagic CD146 promotes foam cell formation and retention during atherosclerosis

    PubMed Central

    Luo, Yongting; Duan, Hongxia; Qian, Yining; Feng, Liqun; Wu, Zhenzhen; Wang, Fei; Feng, Jing; Yang, Dongling; Qin, Zhihai; Yan, Xiyun

    2017-01-01

    The persistence of cholesterol-engorged macrophages (foam cells) in the artery wall fuels the development of atherosclerosis. However, the mechanism that regulates the formation of macrophage foam cells and impedes their emigration out of inflamed plaques is still elusive. Here, we report that adhesion receptor CD146 controls the formation of macrophage foam cells and their retention within the plaque during atherosclerosis exacerbation. CD146 is expressed on the macrophages in human and mouse atheroma and can be upregulated by oxidized low-density lipoprotein (oxLDL). CD146 triggers macrophage activation by driving the internalization of scavenger receptor CD36 during lipid uptake. In response to oxLDL, macrophages show reduced migratory capacity toward chemokines CCL19 and CCL21; this capacity can be restored by blocking CD146. Genetic deletion of macrophagic CD146 or targeting of CD146 with an antibody result in much less complex plaques in high-fat diet-fed ApoE−/− mice by causing lipid-loaded macrophages to leave plaques. Collectively, our findings identify CD146 as a novel retention signal that traps macrophages within the artery wall, and a promising therapeutic target in atherosclerosis treatment. PMID:28084332

  9. Adipogenic role of alternatively activated macrophages in β-adrenergic remodeling of white adipose tissue.

    PubMed

    Lee, Yun-Hee; Kim, Sang-Nam; Kwon, Hyun-Jung; Maddipati, Krishna Rao; Granneman, James G

    2016-01-01

    De novo brown adipogenesis involves the proliferation and differentiation of progenitors, yet the mechanisms that guide these events in vivo are poorly understood. We previously demonstrated that treatment with a β3-adrenergic receptor (ADRB3) agonist triggers brown/beige adipogenesis in gonadal white adipose tissue following adipocyte death and clearance by tissue macrophages. The close physical relationship between adipocyte progenitors and tissue macrophages suggested that the macrophages that clear dying adipocytes might generate proadipogenic factors. Flow cytometric analysis of macrophages from mice treated with CL 316,243 identified a subpopulation that contained elevated lipid and expressed CD44. Lipidomic analysis of fluorescence-activated cell sorting-isolated macrophages demonstrated that CD44+ macrophages contained four- to five-fold higher levels of the endogenous peroxisome-proliferator activated receptor gamma (PPARγ) ligands 9-hydroxyoctadecadienoic acid (HODE), and 13-HODE compared with CD44- macrophages. Gene expression profiling and immunohistochemistry demonstrated that ADRB3 agonist treatment upregulated expression of ALOX15, the lipoxygenase responsible for generating 9-HODE and 13-HODE. Using an in vitro model of adipocyte efferocytosis, we found that IL-4-primed tissue macrophages accumulated lipid from dying fat cells and upregulated expression of Alox15. Furthermore, treatment of differentiating adipocytes with 9-HODE and 13-HODE potentiated brown/beige adipogenesis. Collectively, these data indicate that noninflammatory removal of adipocyte remnants and coordinated generation of PPARγ ligands by M2 macrophages provides localized adipogenic signals to support de novo brown/beige adipogenesis.

  10. The Elusive Antifibrotic Macrophage

    PubMed Central

    Adhyatmika, Adhyatmika; Putri, Kurnia S. S.; Beljaars, Leonie; Melgert, Barbro N.

    2015-01-01

    Fibrotic diseases, especially of the liver, the cardiovascular system, the kidneys, and the lungs, account for approximately 45% of deaths in Western societies. Fibrosis is a serious complication associated with aging and/or chronic inflammation or injury and cannot be treated effectively yet. It is characterized by excessive deposition of extracellular matrix (ECM) proteins by myofibroblasts and impaired degradation by macrophages. This ultimately destroys the normal structure of an organ, which leads to loss of function. Most efforts to develop drugs have focused on inhibiting ECM production by myofibroblasts and have not yielded many effective drugs yet. Another option is to stimulate the cells that are responsible for degradation and uptake of excess ECM, i.e., antifibrotic macrophages. However, macrophages are plastic cells that have many faces in fibrosis, including profibrotic behavior-stimulating ECM production. This can be dependent on their origin, as the different organs have tissue-resident macrophages with different origins and a various influx of incoming monocytes in steady-state conditions and during fibrosis. To be able to pharmacologically stimulate the right kind of behavior in fibrosis, a thorough characterization of antifibrotic macrophages is necessary, as well as an understanding of the signals they need to degrade ECM. In this review, we will summarize the current state of the art regarding the antifibrotic macrophage phenotype and the signals that stimulate its behavior. PMID:26618160

  11. Macrophage polarization in pathology.

    PubMed

    Sica, Antonio; Erreni, Marco; Allavena, Paola; Porta, Chiara

    2015-11-01

    Macrophages are cells of the innate immunity constituting the mononuclear phagocyte system and endowed with remarkable different roles essential for defense mechanisms, development of tissues, and homeostasis. They derive from hematopoietic precursors and since the early steps of fetal life populate peripheral tissues, a process continuing throughout adult life. Although present essentially in every organ/tissue, macrophages are more abundant in the gastro-intestinal tract, liver, spleen, upper airways, and brain. They have phagocytic and bactericidal activity and produce inflammatory cytokines that are important to drive adaptive immune responses. Macrophage functions are settled in response to microenvironmental signals, which drive the acquisition of polarized programs, whose extremes are simplified in the M1 and M2 dichotomy. Functional skewing of monocyte/macrophage polarization occurs in physiological conditions (e.g., ontogenesis and pregnancy), as well as in pathology (allergic and chronic inflammation, tissue repair, infection, and cancer) and is now considered a key determinant of disease development and/or regression. Here, we will review evidence supporting a dynamic skewing of macrophage functions in disease, which may provide a basis for macrophage-centered therapeutic strategies.

  12. Paraoxsonase2 (PON2) and oxidative stress involvement in pomegranate juice protection against cigarette smoke-induced macrophage cholesterol accumulation.

    PubMed

    Rom, Oren; Aviram, Michael

    2016-11-25

    Exposure to cigarette smoke (CS) promotes various stages of atherosclerosis development. Macrophages are the predominant cells in early atherogenesis, and the polyphenolic-rich pomegranate juice (PJ) is known for its protective role against macrophage atherogenicity. The aim of the current study was to examine the atherogenic effects of CS on macrophages, and to evaluate the protective effects of PJ against CS-induced macrophage atherogenicity. Murine J774A.1 macrophages were treated with CS-exposed medium in the absence or presence of PJ. Parameters of lipid peroxidation in CS-exposed medium were measured by the lipid peroxides and thiobarbituric acid reactive substances (TBARS) assays. Atherogenicity of macrophages incubated with increasing concentrations of CS-exposed medium was assessed by cytotoxicity, oxidative stress determined by generation of reactive oxygen species (ROS) using DCFH-DA, activity of the cellular anti-oxidant paraoxonase2 (PON2), macrophage accumulation of cholesterol and triglycerides, as well as through high density lipoprotein (HDL)-mediated cholesterol efflux from the cells. CS exposure resulted in significant and dose-dependent increases in lipid peroxides and TBARS medium levels (up to 3 and 8-fold, respectively). Incubation of macrophages with CS-exposed medium resulted in dose-dependent increases in macrophage damage/injury (up to 6-fold), intracellular ROS levels (up to 31%), PON2 activity (up to 2-fold), and macrophage cholesterol content (up to 24%). The latter might be explained by reduced HDL-mediated cholesterol efflux from CS-exposed macrophages (by 21%). PJ protected macrophages from CS-induced increases in intracellular ROS levels and cholesterol accumulation, as well as the attenuated efflux of cholesterol. These data indicate that CS stimulates macrophage oxidation and activates PON2 as a possible compensatory response to the oxidative burden. CS impairs HDL-mediated cholesterol efflux from macrophages leading to cellular

  13. The chaperone like function of the nonhistone protein HMGB1

    SciTech Connect

    Osmanov, Taner; Ugrinova, Iva; Pasheva, Evdokia

    2013-03-08

    Highlights: ► The HMGB1 protein strongly enhanced the formation of nucleosome particles. ► The target of HMGB1 action as a chaperone is the DNA not the histone octamer. ► The acetylation of HMGB1 decreases the stimulating effect of the protein. -- Abstract: Almost all essential nuclear processes as replication, repair, transcription and recombination require the chromatin template to be correctly unwound and than repackaged. The major strategy that the cell uses to overcome the nucleosome barrier is the proper removal of the histone octamer and subsequent deposition onto DNA. Important factors in this multi step phenomenon are the histone chaperones that can assemble nucleosome arrays in vitro in the absence of ATP. The nonhistone protein HMGB1 is a good candidate for a chaperone as its molecule consists of two DNA binding motives, Box’s A and B, and a long nonstructured C tail highly negatively charged. HMGB1 protein is known as a nuclear “architectural” factor for its property to bind preferentially to distorted DNA structures and was reported to kink the double helix. Our experiments show that in the classical stepwise dialysis method for nucleosome assembly the addition of HMGB1 protein stimulates more than two times the formation of middle-positioned nucleosomes. The stimulation effect persists in dialysis free experiment when the reconstitution is possible only in the presence of a chaperone. The addition of HMGB1 protein strongly enhanced the formation of a nucleosome in a dose dependant manner. Our results show that the target of HMGB1 action as a chaperone is the DNA fragment not the histone octamer. One possible explanation for the stimulating effect of HMGB1 is the “architectural” property of the protein to associate with the middle of the DNA fragment and to kink it. The acquired V shaped DNA structure is probably conformationals more favorable to wrap around the prefolded histone octamer. We tested also the role of the post

  14. Macrophage polarization in inflammatory diseases.

    PubMed

    Liu, Yan-Cun; Zou, Xian-Biao; Chai, Yan-Fen; Yao, Yong-Ming

    2014-01-01

    Diversity and plasticity are two hallmarks of macrophages. M1 macrophages (classically activated macrophages) are pro-inflammatory and have a central role in host defense against infection, while M2 macrophages (alternatively activated macrophages) are associated with responses to anti-inflammatory reactions and tissue remodeling, and they represent two terminals of the full spectrum of macrophage activation. Transformation of different phenotypes of macrophages regulates the initiation, development, and cessation of inflammatory diseases. Here we reviewed the characters and functions of macrophage polarization in infection, atherosclerosis, obesity, tumor, asthma, and sepsis, and proposed that targeting macrophage polarization and skewing their phenotype to adapt to the microenvironment might hold great promise for the treatment of inflammatory diseases.

  15. The impact of impaired macrophage functions in cystic fibrosis disease progression.

    PubMed

    Lévêque, Manuella; Le Trionnaire, Sophie; Del Porto, Paola; Martin-Chouly, Corinne

    2016-11-14

    The underlying cause of morbidity in cystic fibrosis (CF) is the decline in lung function, which results in part from chronic inflammation. Inflammation and infection occur early in infancy in CF and the role of innate immune defense in CF has been highlighted in the last years. Once thought simply to be consumers of bacteria, macrophages have emerged as highly sensitive immune cells that are located at the balance point between inflammation and resolution of this inflammation in CF pathophysiology. In order to assess the potential role of macrophage in CF, we review the evidence that: (1) CF macrophage has a dysregulated inflammatory phenotype; (2) CF macrophage presents altered phagocytosis capacity and bacterial killing; and (3) lipid disorders in CF macrophage affect its function. These alterations of macrophage weaken innate defense of CF patients and may be involved in CF disease progression and lung damage.

  16. Chaperone-mediated specificity in Ras and Rap signaling.

    PubMed

    Azoulay-Alfaguter, Inbar; Strazza, Marianne; Mor, Adam

    2015-01-01

    Ras and Rap proteins are closely related small guanosine triphosphatase (GTPases) that share similar effector-binding domains but operate in a very different signaling networks; Ras has a dominant role in cell proliferation, while Rap mediates cell adhesion. Ras and Rap proteins are regulated by several shared processes such as post-translational modification, phosphorylation, activation by guanine exchange factors and inhibition by GTPase-activating proteins. Sub-cellular localization and trafficking of these proteins to and from the plasma membrane are additional important regulatory features that impact small GTPases function. Despite its importance, the trafficking mechanisms of Ras and Rap proteins are not completely understood. Chaperone proteins play a critical role in trafficking of GTPases and will be the focus of the discussion in this work. We will review several aspects of chaperone biology focusing on specificity toward particular members of the small GTPase family. Understanding this specificity should provide key insights into drug development targeting individual small GTPases.

  17. Chaperoning osteogenesis: new protein-folding disease paradigms.

    PubMed

    Makareeva, Elena; Aviles, Nydea A; Leikin, Sergey

    2011-03-01

    Recent discoveries of severe bone disorders in patients with deficiencies in several endoplasmic reticulum chaperones are reshaping the discussion of type I collagen folding and related diseases. Type I collagen is the most abundant protein in all vertebrates and a crucial structural molecule for bone and other connective tissues. Its misfolding causes bone fragility, skeletal deformity and other tissue failures. Studies of newly discovered bone disorders indicate that collagen folding, chaperones involved in the folding process, cellular responses to misfolding and related bone pathologies might not follow conventional protein folding paradigms. In this review, we examine the features that distinguish collagen folding from that of other proteins and describe the findings that are beginning to reveal how cells manage collagen folding and misfolding. We discuss implications of these studies for general protein folding paradigms, unfolded protein response in cells and protein folding diseases.

  18. Evaluation of Quinazoline analogues as Glucocerebrosidase Inhibitors with Chaperone activity

    PubMed Central

    Marugan, Juan J.; Zheng, Wei; Motabar, Omid; Southall, Noel; Goldin, Ehud; Westbroek, Wendy; K.Stubblefield, Barbara; Sidransky, Ellen; Aungst, Ronald A.; Lea, Wendy A.; Simeonov, Anton; Leister, William; Austin, Christopher P.

    2011-01-01

    Gaucher disease is a Lysosomal Storage Disorder (LSD) caused by deficiency in the enzyme glucocerebrosidase (GC). Small molecule chaperones of protein folding and translocation have been proposed as a promising therapeutic approach to this LSD. Most small molecule chaperones described in the literature contain an iminosugar scaffold. Here we present the discovery and evaluation of a new series of GC inhibitors with a quinazoline core. We demonstrate that this series can improve the translocation of GC to the lysosome in patient-derived cells. To optimize this chemical series, systematic synthetic modifications were performed and the SAR was evaluated and compared using three different readouts of compound activity – enzymatic inhibition, enzyme thermostabilization, and lysosomal translocation of GC. PMID:21250698

  19. Co-chaperones of the mammalian endoplasmic reticulum.

    PubMed

    Melnyk, Armin; Rieger, Heiko; Zimmermann, Richard

    2015-01-01

    In mammalian cells, the rough endoplasmic reticulum or ER plays a central role in the biogenesis of most extracellular plus many organellar proteins and in cellular calcium homeostasis. Therefore, this organelle comprises molecular chaperones that are involved in import, folding/assembly, export, and degradation of polypeptides in millimolar concentrations. In addition, there are calcium channels/pumps and signal transduction components present in the ER membrane that affect and are affected by these processes. The ER lumenal Hsp70, termed immunoglobulin-heavy chain binding protein or BiP, is the central player in all these activities and involves up to seven different co-chaperones, i.e. ER-membrane integrated as well as ER-lumenal Hsp40s, which are termed ERj or ERdj, and two nucleotide exchange factors.

  20. Integrating Immunometabolism and Macrophage Diversity

    PubMed Central

    Artyomov, Maxim; Sergushichev, Alexey; Schilling, Joel D.

    2017-01-01

    Macrophages are heterogeneous cells that play a key role in inflammatory and tissue reparative responses. Over the past decade it has become clear that shifts in cellular metabolism are important determinants of macrophage function and phenotype. At the same time, our appreciation of macrophage diversity in vivo has also been increasing. Factors such as cell origin and tissue localization are now recognized as important variables that influence macrophage biology. Whether different macrophage populations also have unique metabolic phenotypes has not been extensively explored. In this article, we will discuss the importance of understanding how macrophage origin can modulate metabolic programming and influence inflammatory responses. PMID:27771140

  1. Crystal Structures of Cisplatin Bound to a Human Copper Chaperone

    SciTech Connect

    Boal, Amie K.; Rosenzweig, Amy C.

    2010-08-16

    Copper trafficking proteins, including the chaperone Atox1 and the P{sub 1B}-type ATPase ATP7B, have been implicated in cellular resistance to the anticancer drug cisplatin. We have determined two crystal structures of cisplatin-Atox1 adducts that reveal platinum coordination by the conserved CXXC copper-binding motif. Direct interaction of cisplatin with this functionally relevant site has significant implications for understanding the molecular basis for resistance mediated by copper transport pathways.

  2. Generalized iterative annealing model for the action of RNA chaperones

    NASA Astrophysics Data System (ADS)

    Hyeon, Changbong; Thirumalai, D.

    2013-09-01

    As a consequence of the rugged landscape of RNA molecules their folding is described by the kinetic partitioning mechanism according to which only a small fraction (ϕF) reaches the folded state while the remaining fraction of molecules is kinetically trapped in misfolded intermediates. The transition from the misfolded states to the native state can far exceed biologically relevant time. Thus, RNA folding in vivo is often aided by protein cofactors, called RNA chaperones, that can rescue RNAs from a multitude of misfolded structures. We consider two models, based on chemical kinetics and chemical master equation, for describing assisted folding. In the passive model, applicable for class I substrates, transient interactions of misfolded structures with RNA chaperones alone are sufficient to destabilize the misfolded structures, thus entropically lowering the barrier to folding. For this mechanism to be efficient the intermediate ribonucleoprotein complex between collapsed RNA and protein cofactor should have optimal stability. We also introduce an active model (suitable for stringent substrates with small ϕF), which accounts for the recent experimental findings on the action of CYT-19 on the group I intron ribozyme, showing that RNA chaperones do not discriminate between the misfolded and the native states. In the active model, the RNA chaperone system utilizes chemical energy of adenosine triphosphate hydrolysis to repeatedly bind and release misfolded and folded RNAs, resulting in substantial increase of yield of the native state. The theory outlined here shows, in accord with experiments, that in the steady state the native state does not form with unit probability.

  3. Transcriptional profiling of Bordetella pertussis reveals requirement of RNA chaperone Hfq for Type III secretion system functionality.

    PubMed

    Bibova, Ilona; Hot, David; Keidel, Kristina; Amman, Fabian; Slupek, Stephanie; Cerny, Ondrej; Gross, Roy; Vecerek, Branislav

    2015-01-01

    Bordetella pertussis, the causative agent of human whooping cough (pertussis) produces a complex array of virulence factors in order to establish efficient infection in the host. The RNA chaperone Hfq and small regulatory RNAs are key players in posttranscriptional regulation in bacteria and have been shown to play an essential role in virulence of a broad spectrum of bacterial pathogens. This study represents the first attempt to characterize the Hfq regulon of the human pathogen B. pertussis under laboratory conditions as well as upon passage in the host and indicates that loss of Hfq has a profound effect on gene expression in B. pertussis. Comparative transcriptional profiling revealed that Hfq is required for expression of several virulence factors in B. pertussis cells including the Type III secretion system (T3SS). In striking contrast to the wt strain, T3SS did not become operational in the hfq mutant passaged either through mice or macrophages thereby proving that Hfq is required for the functionality of the B. pertussis T3SS. Likewise, expression of virulence factors vag8 and tcfA encoding autotransporter and tracheal colonization factor, respectively, was strongly reduced in the hfq mutant. Importantly, for the first time we demonstrate that B. pertussis T3SS can be activated upon contact with macrophage cells in vitro.

  4. A histone chaperone, DEK, transcriptionally coactivates a nuclear receptor

    PubMed Central

    Sawatsubashi, Shun; Murata, Takuya; Lim, Jinseon; Fujiki, Ryoji; Ito, Saya; Suzuki, Eriko; Tanabe, Masahiko; Zhao, Yue; Kimura, Shuhei; Fujiyama, Sally; Ueda, Takashi; Umetsu, Daiki; Ito, Takashi; Takeyama, Ken-ichi; Kato, Shigeaki

    2010-01-01

    Chromatin reorganization is essential for transcriptional control by sequence-specific transcription factors. However, the molecular link between transcriptional control and chromatin reconfiguration remains unclear. By colocalization of the nuclear ecdysone receptor (EcR) on the ecdysone-induced puff in the salivary gland, Drosophila DEK (dDEK) was genetically identified as a coactivator of EcR in both insect cells and intact flies. Biochemical purification and characterization of the complexes containing fly and human DEKs revealed that DEKs serve as histone chaperones via phosphorylation by forming complexes with casein kinase 2. Consistent with the preferential association of the DEK complex with histones enriched in active epigenetic marks, dDEK facilitated H3.3 assembly during puff formation. In some human myeloid leukemia patients, DEK was fused to CAN by chromosomal translocation. This mutation significantly reduced formation of the DEK complex, which is required for histone chaperone activity. Thus, the present study suggests that at least one histone chaperone can be categorized as a type of transcriptional coactivator for nuclear receptors. PMID:20040570

  5. Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins

    PubMed Central

    Costello, Shawn M.; Plummer, Ashlee M.; Fleming, Patrick J.; Fleming, Karen G.

    2016-01-01

    Outer membrane protein (OMP) biogenesis is critical to bacterial physiology because the cellular envelope is vital to bacterial pathogenesis and antibiotic resistance. The process of OMP biogenesis has been studied in vivo, and each of its components has been studied in isolation in vitro. This work integrates parameters and observations from both in vivo and in vitro experiments into a holistic computational model termed “Outer Membrane Protein Biogenesis Model” (OMPBioM). We use OMPBioM to assess OMP biogenesis mathematically in a global manner. Using deterministic and stochastic methods, we are able to simulate OMP biogenesis under varying genetic conditions, each of which successfully replicates experimental observations. We observe that OMPs have a prolonged lifetime in the periplasm where an unfolded OMP makes, on average, hundreds of short-lived interactions with chaperones before folding into its native state. We find that some periplasmic chaperones function primarily as quality-control factors; this function complements the folding catalysis function of other chaperones. Additionally, the effective rate for the β-barrel assembly machinery complex necessary for physiological folding was found to be higher than has currently been observed in vitro. Overall, we find a finely tuned balance between thermodynamic and kinetic parameters maximizes OMP folding flux and minimizes aggregation and unnecessary degradation. In sum, OMPBioM provides a global view of OMP biogenesis that yields unique insights into this essential pathway. PMID:27482090

  6. Enhancement of macrophage-mediated tumor cell killing by bacterial outer membrane proteins (porins).

    PubMed Central

    Weinberg, J B; Ribi, E; Wheat, R W

    1983-01-01

    Various microbial products are known to influence the function of mouse peritoneal macrophages. Lipopolysaccharide (LPS) and certain lipid A-associated proteins are known to enhance the tumoricidal effects of macrophages. The purpose of this study was to determine whether porins (outer membrane proteins) of Salmonella typhimurium G30/C21 would influence the activity of macrophages from lipid A-responsive and -unresponsive mice. Porins, extracted by a combined sodium dodecyl sulfate-EDTA method from cell walls, were free of LPS as determined by Limulus amebocyte lysate assay and appeared as a band at approximately 36,000 molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In tumor cell killing assays done under LPS-free conditions, the porins in doses of 1 to 10 ng/ml enhanced the tumoricidal effect of macrophages from bacillus Calmette-Guérin-infected C3H/HeN or C3H/HeJ mice. Protein-free LPS enhanced the tumoricidal activity of macrophages from bacillus Calmette-Guérin-infected C3H/HeN but not C3H/HeJ mice. The tumoricidal-enhancing activity of protein-free LPS was blocked by the lipid A-binding antibiotic polymyxin B sulfate, but the effects of porins were not altered by the polymyxin B sulfate. These results suggest that porins, proteins known to alter membrane function, may alter macrophage function by interaction with macrophage membranes. Images PMID:6311745

  7. Macrophages interaction with pulmonary surfactant using coherent anti-Stokes Raman scattering (CARS) microscopy

    NASA Astrophysics Data System (ADS)

    Ocampo, Minette; Telesford, Dana Marie; Allen, Heather

    2012-04-01

    Alveolar pulmonary surfactant, composed mostly of phospholipids, is essential for maintenance of normal lung function. However, increased production of lung surfactant can lead to many pulmonary inflammatory disorders. Alveolar macrophages are responsible for the degradation of the surfactant and exhibit increased lipid uptake in inflamated lungs. Owing to their limited clearance capability, excessive accumulation of surfactant may impair their phagocytic function. In this study, the interaction of the macrophages with different lipid components was studied using coherent anti-Stokes Raman scattering (CARS) microscopy. CARS microscopy, a nonlinear vibrational technique which combines spectroscopy and microscopy, allows noninvasive characterization and imaging of chemical species without preparation or labeling. A monolayer of THP-1 macrophages and palmitic acid-d31 on phosphate buffer solution was transferred to a coverslip using the Langmuir-Blodgett method and then imaged using CARS by mapping the CH2 stretch signal of the lipid membrane of the macrophage and C-D stretch signal from palmitic acid-d31. Preliminary results showed CARS images of the macrophage on the solid substrate and thermal degradation of the sample due to long exposure to high laser power. A contrast image is expected to be observed by mapping the CH2 and C-D signals, which can show the lipid interaction and phagocytosis of the macrophage.

  8. [Macrophages in asthma].

    PubMed

    Medina Avalos, M A; Orea Solano, M

    1997-01-01

    Every time they exist more demonstrations of the paper than performs the line monocytes-macrophage in the patogenesis of the bronchial asthma. The mononuclear phagocytes cells, as the alveolar macrophages, also they can be activated during allergic methods. The monocytes macrophages are possible efficient inductors of the inflammation; this due to the fact that they can secrete inflammatory mediators, between those which are counted the pre-forming granules of peptides, metabolites of oxidation activation, activator of platelets activator and metabolites of the arachidonic acid. The identification of IL-1 in the liquidate of the bronchial ablution of sick asthmatic, as well as the identification of IL-1 in the I bronchioalveolar washing of places of allergens cutaneous prick, supports the activation concept mononuclear of phagocytic cells in allergic sufferings.

  9. Targeting macrophage Histone deacetylase 3 stabilizes atherosclerotic lesions

    PubMed Central

    Hoeksema, Marten A; Gijbels, Marion JJ; Van den Bossche, Jan; van der Velden, Saskia; Sijm, Ayestha; Neele, Annette E; Seijkens, Tom; Stöger, J Lauran; Meiler, Svenja; Boshuizen, Marieke CS; Dallinga-Thie, Geesje M; Levels, Johannes HM; Boon, Louis; Mullican, Shannon E; Spann, Nathanael J; Cleutjens, Jack P; Glass, Chris K; Lazar, Mitchell A; de Vries, Carlie JM; Biessen, Erik AL; Daemen, Mat JAP; Lutgens, Esther; de Winther, Menno PJ

    2014-01-01

    Macrophages are key immune cells found in atherosclerotic plaques and critically shape atherosclerotic disease development. Targeting the functional repertoire of macrophages may hold novel approaches for future atherosclerosis management. Here, we describe a previously unrecognized role of the epigenomic enzyme Histone deacetylase 3 (Hdac3) in regulating the atherosclerotic phenotype of macrophages. Using conditional knockout mice, we found that myeloid Hdac3 deficiency promotes collagen deposition in atherosclerotic lesions and thus induces a stable plaque phenotype. Also, macrophages presented a switch to anti-inflammatory wound healing characteristics and showed improved lipid handling. The pro-fibrotic phenotype was directly linked to epigenetic regulation of the Tgfb1 locus upon Hdac3 deletion, driving smooth muscle cells to increased collagen production. Moreover, in humans, HDAC3 was the sole Hdac upregulated in ruptured atherosclerotic lesions, Hdac3 associated with inflammatory macrophages, and HDAC3 expression inversely correlated with pro-fibrotic TGFB1 expression. Collectively, we show that targeting the macrophage epigenome can improve atherosclerosis outcome and we identify Hdac3 as a potential novel therapeutic target in cardiovascular disease. PMID:25007801

  10. Membrane fusion catalyzed by a Rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis

    PubMed Central

    Zucchi, Paola C.; Zick, Michael

    2011-01-01

    The fusion of sealed biological membranes joins their enclosed aqueous compartments while mixing their membrane bilayers. Reconstituted fusion reactions are commonly assayed by lipid mixing, which can result from either true fusion or from lysis and its attendant reannealing of membranes. Fusion is also frequently assayed by the mixing of lumenal aqueous compartments, using probes of low molecular weight. With several probes (biotin, methylumbelliferyl-N-acetyl-α-d-neuraminic acid, and dithionite), we find that yeast vacuolar SNAREs (SNAP [Soluble NSF attachment protein] Receptors) increase the permeability of membranes to small molecules and that this permeabilization is enhanced by homotypic fusion and vacuole protein sorting complex (HOPS) and Sec17p/Sec18p, the vacuolar tethering and SNARE chaperone proteins. We now report the development of a novel assay that allows the parallel assessment of lipid mixing, the mixing of intact lumenal compartments, any lysis that occurs, and the membrane permeation of small molecules. Applying this assay to an all-purified reconstituted system consisting of vacuolar lipids, the four vacuolar SNAREs, the SNARE disassembly chaperones Sec17p and Sec18p, the Rab Ypt7p, and the Rab effector/SM protein complex HOPS, we show that true fusion is accompanied by strongly enhanced membrane permeability to small molecules and a measurable rate of lysis. PMID:21976702

  11. Methods to study histone chaperone function in nucleosome assembly and chromatin transcription.

    PubMed

    Senapati, Parijat; Sudarshan, Deepthi; Gadad, Shrikanth S; Shandilya, Jayasha; Swaminathan, Venkatesh; Kundu, Tapas K

    2015-01-01

    Histone chaperones are histone interacting proteins that are involved in various stages of histone metabolism in the cell such as histone storage, transport, nucleosome assembly and disassembly. Histone assembly and disassembly are essential processes in certain DNA-templated phenomena such as replication, repair and transcription in eukaryotes. Since the first histone chaperone Nucleoplasmin was discovered in Xenopus, a plethora of histone chaperones have been identified, characterized and their functional significance elucidated in the last 35 years or so. Some of the histone chaperone containing complexes such as FACT have been described to play a significant role in nucleosome disassembly during transcription elongation. We have reported earlier that human Nucleophosmin (NPM1), a histone chaperone belonging to the Nucleoplasmin family, is a co-activator of transcription. In this chapter, we describe several methods that are used to study the histone chaperone activity of proteins and their role in transcription.

  12. Reactivation of Aggregated Proteins by the ClpB/DnaK Bi-chaperone System

    PubMed Central

    Zolkiewski, Michal; Chesnokova, Liudmila S.; Witt, Stephan N.

    2016-01-01

    Protein aggregation is a common problem in protein biochemistry and is linked to many cellular pathologies and human diseases. The molecular chaperone ClpB can resolubilize and reactivate aggregated proteins. This unit describes the procedure for following reactivation of an aggregated enzyme glucose-6-phosphate dehydrogenase mediated by ClpB from Escherichia coli in cooperation with another molecular chaperone DnaK. The procedures for purification of these chaperones are also described. PMID:26836408

  13. MicroRNA 21 Is a Homeostatic Regulator of Macrophage Polarization and Prevents Prostaglandin E2-Mediated M2 Generation

    PubMed Central

    Wang, Zhuo; Brandt, Stephanie; Medeiros, Alexandra; Wang, Soujuan; Wu, Hao; Dent, Alexander; Serezani, C. Henrique

    2015-01-01

    Macrophages dictate both initiation and resolution of inflammation. During acute inflammation classically activated macrophages (M1) predominate, and during the resolution phase alternative macrophages (M2) are dominant. The molecular mechanisms involved in macrophage polarization are understudied. MicroRNAs are differentially expressed in M1 and M2 macrophages that influence macrophage polarization. We identified a role of miR-21 in macrophage polarization, and found that cross-talk between miR-21 and the lipid mediator prostaglandin E2 (PGE2) is a determining factor in macrophage polarization. miR-21 inhibition impairs expression of M2 signature genes but not M1 genes. PGE2 and its downstream effectors PKA and Epac inhibit miR-21 expression and enhance expression of M2 genes, and this effect is more pronounced in miR-21-/- cells. Among potential targets involved in macrophage polarization, we found that STAT3 and SOCS1 were enhanced in miR-21-/- cells and further enhanced by PGE2. We found that STAT3 was a direct target of miR-21 in macrophages. Silencing the STAT3 gene abolished PGE2-mediated expression of M2 genes in miR-21-/- macrophages. These data shed light on the molecular brakes involved in homeostatic macrophage polarization and suggest new therapeutic strategies to prevent inflammatory responses. PMID:25706647

  14. Therapeutic uses of drug-carrier systems for imidazole-containing dipeptide compounds that act as pharmacological chaperones and have significant impact on the treatment of chronic diseases associated with increased oxidative stress and the formation of advanced glycation end products.

    PubMed

    Babizhayev, Mark A; Yegorov, Yegor E

    2010-01-01

    The purpose of this study was to determine how the naturally occurring molecules N-acetylcarnosine, L-carnosine, and carcinine, which are chemical or pharmacological chaperones, affect the cells and biomolecules of patients with skin diseases, cosmetic skin lesions, or underlying clinically significant visual impairment such as age-related cataracts, age-related retinal degeneration, and ocular complications of diabetes. We evaluated and characterized the effects of cited pharmacological chaperones on enzyme activity, protein structure in tissues, and other biomarkers of diseases in skin cells and tissues or in ocular tissues (human cataractous and normal lenses) derived from ophthalmic patients or age-matched donors. The samples were used to test imidazole-containing peptidomimetic chemical/pharmacological chaperones in relation to oxidative stress induced by reaction with lipid peroxides or advanced non-enzymatic glycation processes. Chaperone function is characterized by interaction with other proteins, mediating their folding, transport, and interaction with other molecules, lipid peroxidation products, and membranes. Although these therapies remain on hold pending further investigation, we present growing evidence demonstrating the ability of N-acetylcarnosine (lubricant eye drops) or carcinine pharmacological chaperone therapy to act as novel treatments for age-related cataracts, age-related macular degeneration, and ocular complications of diabetes. Finally, we examine strategies for identifying potential chaperone compounds and for experimentally demonstrating chaperone and transglycating (de-glycation) types of activity in in vitro and in vivo models of human age-related eye diseases, such as cataracts, and advanced glycation tissue protein-engineered systems.

  15. The right place at the right time: chaperoning core histone variants.

    PubMed

    Mattiroli, Francesca; D'Arcy, Sheena; Luger, Karolin

    2015-11-01

    Histone proteins dynamically regulate chromatin structure and epigenetic signaling to maintain cell homeostasis. These processes require controlled spatial and temporal deposition and eviction of histones by their dedicated chaperones. With the evolution of histone variants, a network of functionally specific histone chaperones has emerged. Molecular details of the determinants of chaperone specificity for different histone variants are only slowly being resolved. A complete understanding of these processes is essential to shed light on the genuine biological roles of histone variants, their chaperones, and their impact on chromatin dynamics.

  16. The right place at the right time: chaperoning core histone variants

    PubMed Central

    Mattiroli, Francesca; D’Arcy, Sheena; Luger, Karolin

    2015-01-01

    Histone proteins dynamically regulate chromatin structure and epigenetic signaling to maintain cell homeostasis. These processes require controlled spatial and temporal deposition and eviction of histones by their dedicated chaperones. With the evolution of histone variants, a network of functionally specific histone chaperones has emerged. Molecular details of the determinants of chaperone specificity for different histone variants are only slowly being resolved. A complete understanding of these processes is essential to shed light on the genuine biological roles of histone variants, their chaperones, and their impact on chromatin dynamics. PMID:26459557

  17. Loss of hepatic chaperone-mediated autophagy accelerates proteostasis failure in aging

    PubMed Central

    Schneider, Jaime L; Villarroya, Joan; Diaz-Carretero, Antonio; Patel, Bindi; Urbanska, Aleksandra M; Thi, Mia M; Villarroya, Francesc; Santambrogio, Laura; Cuervo, Ana Maria

    2015-01-01

    Chaperone-mediated autophagy (CMA), a cellular process that contributes to protein quality control through targeting of a subset of cytosolic proteins to lysosomes for degradation, undergoes a functional decline with age. We have used a mouse model with liver-specific defective CMA to identify changes in proteostasis attributable to reduced CMA activity in this organ with age. We have found that other proteolytic systems compensate for CMA loss in young mice which helps to preserve proteostasis. However, these compensatory responses are not sufficient for protection against proteotoxicity induced by stress (oxidative stress, lipid challenges) or associated with aging. Livers from old mice with CMA blockage exhibit altered protein homeostasis, enhanced susceptibility to oxidative stress and hepatic dysfunction manifested by a diminished ability to metabolize drugs, and a worsening of the metabolic dysregulation identified in young mice. Our study reveals that while the regulatory function of CMA cannot be compensated for in young organisms, its contribution to protein homeostasis can be handled by other proteolytic systems. However, the decline in the compensatory ability identified with age explains the more severe consequences of CMA impairment in older organisms and the contribution of CMA malfunction to the gradual decline in proteostasis and stress resistance observed during aging. PMID:25620427

  18. Polydatin Inhibits Formation of Macrophage-Derived Foam Cells

    PubMed Central

    Wu, Min; Liu, Meixia; Guo, Gang; Zhang, Wengao; Liu, Longtao

    2015-01-01

    Rhizoma Polygoni Cuspidati, a Chinese herbal medicine, has been widely used in traditional Chinese medicine for a long time. Polydatin, one of the major active ingredients in Rhizoma Polygoni Cuspidati, has been recently shown to possess extensive cardiovascular pharmacological activities. In present study, we examined the effects of Polydatin on the formation of peritoneal macrophage-derived foam cells in Apolipoprotein E gene knockout mice (ApoE−/−) and explored the potential underlying mechanisms. Peritoneal macrophages were collected from ApoE−/− mice and cultured in vitro. These cells sequentially were divided into four groups: Control group, Model group, Lovastatin group, and Polydatin group. Our results demonstrated that Polydatin significantly inhibits the formation of foam cells derived from peritoneal macrophages. Further studies indicated that Polydatin regulates the metabolism of intracellular lipid and possesses anti-inflammatory effects, which may be regulated through the PPAR-γ signaling pathways. PMID:26557864

  19. Macrophage in chronic kidney disease

    PubMed Central

    Flaquer, Maria; Cruzado, Josep M.

    2016-01-01

    Chronic kidney disease (CKD) has become a major health problem worldwide. This review describes the role of macrophages in CKD and highlights the importance of anti-inflammatory M2 macrophage activation in both renal fibrosis and wound healing processes. Furthermore, the mechanisms by which M2 macrophages induce renal repair and regeneration are still under debate and currently demand more attention. The M1/M2 macrophage balance is related to the renal microenvironment and could influence CKD progression. In fact, an inflammatory renal environment and M2 plasticity can be the major hurdles to establishing macrophage cell-based therapies in CKD. M2 macrophage cell-based therapy is promising if the M2 phenotype remains stable and is ‘fixed’ by in vitro manipulation. However, a greater understanding of phenotype polarization is still required. Moreover, better strategies and targets to induce reparative macrophages in vivo should guide future investigations in order to abate kidney diseases. PMID:27994852

  20. Transcriptional Regulation and Macrophage Differentiation.

    PubMed

    Hume, David A; Summers, Kim M; Rehli, Michael

    2016-06-01

    Monocytes and macrophages are professional phagocytes that occupy specific niches in every tissue of the body. Their survival, proliferation, and differentiation are controlled by signals from the macrophage colony-stimulating factor receptor (CSF-1R) and its two ligands, CSF-1 and interleukin-34. In this review, we address the developmental and transcriptional relationships between hematopoietic progenitor cells, blood monocytes, and tissue macrophages as well as the distinctions from dendritic cells. A huge repertoire of receptors allows monocytes, tissue-resident macrophages, or pathology-associated macrophages to adapt to specific microenvironments. These processes create a broad spectrum of macrophages with different functions and individual effector capacities. The production of large transcriptomic data sets in mouse, human, and other species provides new insights into the mechanisms that underlie macrophage functional plasticity.

  1. Macrophage polarization in kidney diseases.

    PubMed

    Tian, Shaojiang; Chen, Shi-You

    Macrophage accumulation associates closely with the degree of renal structural injury and renal dysfunction in human kidney diseases. Depletion of macrophages reduces while adoptive transfer of macrophages worsens inflammation in animal models of the renal injury. However, emerging evidence support that macrophage polarization plays a critical role in the progression of a number of kidney diseases including obstructive nephropathy, ischemia-reperfusion injury, glomerulonephritis, diabetic nephropathy, and other kidney diseases. In this mini-review, we briefly summarize the macrophage infiltration and polarization in these inflammatory and fibrotic kidney diseases, discussing the results mostly from studies in animal models. In view of the critical role of macrophage in the progression of these diseases, manipulating macrophage phenotype may be a potential effective strategy to treat various kidney diseases.

  2. Catapult mechanism renders the chaperone action of Hsp70 unidirectional.

    PubMed

    Gisler, S M; Pierpaoli, E V; Christen, P

    1998-06-19

    Molecular chaperones of the Hsp70 type promote the folding and membrane translocation of proteins. The interaction of Hsp70s with polypeptides is linked to ATP binding and hydrolysis. We formed complexes of seven different fluorescence-labeled peptides with DnaK, the Hsp70 homolog of Escherichia coli, and determined the rate of peptide release under two different sets of conditions. (1) Upon addition of ATP to nucleotide-free peptide.DnaK complexes, all tested peptides were released with similar rate constants (2.2 s-1 to 6.7 s-1). (2) In the binding equilibrium of peptide and ATP-liganded DnaK, the dissociation followed one or two-step reactions, depending on the amino acid sequence of the peptide. For the monophasic reactions, the dissociation rate constants diverged by four orders of magnitude from 0.0004 s-1 to 5.7 s-1; for the biphasic reactions, the rate constants of the second, slower isomerization step were in the range from 0.3 s-1 to 0.0005 s-1. The release of the different peptides in case (1) is 1.4 to 14,000 times faster than in case (2). Apparently, binding of ATP induces a transient state of the chaperone which ejects target peptides before the final state of ATP-liganded DnaK is reached. This "catapult" mechanism provides the chaperone cycle with a mode of peptide release that does not correspond with the reverse of peptide binding. By allowing the conformation of the outgoing polypeptide to differ from that of the incoming polypeptide, a futile cycle with respect to conformational work exerted on the target protein is obviated.

  3. Structure of Vps75 and Implications for Histone Chaperone Function

    SciTech Connect

    Tang,Y.; Meeth, K.; Jiang, E.; Luo, c.; Marmostein, R.

    2008-01-01

    The vacuolar protein sorting 75 (Vps75) histone chaperone participates in chromatin assembly and disassembly at both active and inactive genes through the preferential binding to histone H3-H4. Vps75 is also one of two histone chaperones, along with antisilencing factor 1, that promotes histone H3-Lys-56 acetylation by the regulation of Ty1 transposition protein 109 (Rtt109) histone acetyltransferase. Here, we report the x-ray crystal structure of Vps75 and carry out biochemical studies to characterize its interaction with Rtt109. We find that the Vps75 structure forms a homodimeric 'headphone' architecture that includes an extended helical dimerization domain and earmuff domains at opposite ends and sides of the dimerization domain. Despite the similar overall architecture with the yeast nucleosome assembly protein 1 and human SET/TAF-1{beta}/INHAT histone chaperones, Vps75 shows several unique features including the relative disposition of the earmuff domains to the dimerization domain, characteristics of the earmuff domains, and a pronounced cleft at the center of the Vps75 dimer. These differences appear to correlate with the unique function of Vps75 to interact with Rtt109 for histone acetylation. Our biochemical studies reveal that two surfaces on the earmuff domain of Vps75 participate in Rtt109 interaction with a stoichiometry of 2:1, thus leaving the pronounced central cleft of the Vps75 dimer largely accessible for histone binding. Taken together, our data provide a structural framework for understanding how Vps75 mediates both nucleosome assembly and histone acetylation by Rtt109.

  4. A chemical chaperone induces inhomogeneous conformational changes in flexible proteins.

    PubMed

    Hamdane, Djemel; Velours, Christophe; Cornu, David; Nicaise, Magali; Lombard, Murielle; Fontecave, Marc

    2016-07-27

    Organic osmolytes also known as chemical chaperones are major cellular compounds that favor, by an unclear mechanism, protein's compaction and stabilization of the native state. Here, we have examined the chaperone effect of the naturally occurring trimethylamine N-oxide (TMAO) osmolyte on a loosely packed protein (LPP), known to be a highly flexible form, using an apoprotein mutant of the flavin-dependent RNA methyltransferase as a model. Thermal and chemical denaturation experiments showed that TMAO stabilizes the structural integrity of the apoprotein dramatically. The denaturation reaction is irreversible indicating that the stability of the apoprotein is under kinetic control. This result implies that the stabilization is due to a TMAO-induced reconfiguration of the flexible LPP state, which leads to conformational limitations of the apoprotein likely driven by favorable entropic contribution. Evidence for the conformational perturbation of the apoprotein had been obtained through several biophysical approaches notably analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, labelling experiments and proteolysis coupled to mass spectrometry. Unexpectedly, TMAO promotes an overall elongation or asymmetrical changes of the hydrodynamic shape of the apoprotein without alteration of the secondary structure. The modulation of the hydrodynamic properties of the protein is associated with diverse inhomogenous conformational changes: loss of the solvent accessible cavities resulting in a dried protein matrix; some side-chain residues initially buried become solvent exposed while some others become hidden. Consequently, the TMAO-induced protein state exhibits impaired capability in the flavin binding process. Our study suggests that the nature of protein conformational changes induced by the chemical chaperones may be specific to protein packing and plasticity. This could be an efficient mechanism by which the cell controls and finely tunes the

  5. Chaperone proteins and brain tumors: Potential targets and possible therapeutics1

    PubMed Central

    Graner, Michael W.; Bigner, Darell D.

    2005-01-01

    Chaperone proteins are most notable for the proteo- and cyotoprotective capacities they afford during cellular stress. Under conditions of cellular normalcy, chaperones still play integral roles in the folding of nascent polypeptides into functional entities, in assisting in intracellular/intraorganellar transport, in assembly and maintenance of multi-subunit protein complexes, and in aiding and abetting the degradation of senescent proteins. Tumors frequently have relatively enhanced needs for chaperone number and activity because of the stresses of rapid proliferation, increased metabolism, and overall genetic instability. Thus, it may be possible to take advantage of this reliance that tumor cells have on chaperones by pharmacologic and biologic means. Certain chaperones are abundant in the brain, which implies important roles for them. While it is presumed that the requirements of brain tumors for chaperone proteins are similar to those of any other cell type, tumor or otherwise, very little inquiry has been directed at the possibility of using chaperone proteins as therapeutic targets or even as therapeutic agents against central nervous system malignancies. This review highlights some of the research on the functions of chaperone proteins, on what can be done to modify those functions, and on the physiological responses that tumors and organisms can have to chaperone-targeted or chaperone-based therapies. In particular, this review will also underscore areas of research where brain tumors have been part of the field, although in general those instances are few and far between. This relative dearth of research devoted to chaperone protein targets and therapeutics in brain tumors reveals much untrodden turf to explore for potential treatments of these dreadfully refractive diseases. PMID:16053701

  6. Thanks for asking: Adolescent attitudes and preferences regarding the use of chaperones during physical examinations

    PubMed Central

    Morgan, Renee; Katzman, Debra K; Kaufman, Miriam; Goldberg, Eudice; Toulany, Alene

    2016-01-01

    BACKGROUND: There is no uniformity as to how and when chaperones should be used for general and intimate (genitalia and/or breasts) physical examinations of adolescents. OBJECTIVE: To explore adolescents’ attitudes and preferences regarding the use of medical chaperones during physical examinations. METHODS: The present analysis was a cross-sectional descriptive study performed as part of a quality improvement project in the Adolescent Medicine Clinics at The Hospital for Sick Children (Toronto, Ontario) between January 1 and April 30, 2011. Adolescents 13 to 18 years of age completed an anonymous 10-item, self-administered questionnaire regarding their thoughts on chaperones during physical examinations. Demographic and descriptive data were collected. RESULTS: A total of 127 adolescents participated in the present study. The mean (± SD) age was 16.3±1.5 years and the majority (93.7%) were female. More than one-half (61%) of female adolescents had previous experience with an intimate examination; however, a chaperone was present only 36% of the time. Seventy percent of female adolescents wanted the choice of a chaperone for a general examination compared with 61% for an intimate examination. Among female adolescents with past chaperone experience, 78% wanted the choice of a chaperone for subsequent intimate examinations, compared with 55% among those with no previous chaperone experience. Only 21% believed they would ask for a chaperone if one were not offered. CONCLUSIONS: Although there was variation in adolescents’ attitudes and preferences regarding the use of chaperones, many females indicated a desire to discuss the option of a chaperone for all types of examinations. PMID:27429571

  7. Schistosomal-derived lysophosphatidylcholine triggers M2 polarization of macrophages through PPARγ dependent mechanisms.

    PubMed

    Assunção, Leonardo Santos; Magalhães, Kelly G; Carneiro, Alan Brito; Molinaro, Raphael; Almeida, Patrícia E; Atella, Georgia C; Castro-Faria-Neto, Hugo C; Bozza, Patrícia T

    2017-02-01

    Mansonic schistosomiasis is a disease caused by the trematode Schistosoma mansoni, endemic to tropical countries. S. mansoni infection induces the formation of granulomas and potent polarization of Th2-type immune response. There is great interest in understanding the mechanisms used by this parasite that causes a modulation of the immune system. Recent studies from our group demonstrated that lipids of S. mansoni, including lysophosphatidylcholine (LPC) have immunomodulatory activity. In the present study, our aim was to investigate the role of lipids derived from S. mansoni in the activation and polarization of macrophages and to characterize the mechanisms involved in this process. Peritoneal macrophages obtained from wild type C57BL/6mice or bone marrow derived macrophages were stimulated in vitro with lipids extracted from adult worms of S. mansoni. We demonstrated that total schistosomal-derived lipids as well as purified LPC induced alternatively activated macrophages/M2 profile observed by increased expression of arginase-1, mannose receptor, Chi3l3, TGFβ and production of IL-10 and PGE2 24h after stimulation. The involvement of the nuclear receptor PPARγ in macrophage response against LPC was investigated. Through Western blot and immunofluorescence confocal microscopy we demonstrated that schistosomal-derived LPC induces increased expression of PPARγ in macrophages. The LPC-induced increased expression of arginase-1 were significantly inhibited by the PPAR-γ antagonist GW9662. Together, these results demonstrate an immunomodulatory role of schistosomal-derived LPC in activating macrophages to a profile of the type M2 through PPARγ-dependent mechanisms, indicating a novel pathway for macrophage polarization triggered by parasite-derived LPC with potential implications to disease pathogenesis.

  8. Exocytosis of macrophage lysosomes leads to digestion of apoptotic adipocytes and foam cell formation[S

    PubMed Central

    Haka, Abigail S.; Barbosa-Lorenzi, Valéria C.; Lee, Hyuek Jong; Falcone, Domenick J.; Hudis, Clifford A.; Dannenberg, Andrew J.

    2016-01-01

    Many types of apoptotic cells are phagocytosed and digested by macrophages. Adipocytes can be hundreds of times larger than macrophages, so they are too large to be digested by conventional phagocytic processes. The nature of the interaction between macrophages and apoptotic adipocytes has not been studied in detail. We describe a cellular process, termed exophagy, that is important for macrophage clearance of dead adipocytes and adipose tissue homeostasis. Using mouse models of obesity, human tissue, and a cell culture model, we show that macrophages form hydrolytic extracellular compartments at points of contact with dead adipocytes using local actin polymerization. These compartments are acidic and contain lysosomal enzymes delivered by exocytosis. Uptake and complete degradation of adipocyte fragments, which are released by extracellular hydrolysis, leads to macrophage foam cell formation. Exophagy-mediated foam cell formation is a highly efficient means by which macrophages internalize large amounts of lipid, which may ultimately overwhelm the metabolic capacity of the macrophage. This process provides a mechanism for degradation of objects, such as dead adipocytes, that are too large to be phagocytosed by macrophages. PMID:27044658

  9. Prostaglandin E2 restrains macrophage maturation via E prostanoid receptor 2/protein kinase A signaling

    PubMed Central

    Zasłona, Zbigniew; Serezani, Carlos H.; Okunishi, Katsuhide; Aronoff, David M.

    2012-01-01

    Prostaglandin E2 (PGE2) is a lipid mediator that acts by ligating 4 distinct G protein–coupled receptors, E prostanoid (EP) 1 to 4. Previous studies identified the importance of PGE2 in regulating macrophage functions, but little is known about its effect on macrophage maturation. Macrophage maturation was studied in vitro in bone marrow cell cultures, and in vivo in a model of peritonitis. EP2 was the most abundant PGE2 receptor expressed by bone marrow cells, and its expression further increased during macrophage maturation. EP2-deficient (EP2−/−) macrophages exhibited enhanced in vitro maturation compared with wild-type cells, as evidenced by higher F4/80 expression. An EP2 antagonist also increased maturation. In the peritonitis model, EP2−/− mice exhibited a higher percentage of F4/80high/CD11bhigh cells and greater expression of macrophage colony-stimulating factor receptor (M-CSFR) in both the blood and the peritoneal cavity. Subcutaneous injection of the PGE2 analog misoprostol decreased M-CSFR expression in bone marrow cells and reduced the number of peritoneal macrophages in wild-type mice but not EP2−/− mice. The suppressive effect of EP2 ligation on in vitro macrophage maturation was mimicked by a selective protein kinase A agonist. Our findings reveal a novel role for PGE2/EP2/protein kinase A signaling in the suppression of macrophage maturation. PMID:22234697

  10. Macrophage infection models for Mycobacterium tuberculosis.

    PubMed

    Johnson, Benjamin K; Abramovitch, Robert B

    2015-01-01

    Mycobacterium tuberculosis colonizes, survives, and grows inside macrophages. In vitro macrophage infection models, using both primary macrophages and cell lines, enable the characterization of the pathogen response to macrophage immune pressure and intracellular environmental cues. We describe methods to propagate and infect primary murine bone marrow-derived macrophages and J774 and THP-1 macrophage-like cell lines. We also present methods on the characterization of M. tuberculosis intracellular survival and the preparation of infected macrophages for imaging.

  11. Regulation of Macrophage Motility by the Water Channel Aquaporin-1: Crucial Role of M0/M2 Phenotype Switch

    PubMed Central

    Tyteca, Donatienne; Nishino, Tomoya; Debaix, Huguette; Van Der Smissen, Patrick; N'Kuli, Francisca; Hoffmann, Delia; Cnops, Yvette; Rabolli, Virginie; van Loo, Geert; Beyaert, Rudi; Huaux, François; Devuyst, Olivier; Courtoy, Pierre J.

    2015-01-01

    The water channel aquaporin-1 (AQP1) promotes migration of many cell types. Although AQP1 is expressed in macrophages, its potential role in macrophage motility, particularly in relation with phenotype polarization, remains unknown. We here addressed these issues in peritoneal macrophages isolated from AQP1-deficient mice, either undifferentiated (M0) or stimulated with LPS to orientate towards pro-inflammatory phenotype (classical macrophage activation; M1). In non-stimulated macrophages, ablation of AQP1 (like inhibition by HgCl2) increased by 2–3 fold spontaneous migration in a Src/PI3K/Rac-dependent manner. This correlated with cell elongation and formation of lamellipodia/ruffles, resulting in membrane lipid and F4/80 recruitment to the leading edge. This indicated that AQP1 normally suppresses migration of resting macrophages, as opposed to other cell types. Resting Aqp1-/- macrophages exhibited CD206 redistribution into ruffles and increased arginase activity like IL4/IL13 (alternative macrophage activation; M2), indicating a M0-M2 shift. In contrast, upon M1 orientation by LPS in vitro or peritoneal inflammation in vivo, migration of Aqp1-/- macrophages was reduced. Taken together, these data indicate that AQP1 oppositely regulates macrophage migration, depending on stimulation or not by LPS, and that macrophage phenotypic and migratory changes may be regulated independently of external cues. PMID:25719758

  12. The Molecular Chaperone DnaK Is a Source of Mutational Robustness.

    PubMed

    Aguilar-Rodríguez, José; Sabater-Muñoz, Beatriz; Montagud-Martínez, Roser; Berlanga, Víctor; Alvarez-Ponce, David; Wagner, Andreas; Fares, Mario A

    2016-10-05

    Molecular chaperones, also known as heat-shock proteins, refold misfolded proteins and help other proteins reach their native conformation. Thanks to these abilities, some chaperones, such as the Hsp90 protein or the chaperonin GroEL, can buffer the deleterious phenotypic effects of mutations that alter protein structure and function. Hsp70 chaperones use a chaperoning mechanism different from that of Hsp90 and GroEL, and it is not known whether they can also buffer mutations. Here, we show that they can. To this end, we performed a mutation accumulation experiment in Escherichia coli, followed by whole-genome resequencing. Overexpression of the Hsp70 chaperone DnaK helps cells cope with mutational load and completely avoid the extinctions we observe in lineages evolving without chaperone overproduction. Additionally, our sequence data show that DnaK overexpression increases mutational robustness, the tolerance of its clients to nonsynonymous nucleotide substitutions. We also show that this elevated mutational buffering translates into differences in evolutionary rates on intermediate and long evolutionary time scales. Specifically, we studied the evolutionary rates of DnaK clients using the genomes of E. coli, Salmonella enterica, and 83 other gamma-proteobacteria. We find that clients that interact strongly with DnaK evolve faster than weakly interacting clients. Our results imply that all three major chaperone classes can buffer mutations and affect protein evolution. They illustrate how an individual protein like a chaperone can have a disproportionate effect on the evolution of a proteome.

  13. Information encoded in non-native states drives substrate-chaperone pairing.

    PubMed

    Mapa, Koyeli; Tiwari, Satyam; Kumar, Vignesh; Jayaraj, Gopal Gunanathan; Maiti, Souvik

    2012-09-05

    Many proteins refold in vitro through kinetic folding intermediates that are believed to be by-products of native-state centric evolution. These intermediates are postulated to play only minor roles, if any, in vivo because they lack any information related to translation-associated vectorial folding. We demonstrate that refolding intermediate of a test protein, generated in vitro, is able to find its cognate chaperone, from the whole complement of Escherichia coli soluble chaperones. Cognate chaperone-binding uniquely alters the conformation of non-native substrate. Importantly, precise chaperone targeting of substrates are maintained as long as physiological molar ratios of chaperones remain unaltered. Using a library of different chaperone substrates, we demonstrate that kinetically trapped refolding intermediates contain sufficient structural features for precise targeting to cognate chaperones. We posit that evolution favors sequences that, in addition to coding for a functional native state, encode folding intermediates with higher affinity for cognate chaperones than noncognate ones.

  14. Epigenome-Guided Analysis of the Transcriptome of Plaque Macrophages during Atherosclerosis Regression Reveals Activation of the Wnt Signaling Pathway

    PubMed Central

    Menon, Prashanthi; Podolsky, Irina; Feig, Jonathan E.; Aderem, Alan; Fisher, Edward A.; Gold, Elizabeth S.

    2014-01-01

    We report the first systems biology investigation of regulators controlling arterial plaque macrophage transcriptional changes in response to lipid lowering in vivo in two distinct mouse models of atherosclerosis regression. Transcriptome measurements from plaque macrophages from the Reversa mouse were integrated with measurements from an aortic transplant-based mouse model of plaque regression. Functional relevance of the genes detected as differentially expressed in plaque macrophages in response to lipid lowering in vivo was assessed through analysis of gene functional annotations, overlap with in vitro foam cell studies, and overlap of associated eQTLs with human atherosclerosis/CAD risk SNPs. To identify transcription factors that control plaque macrophage responses to lipid lowering in vivo, we used an integrative strategy – leveraging macrophage epigenomic measurements – to detect enrichment of transcription factor binding sites upstream of genes that are differentially expressed in plaque macrophages during regression. The integrated analysis uncovered eight transcription factor binding site elements that were statistically overrepresented within the 5′ regulatory regions of genes that were upregulated in plaque macrophages in the Reversa model under maximal regression conditions and within the 5′ regulatory regions of genes that were upregulated in the aortic transplant model during regression. Of these, the TCF/LEF binding site was present in promoters of upregulated genes related to cell motility, suggesting that the canonical Wnt signaling pathway may be activated in plaque macrophages during regression. We validated this network-based prediction by demonstrating that β-catenin expression is higher in regressing (vs. control group) plaques in both regression models, and we further demonstrated that stimulation of canonical Wnt signaling increases macrophage migration in vitro. These results suggest involvement of canonical Wnt signaling in

  15. Imaging macrophages with nanoparticles

    NASA Astrophysics Data System (ADS)

    Weissleder, Ralph; Nahrendorf, Matthias; Pittet, Mikael J.

    2014-02-01

    Nanomaterials have much to offer, not only in deciphering innate immune cell biology and tracking cells, but also in advancing personalized clinical care by providing diagnostic and prognostic information, quantifying treatment efficacy and designing better therapeutics. This Review presents different types of nanomaterial, their biological properties and their applications for imaging macrophages in human diseases, including cancer, atherosclerosis, myocardial infarction, aortic aneurysm, diabetes and other conditions. We anticipate that future needs will include the development of nanomaterials that are specific for immune cell subsets and can be used as imaging surrogates for nanotherapeutics. New in vivo imaging clinical tools for noninvasive macrophage quantification are thus ultimately expected to become relevant to predicting patients' clinical outcome, defining treatment options and monitoring responses to therapy.

  16. Macrophage polarization following chitosan implantation.

    PubMed

    Vasconcelos, Daniela P; Fonseca, Ana C; Costa, Madalena; Amaral, Isabel F; Barbosa, Mário A; Águas, Artur P; Barbosa, Judite N

    2013-12-01

    Macrophages are a key cell in the host response to implants and can be polarized into different phenotypes capable of inducing both detrimental and beneficial outcomes in tissue repair and remodeling, being important in tissue engineering and regenerative medicine. The objective of this study was to evaluate the macrophage response to 3D porous chitosan (Ch) scaffolds with different degrees of acetylation (DA, 5% and 15%). The M1/M2 phenotypic polarization profile of macrophages was investigated in vivo using a rodent air-pouch model. Our results show that the DA affects the macrophage response. Ch scaffolds with DA 5% induced the adhesion of lower numbers of inflammatory cells, being the M2 the predominant phenotypic profile among the adherent macrophages. In the inflammatory exudates F4/80(+)/CD206(+) cells (M2 macrophages) appeared in higher numbers then F4/80(+)/CCR7(+) cells (M1 macrophages), in addition, lower levels of pro-inflammatory cytokines together with higher levels of anti-inflammatory cytokines were found. Ch scaffolds with DA 15% showed opposite results, since M1 were the predominant macrophages both adherent to the scaffold and in the exudates, together with high levels of pro-inflammatory cytokines. In conclusion, Ch scaffolds with DA 5% induced a benign M2 anti-inflammatory macrophage response, whereas Ch scaffolds with DA 15% caused a macrophage M1 pro-inflammatory response.

  17. Degradation of AF1Q by chaperone-mediated autophagy

    SciTech Connect

    Li, Peng; Ji, Min; Lu, Fei; Zhang, Jingru; Li, Huanjie; Cui, Taixing; Li Wang, Xing; Tang, Dongqi; Ji, Chunyan

    2014-09-10

    AF1Q, a mixed lineage leukemia gene fusion partner, is identified as a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetic and adult myelodysplastic syndrome. AF1Q is highly regulated during hematopoietic progenitor differentiation and development but its regulatory mechanism has not been defined clearly. In the present study, we used pharmacological and genetic approaches to influence chaperone-mediated autophagy (CMA) and explored the degradation mechanism of AF1Q. Pharmacological inhibitors of lysosomal degradation, such as chloroquine, increased AF1Q levels, whereas activators of CMA, including 6-aminonicotinamide and nutrient starvation, decreased AF1Q levels. AF1Q interacts with HSPA8 and LAMP-2A, which are core components of the CMA machinery. Knockdown of HSPA8 or LAMP-2A increased AF1Q protein levels, whereas overexpression showed the opposite effect. Using an amino acid deletion AF1Q mutation plasmid, we identified that AF1Q had a KFERQ-like motif which was recognized by HSPA8 for CMA-dependent proteolysis. In conclusion, we demonstrate for the first time that AF1Q can be degraded in lysosomes by CMA. - Highlights: • Chaperone-mediated autophagy (CMA) is involved in the degradation of AF1Q. • Macroautophagy does not contribute to the AF1Q degradation. • AF1Q has a KFERQ-like motif that is recognized by CMA core components.

  18. Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase.

    PubMed

    Bendikov-Bar, Inna; Maor, Gali; Filocamo, Mirella; Horowitz, Mia

    2013-02-01

    Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase β-glucocerebrosidase (GCase). The disease has a broad spectrum of phenotypes, which were divided into three different Types; Type 1 GD is not associated with primary neurological disease while Types 2 and 3 are associated with central nervous system disease. GCase molecules are synthesized on endoplasmic reticulum (ER)-bound polyribosomes, translocated into the ER and following modifications and correct folding, shuttle to the lysosomes. Mutant GCase molecules, which fail to fold correctly, undergo ER associated degradation (ERAD) in the proteasomes, the degree of which is one of the factors that determine GD severity. Several pharmacological chaperones have already been shown to assist correct folding of mutant GCase molecules in the ER, thus facilitating their trafficking to the lysosomes. Ambroxol, a known expectorant, is one such chaperone. Here we show that ambroxol increases both the lysosomal fraction and the enzymatic activity of several mutant GCase variants in skin fibroblasts derived from Type 1 and Type 2 GD patients.

  19. Chaperones in Polyglutamine Aggregation: Beyond the Q-Stretch

    PubMed Central

    Kuiper, E. F. E.; de Mattos, Eduardo P.; Jardim, Laura B.; Kampinga, Harm H.; Bergink, Steven

    2017-01-01

    Expanded polyglutamine (polyQ) stretches in at least nine unrelated proteins lead to inherited neuronal dysfunction and degeneration. The expansion size in all diseases correlates with age at onset (AO) of disease and with polyQ protein aggregation, indicating that the expanded polyQ stretch is the main driving force for the disease onset. Interestingly, there is marked interpatient variability in expansion thresholds for a given disease. Between different polyQ diseases the repeat length vs. AO also indicates the existence of modulatory effects on aggregation of the upstream and downstream amino acid sequences flanking the Q expansion. This can be either due to intrinsic modulation of aggregation by the flanking regions, or due to differential interaction with other proteins, such as the components of the cellular protein quality control network. Indeed, several lines of evidence suggest that molecular chaperones have impact on the handling of different polyQ proteins. Here, we review factors differentially influencing polyQ aggregation: the Q-stretch itself, modulatory flanking sequences, interaction partners, cleavage of polyQ-containing proteins, and post-translational modifications, with a special focus on the role of molecular chaperones. By discussing typical examples of how these factors influence aggregation, we provide more insight on the variability of AO between different diseases as well as within the same polyQ disorder, on the molecular level. PMID:28386214

  20. Structural Basis of Pharmacological Chaperoning for Human β-Galactosidase*

    PubMed Central

    Suzuki, Hironori; Ohto, Umeharu; Higaki, Katsumi; Mena-Barragán, Teresa; Aguilar-Moncayo, Matilde; Ortiz Mellet, Carmen; Nanba, Eiji; Garcia Fernandez, Jose M.; Suzuki, Yoshiyuki; Shimizu, Toshiyuki

    2014-01-01

    GM1 gangliosidosis and Morquio B disease are autosomal recessive diseases caused by the defect in the lysosomal β-galactosidase (β-Gal), frequently related to misfolding and subsequent endoplasmic reticulum-associated degradation. Pharmacological chaperone (PC) therapy is a newly developed molecular therapeutic approach by using small molecule ligands of the mutant enzyme that are able to promote the correct folding and prevent endoplasmic reticulum-associated degradation and promote trafficking to the lysosome. In this report, we describe the enzymological properties of purified recombinant human β-GalWT and two representative mutations in GM1 gangliosidosis Japanese patients, β-GalR201C and β-GalI51T. We have also evaluated the PC effect of two competitive inhibitors of β-Gal. Moreover, we provide a detailed atomic view of the recognition mechanism of these compounds in comparison with two structurally related analogues. All compounds bind to the active site of β-Gal with the sugar-mimicking moiety making hydrogen bonds to active site residues. Moreover, the binding affinity, the enzyme selectivity, and the PC potential are strongly affected by the mono- or bicyclic structure of the core as well as the orientation, nature, and length of the exocyclic substituent. These results provide understanding on the mechanism of action of β-Gal selective chaperoning by newly developed PC compounds. PMID:24737316

  1. Synthetic cation-selective nanotube: Permeant cations chaperoned by anions

    NASA Astrophysics Data System (ADS)

    Hilder, Tamsyn A.; Gordon, Dan; Chung, Shin-Ho

    2011-01-01

    The ability to design ion-selective, synthetic nanotubes which mimic biological ion channels may have significant implications for the future treatment of bacteria, diseases, and as ultrasensitive biosensors. We present the design of a synthetic nanotube made from carbon atoms that selectively allows monovalent cations to move across and rejects all anions. The cation-selective nanotube mimics some of the salient properties of biological ion channels. Before practical nanodevices are successfully fabricated it is vital that proof-of-concept computational studies are performed. With this in mind we use molecular and stochastic dynamics simulations to characterize the dynamics of ion permeation across a single-walled (10, 10), 36 Å long, carbon nanotube terminated with carboxylic acid with an effective radius of 5.08 Å. Although cations encounter a high energy barrier of 7 kT, its height is drastically reduced by a chloride ion in the nanotube. The presence of a chloride ion near the pore entrance thus enables a cation to enter the pore and, once in the pore, it is chaperoned by the resident counterion across the narrow pore. The moment the chaperoned cation transits the pore, the counterion moves back to the entrance to ferry another ion. The synthetic nanotube has a high sodium conductance of 124 pS and shows linear current-voltage and current-concentration profiles. The cation-anion selectivity ratio ranges from 8 to 25, depending on the ionic concentrations in the reservoirs.

  2. Anticancer Gold(III) Porphyrins Target Mitochondrial Chaperone Hsp60.

    PubMed

    Hu, Di; Liu, Yungen; Lai, Yau-Tsz; Tong, Ka-Chung; Fung, Yi-Man; Lok, Chun-Nam; Che, Chi-Ming

    2016-01-22

    Identification of the molecular target(s) of anticancer metal complexes is a formidable challenge since most of them are unstable toward ligand exchange reaction(s) or biological reduction under physiological conditions. Gold(III) meso-tetraphenylporphyrin (gold-1 a) is notable for its high stability in biological milieux and potent in vitro and in vivo anticancer activities. Herein, extensive chemical biology approaches employing photo-affinity labeling, click chemistry, chemical proteomics, cellular thermal shift, saturation-transfer difference NMR, protein fluorescence quenching, and protein chaperone assays were used to provide compelling evidence that heat-shock protein 60 (Hsp60), a mitochondrial chaperone and potential anticancer target, is a direct target of gold-1 a in vitro and in cells. Structure-activity studies with a panel of non-porphyrin gold(III) complexes and other metalloporphyrins revealed that Hsp60 inhibition is specifically dependent on both the gold(III) ion and the porphyrin ligand.

  3. Lipid Nanotechnology

    PubMed Central

    Mashaghi, Samaneh; Jadidi, Tayebeh; Koenderink, Gijsje; Mashaghi, Alireza

    2013-01-01

    Nanotechnology is a multidisciplinary field that covers a vast and diverse array of devices and machines derived from engineering, physics, materials science, chemistry and biology. These devices have found applications in biomedical sciences, such as targeted drug delivery, bio-imaging, sensing and diagnosis of pathologies at early stages. In these applications, nano-devices typically interface with the plasma membrane of cells. On the other hand, naturally occurring nanostructures in biology have been a source of inspiration for new nanotechnological designs and hybrid nanostructures made of biological and non-biological, organic and inorganic building blocks. Lipids, with their amphiphilicity, diversity of head and tail chemistry, and antifouling properties that block nonspecific binding to lipid-coated surfaces, provide a powerful toolbox for nanotechnology. This review discusses the progress in the emerging field of lipid nanotechnology. PMID:23429269

  4. Epigenomics of macrophages.

    PubMed

    Gosselin, David; Glass, Christopher K

    2014-11-01

    Macrophages play essential roles in tissue homeostasis, pathogen elimination, and tissue repair. A defining characteristic of these cells is their ability to efficiently adapt to a variety of abruptly changing and complex environments. This ability is intrinsically linked to a capacity to quickly alter their transcriptome, and this is tightly associated with the epigenomic organization of these cells and, in particular, their enhancer repertoire. Indeed, enhancers are genomic sites that serve as platforms for the integration of signaling pathways with the mechanisms that regulate mRNA transcription. Notably, transcription is pervasive at active enhancers and enhancer RNAs (eRNAs) are tightly coupled to regulated transcription of protein-coding genes. Furthermore, given that each cell type possesses a defining enhancer repertoire, studies on enhancers provide a powerful method to study how specialization of functions among the diverse macrophage subtypes may arise. Here, we review recent studies providing insights into the distinct mechanisms that contribute to the establishment of enhancers and their role in the regulation of transcription in macrophages.

  5. Pharmacological chaperone approaches for rescuing GPCR mutants: Current state, challenges, and screening strategies.

    PubMed

    Beerepoot, Pieter; Nazari, Reza; Salahpour, Ali

    2017-03-01

    A substantial number of G-protein coupled receptors (GPCRs) genetic disorders are due to mutations that cause misfolding or dysfunction of the receptor product. Pharmacological chaperoning approaches can rescue such mutant receptors by stabilizing protein conformations that behave similar to the wild type protein. For example, this can be achieved by improving folding efficiency and/or interaction with chaperone proteins. Although efficacy of pharmacological chaperones has been demonstrated in vitro for a variety of GPCRs, translation to clinical use has been limited. In this paper we discuss the history of pharmacological chaperones of GPCR's and other membrane proteins, the challenges in translation to the clinic, and the use of different assays for pharmacological chaperone discovery.

  6. Unfolding the Therapeutic Potential of Chemical Chaperones for Age-related Macular Degeneration

    PubMed Central

    Sauer, Theodor; Patel, Mrinali; Chan, Chi-Chao; Tuo, Jingsheng

    2008-01-01

    SUMMARY Recent studies suggest that pathological processes involved in age-related macular degeneration (AMD) might induce endoplasmic reticulum (ER) stress. Growing evidence demonstrates the ability of chemical chaperones to decrease ER stress and ameliorate ER stress-related disease phenotypes, suggesting that the field of chaperone therapy might hold novel treatments for AMD. In this review, we examine the evidence suggesting a role for ER stress in AMD. Furthermore, we discuss the use of chaperone therapy for the treatment of ER stress-associated diseases, including other neurodegenerative diseases and retinopathies. Finally, we examine strategies for identifying potential chaperone compounds and for experimentally demonstrating chaperone activity in in vitro and in vivo models of human disease. PMID:18528533

  7. Regulation of Neuronal Survival Factor MEF2D by Chaperone-Mediated Autophagy

    PubMed Central

    Yang, Qian; She, Hua; Gearing, Marla; Colla, Emanuela; Lee, Michael; Shacka, John J.; Mao, Zixu

    2009-01-01

    Chaperone-mediated autophagy controls the degradation of selective cytosolic proteins and may protect neurons against degeneration. In a neuronal cell line, we found that chaperone-mediated autophagy regulated the activity of myocyte enhancer factor 2D (MEF2D), a transcription factor required for neuronal survival. MEF2D was observed to continuously shuttle to the cytoplasm, interact with the chaperone Hsc70, and undergo degradation. Inhibition of chaperone-mediated autophagy caused accumulation of inactive MEF2D in the cytoplasm. MEF2D levels were increased in the brains of α-synuclein transgenic mice and patients with Parkinson’s disease. Wild-type α-synuclein and a Parkinson’s disease–associated mutant disrupted the MEF2D-Hsc70 binding and led to neuronal death. Thus, chaperone-mediated autophagy modulates the neuronal survival machinery, and dysregulation of this pathway is associated with Parkinson’s disease. PMID:19119233

  8. Effect of the Surface Charge of Artificial Chaperones on the Refolding of Thermally Denatured Lysozymes.

    PubMed

    Huang, Fan; Shen, Liangliang; Wang, Jianzu; Qu, Aoting; Yang, Huiru; Zhang, Zhenkun; An, Yingli; Shi, Linqi

    2016-02-17

    Artificial chaperones are of great interest in fighting protein misfolding and aggregation for the protection of protein bioactivity. A comprehensive understanding of the interaction between artificial chaperones and proteins is critical for the effective utilization of these materials in biomedicine. In this work, we fabricated three kinds of artificial chaperones with different surface charges based on mixed-shell polymeric micelles (MSPMs), and investigated their protective effect for lysozymes under thermal stress. It was found that MSPMs with different surface charges showed distinct chaperone-like behavior, and the neutral MSPM with PEG shell and PMEO2MA hydrophobic domain at high temperature is superior to the negatively and positively charged one, because of the excessive electrostatic interactions between the protein and charged MSPMs. The results may benefit to optimize this kind of artificial chaperone with enhanced properties and expand their application in the future.

  9. Targeting the molecular chaperone SlyD to inhibit bacterial growth with a small molecule

    PubMed Central

    Kumar, Amit; Balbach, Jochen

    2017-01-01

    Molecular chaperones are essential molecules for cell growth, whereby they maintain protein homeostasis. Because of their central cellular function, bacterial chaperones might be potential candidates for drug targets. Antimicrobial resistance is currently one of the greatest threats to human health, with gram-negative bacteria being of major concern. We found that a Cu2+ complex readily crosses the bacterial cell wall and inhibits SlyD, which is a molecular chaperone, cis/trans peptidyl prolyl isomerise (PPIase) and involved in various other metabolic pathways. The Cu2+ complex binds to the active sites of SlyD, which suppresses its PPIase and chaperone activities. Significant cell growth retardation could be observed for pathogenic bacteria (e.g., Staphylococcus aureus and Pseudomonas aeruginosa). We anticipate that rational development of drugs targeting molecular chaperones might help in future control of pathogenic bacterial growth, in an era of rapidly increasing antibiotic resistance. PMID:28176839

  10. Macrophage-targeted photodynamic detection of vulnerable atherosclerotic plaque

    NASA Astrophysics Data System (ADS)

    Hamblin, Michael R.; Tawakol, Ahmed; Castano, Ana P.; Gad, Faten; Zahra, Touqir; Ahmadi, Atosa; Stern, Jeremy; Ortel, Bernhard; Chirico, Stephanie; Shirazi, Azadeh; Syed, Sakeena; Muller, James E.

    2003-06-01

    Rupture of a vulnerable atherosclerotic plaque (VP) leading to coronary thrombosis is the chief cause of sudden cardiac death. VPs are angiographically insignificant lesions, which are excessively inflamed and characterized by dense macrophage infiltration, large necrotic lipid cores, thin fibrous caps, and paucity of smooth muscle cells. We have recently shown that chlorin(e6) conjugated with maleylated albumin can target macrophages with high selectivity via the scavenger receptor. We report the potential of this macrophage-targeted fluorescent probe to localize in VPs in a rabbit model of atherosclerosis, and allow detection and/or diagnosis by fluorescence spectroscopy or imaging. Atherosclerotic lesions were induced in New Zealand White rabbit aortas by balloon injury followed by administration of a high-fat diet. 24-hours after IV injection of the conjugate into atherosclerotic or normal rabbits, the animals were sacrificed, and aortas were removed, dissected and examined for fluorescence localization in plaques by fiber-based spectrofluorimetry and confocal microscopy. Dye uptake within the aortas was also quantified by fluorescence extraction of samples from aorta segments. Biodistribution of the dye was studied in many organs of the rabbits. Surface spectrofluorimetry after conjugate injection was able to distinguish between plaque and adjacent aorta, between atherosclerotic and normal aorta, and balloon-injured and normal iliac arteries with high significance. Discrete areas of high fluorescence (up to 20 times control were detected in the balloon-injured segments, presumably corresponding to macrophage-rich plaques. Confocal microscopy showed red ce6 fluorescence localized in plaques that showed abundant foam cells and macrophages by histology. Extraction data on aortic tissue corroborated the selectivity of the conjugate for plaques. These data support the strategy of employing macrophage-targeted fluorescent dyes to detect VP by intravascular

  11. Targeting the Monocyte–Macrophage Lineage in Solid Organ Transplantation

    PubMed Central

    van den Bosch, Thierry P. P.; Kannegieter, Nynke M.; Hesselink, Dennis A.; Baan, Carla C.; Rowshani, Ajda T.

    2017-01-01

    There is an unmet clinical need for immunotherapeutic strategies that specifically target the active immune cells participating in the process of rejection after solid organ transplantation. The monocyte–macrophage cell lineage is increasingly recognized as a major player in acute and chronic allograft immunopathology. The dominant presence of cells of this lineage in rejecting allograft tissue is associated with worse graft function and survival. Monocytes and macrophages contribute to alloimmunity via diverse pathways: antigen processing and presentation, costimulation, pro-inflammatory cytokine production, and tissue repair. Cross talk with other recipient immune competent cells and donor endothelial cells leads to amplification of inflammation and a cytolytic response in the graft. Surprisingly, little is known about therapeutic manipulation of the function of cells of the monocyte–macrophage lineage in transplantation by immunosuppressive agents. Although not primarily designed to target monocyte–macrophage lineage cells, multiple categories of currently prescribed immunosuppressive drugs, such as mycophenolate mofetil, mammalian target of rapamycin inhibitors, and calcineurin inhibitors, do have limited inhibitory effects. These effects include diminishing the degree of cytokine production, thereby blocking costimulation and inhibiting the migration of monocytes to the site of rejection. Outside the field of transplantation, some clinical studies have shown that the monoclonal antibodies canakinumab, tocilizumab, and infliximab are effective in inhibiting monocyte functions. Indirect effects have also been shown for simvastatin, a lipid lowering drug, and bromodomain and extra-terminal motif inhibitors that reduce the cytokine production by monocytes–macrophages in patients with diabetes mellitus and rheumatoid arthritis. To date, detailed knowledge concerning the origin, the developmental requirements, and functions of diverse specialized monocyte–macrophage

  12. Endoplasmic Reticulum Chaperones and Their Roles in the Immunogenicity of Cancer Vaccines

    PubMed Central

    Graner, Michael W.; Lillehei, Kevin O.; Katsanis, Emmanuel

    2015-01-01

    The endoplasmic reticulum (ER) is a major site of passage for proteins en route to other organelles, to the cell surface, and to the extracellular space. It is also the transport route for peptides generated in the cytosol by the proteasome into the ER for loading onto major histocompatibility complex class I (MHC I) molecules for eventual antigen presentation at the cell surface. Chaperones within the ER are critical for many of these processes; however, outside the ER certain of those chaperones may play important and direct roles in immune responses. In some cases, particular ER chaperones have been utilized as vaccines against tumors or infectious disease pathogens when purified from tumor tissue or recombinantly generated and loaded with antigen. In other cases, the cell surface location of ER chaperones has implications for immune responses as well as possible tumor resistance. We have produced heat-shock protein/chaperone protein-based cancer vaccines called “chaperone-rich cell lysate” (CRCL) that are conglomerates of chaperones enriched from solid tumors by an isoelectric focusing technique. These preparations have been effective against numerous murine tumors, as well as in a canine with an advanced lung carcinoma treated with autologous CRCL. We also published extensive proteomic analyses of CRCL prepared from human surgically resected tumor samples. Of note, these preparations contained at least 10 ER chaperones and a number of other residents, along with many other chaperones/heat-shock proteins. Gene ontology and network analyses utilizing these proteins essentially recapitulate the antigen presentation pathways and interconnections. In conjunction with our current knowledge of cell surface/extracellular ER chaperones, these data collectively suggest that a systems-level view may provide insight into the potent immune stimulatory activities of CRCL with an emphasis on the roles of ER components in those processes. PMID:25610811

  13. The macrophages in rheumatic diseases

    PubMed Central

    Laria, Antonella; Lurati, Alfredomaria; Marrazza, Mariagrazia; Mazzocchi, Daniela; Re, Katia Angela; Scarpellini, Magda

    2016-01-01

    Macrophages belong to the innate immune system giving us protection against pathogens. However it is known that they are also involved in rheumatic diseases. Activated macrophages have two different phenotypes related to different stimuli: M1 (classically activated) and M2 (alternatively activated). M1 macrophages release high levels of pro-inflammatory cytokines, reactive nitrogen and oxygen intermediates killing microorganisms and tumor cells; while M2 macrophages are involved in resolution of inflammation through phagocytosis of apoptotic neutrophils, reduced production of pro-inflammatory cytokines, and increased synthesis of mediators important in tissue remodeling, angiogenesis, and wound repair. The role of macrophages in the different rheumatic diseases is different according to their M1/M2 macrophages phenotype. PMID:26929657

  14. Bioelectric modulation of macrophage polarization.

    PubMed

    Li, Chunmei; Levin, Michael; Kaplan, David L

    2016-02-12

    Macrophages play a critical role in regulating wound healing and tissue regeneration by changing their polarization state in response to local microenvironmental stimuli. The native roles of polarized macrophages encompass biomaterials and tissue remodeling needs, yet harnessing or directing the polarization response has been largely absent as a potential strategy to exploit in regenerative medicine to date. Recent data have revealed that specific alteration of cells' resting potential (Vmem) is a powerful tool to direct proliferation and differentiation in a number of complex tissues, such as limb regeneration, craniofacial patterning and tumorigenesis. In this study, we explored the bioelectric modulation of macrophage polarization by targeting ATP sensitive potassium channels (KATP). Glibenclamide (KATP blocker) and pinacidil (KATP opener) treatment not only affect macrophage polarization, but also influence the phenotype of prepolarized macrophages. Furthermore, modulation of cell membrane electrical properties can fine-tune macrophage plasticity. Glibenclamide decreased the secretion and gene expression of selected M1 markers, while pinacidil augmented M1 markers. More interestingly, glibencalmide promoted macrophage alternative activation by enhancing certain M2 markers during M2 polarization. These findings suggest that control of bioelectric properties of macrophages could offer a promising approach to regulate macrophage phenotype as a useful tool in regenerative medicine.

  15. Macrophages in homeostatic immune function

    PubMed Central

    Jantsch, Jonathan; Binger, Katrina J.; Müller, Dominik N.; Titze, Jens

    2014-01-01

    Macrophages are not only involved in inflammatory and anti-infective processes, but also play an important role in maintaining tissue homeostasis. In this review, we summarize recent evidence investigating the role of macrophages in controlling angiogenesis, metabolism as well as salt and water balance. Particularly, we summarize the importance of macrophage tonicity enhancer binding protein (TonEBP, also termed nuclear factor of activated T-cells 5 [NFAT5]) expression in the regulation of salt and water homeostasis. Further understanding of homeostatic macrophage function may lead to new therapeutic approaches to treat ischemia, hypertension and metabolic disorders. PMID:24847274

  16. Bioelectric modulation of macrophage polarization

    NASA Astrophysics Data System (ADS)

    Li, Chunmei; Levin, Michael; Kaplan, David L.

    2016-02-01

    Macrophages play a critical role in regulating wound healing and tissue regeneration by changing their polarization state in response to local microenvironmental stimuli. The native roles of polarized macrophages encompass biomaterials and tissue remodeling needs, yet harnessing or directing the polarization response has been largely absent as a potential strategy to exploit in regenerative medicine to date. Recent data have revealed that specific alteration of cells’ resting potential (Vmem) is a powerful tool to direct proliferation and differentiation in a number of complex tissues, such as limb regeneration, craniofacial patterning and tumorigenesis. In this study, we explored the bioelectric modulation of macrophage polarization by targeting ATP sensitive potassium channels (KATP). Glibenclamide (KATP blocker) and pinacidil (KATP opener) treatment not only affect macrophage polarization, but also influence the phenotype of prepolarized macrophages. Furthermore, modulation of cell membrane electrical properties can fine-tune macrophage plasticity. Glibenclamide decreased the secretion and gene expression of selected M1 markers, while pinacidil augmented M1 markers. More interestingly, glibencalmide promoted macrophage alternative activation by enhancing certain M2 markers during M2 polarization. These findings suggest that control of bioelectric properties of macrophages could offer a promising approach to regulate macrophage phenotype as a useful tool in regenerative medicine.

  17. Macrophage Cryptococcus interactions: an update

    PubMed Central

    Mansour, Michael K.; Reedy, Jennifer L.; Tam, Jenny M.; Vyas, Jatin M.

    2014-01-01

    Cryptococcus species are fungal pathogens that are a leading cause of mortality. Initial inoculation is through the pulmonary route and, if disseminated, results in severe invasive infection including meningoencephalitis. Macrophages are the dominant phagocytic cell that interacts with Cryptococcus. Emerging theories suggest that Cryptococcus microevolution in macrophages is linked to survival and virulence within the host. In addition, Cryptococcus elaborates virulence factors as well as usurps host machinery to establish macrophage activation states that are permissive to intracellular survival and replication. In this review, we provide an update of the recent findings pertaining to macrophage interaction with Cryptococcus and focus on new avenues for biomedical research. PMID:24660045

  18. The Hsc66-Hsc20 Chaperone System in Escherichia coli: Chaperone Activity and Interactions with the DnaK-DnaJ-GrpE System

    PubMed Central

    Silberg, Jonathan J.; Hoff, Kevin G.; Vickery, Larry E.

    1998-01-01

    Hsc66, a stress-70 protein, and Hsc20, a J-type accessory protein, comprise a newly described Hsp70-type chaperone system in addition to DnaK-DnaJ-GrpE in Escherichia coli. Because endogenous substrates for the Hsc66-Hsc20 system have not yet been identified, we investigated chaperone-like activities of Hsc66 and Hsc20 by their ability to suppress aggregation of denatured model substrate proteins, such as rhodanese, citrate synthase, and luciferase. Hsc66 suppressed aggregation of rhodanese and citrate synthase, and ATP caused effects consistent with complex destabilization typical of other Hsp70-type chaperones. Differences in the activities of Hsc66 and DnaK, however, suggest that these chaperones have dissimilar substrate specificity profiles. Hsc20, unlike DnaJ, did not exhibit intrinsic chaperone activity and appears to function solely as a regulatory cochaperone protein for Hsc66. Possible interactions between the Hsc66-Hsc20 and DnaK-DnaJ-GrpE chaperone systems were also investigated by measuring the effects of cochaperone proteins on Hsp70 ATPase activities. The nucleotide exchange factor GrpE did not stimulate the ATPase activity of Hsc66 and thus appears to function specifically with DnaK. Cross-stimulation by the cochaperones Hsc20 and DnaJ was observed, but the requirement for supraphysiological concentrations makes it unlikely that these interactions occur significantly in vivo. Together these results suggest that Hsc66-Hsc20 and DnaK-DnaJ-GrpE comprise separate molecular chaperone systems with distinct, nonoverlapping cellular functions. PMID:9852006

  19. Macrophages programmed by apoptotic cells promote angiogenesis via prostaglandin E2.

    PubMed

    Brecht, Kerstin; Weigert, Andreas; Hu, Jiong; Popp, Rüdiger; Fisslthaler, Beate; Korff, Thomas; Fleming, Ingrid; Geisslinger, Gerd; Brüne, Bernhard

    2011-07-01

    Macrophages contribute to tissue homeostasis in the developing as well as the adult organism. They promote tissue regeneration and remodeling after injury, which requires efficient neoangiogenesis. Signaling pathways activating an angiogenic program in macrophages are still poorly defined. We report that apoptotic cells (ACs), which originate from stressed or damaged tissues, can induce angiogenic properties in primary human macrophages. The signal originating from ACs is the lipid mediator sphingosine-1-phosphate (S1P), which activates S1P1/3 on macrophages to up-regulate cyclooxygenase-2. The formation and liberation of prostaglandin E(2) (PGE(2)) then stimulates migration of endothelial cells. This is demonstrated by using PGE(2) receptor antagonists or a neutralizing PGE(2) antibody in vitro, thereby attenuating endothelial cell migration using a Boyden chamber assay. In vivo, neutralization of PGE(2) from proangiogenic macrophage supernatants blocked vessel formation into Matrigel plugs. In particular, apoptotic cancer cells shifted prostanoid formation in macrophages selectively toward PGE(2) by up-regulating cyclooxygenase-2 and microsomal prostaglandin E synthase-1 (mPGES1), while down-regulating the PGE(2)-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or prostaglandin-D synthase (PGDS). Angiogenic programming of macrophages by ACs, therefore, may control responses to tissue stress such as in tumors, where macrophages support cancer progression.

  20. M1 of Murine Gamma-Herpesvirus 68 Induces Endoplasmic Reticulum Chaperone Production

    PubMed Central

    Feng, Jiaying; Gong, Danyang; Fu, Xudong; Wu, Ting-ting; Wang, Jane; Chang, Jennifer; Zhou, Jingting; Lu, Gang; Wang, Yibin; Sun, Ren

    2015-01-01

    Viruses rely on host chaperone network to support their infection. In particular, the endoplasmic reticulum (ER) resident chaperones play key roles in synthesizing and processing viral proteins. Influx of a large amount of foreign proteins exhausts the folding capacity in ER and triggers the unfolded protein response (UPR). A fully-executed UPR comprises signaling pathways that induce ER folding chaperones, increase protein degradation, block new protein synthesis and may eventually activate apoptosis, presenting both opportunities and threats to the virus. Here, we define a role of the MHV-68M1 gene in differential modulation of UPR pathways to enhance ER chaperone production. Ectopic expression of M1 markedly induces ER chaperone genes and expansion of ER. The M1 protein accumulates in ER during infection and this localization is indispensable for its function, suggesting M1 acts from the ER. We found that M1 protein selectively induces the chaperon-producing pathways (IRE1, ATF6) while, interestingly, sparing the translation-blocking arm (PERK). We identified, for the first time, a viral factor capable of selectively intervening the initiation of ER stress signaling to induce chaperon production. This finding provides a unique opportunity of using viral protein as a tool to define the activation mechanisms of individual UPR pathways. PMID:26615759

  1. Catalysis of protein folding by chaperones accelerates evolutionary dynamics in adapting cell populations.

    PubMed

    Cetinbaş, Murat; Shakhnovich, Eugene I

    2013-01-01

    Although molecular chaperones are essential components of protein homeostatic machinery, their mechanism of action and impact on adaptation and evolutionary dynamics remain controversial. Here we developed a physics-based ab initio multi-scale model of a living cell for population dynamics simulations to elucidate the effect of chaperones on adaptive evolution. The 6-loci genomes of model cells encode model proteins, whose folding and interactions in cellular milieu can be evaluated exactly from their genome sequences. A genotype-phenotype relationship that is based on a simple yet non-trivially postulated protein-protein interaction (PPI) network determines the cell division rate. Model proteins can exist in native and molten globule states and participate in functional and all possible promiscuous non-functional PPIs. We find that an active chaperone mechanism, whereby chaperones directly catalyze protein folding, has a significant impact on the cellular fitness and the rate of evolutionary dynamics, while passive chaperones, which just maintain misfolded proteins in soluble complexes have a negligible effect on the fitness. We find that by partially releasing the constraint on protein stability, active chaperones promote a deeper exploration of sequence space to strengthen functional PPIs, and diminish the non-functional PPIs. A key experimentally testable prediction emerging from our analysis is that down-regulation of chaperones that catalyze protein folding significantly slows down the adaptation dynamics.

  2. Selective induction of metabolic activation programs in peritoneal macrophages by lipopolysaccharide substructures.

    PubMed Central

    Lehmann, V; Benninghoff, B; Dröge, W

    1991-01-01

    The structural elements of Salmonella typhimurium lipopolysaccharides (LPS) that are able to stimulate peritoneal macrophages to produce increased amounts of prostaglandin E2, ornithine, and citrulline, agents known to modulate immune responses, are described. Two different incomplete lipid A structures which lack the carbohydrate portion, the nonhydroxylated fatty acids lauric acid and myristic acid (lipid A precursor IB), and additional palmitic acid (lipid A precursor IA) stimulated increased prostaglandin E2 synthesis but were unable to augment ornithine and citrulline production at concentrations of up to 0.5 microgram/ml. Acyl-deficient smooth LPS containing lipid A precursors IA and IB substituted by the complete carbohydrate region were able to augment prostaglandin E2 and ornithine production but failed, even at a high concentration (0.5 microgram/ml), to stimulate citrulline production. Moreover, Re glycolipids and smooth intact LPS containing the lipid A region with 3-acyloxyacyl residues possessed all of the structural requirements to induce increased prostaglandin E2, ornithine, and citrulline synthesis. Finally, all of the LPS structures, including lipid A precursors IA and IB stimulated, in combination with gamma interferon, production of citrulline with similar efficiencies. These results demonstrate that LPS contains various substructures including regions of the carbohydrate and lipid A structure that can deliver signals for the activation of peritoneal macrophages. Signals for partial activation of macrophages to produce prostaglandins and ornithine can be delivered by acyl-deficient LPS structures. In contrast, full activation of macrophages to produce citrulline requires an additional signal that is delivered by 3-acyloxyacyl residues of the lipid A region or gamma interferon. PMID:1906843

  3. Consequences of the selective blockage of chaperone-mediated autophagy

    PubMed Central

    Massey, Ashish C.; Kaushik, Susmita; Sovak, Guy; Kiffin, Roberta; Cuervo, Ana Maria

    2006-01-01

    Chaperone-mediated autophagy (CMA) is a selective pathway for the degradation of cytosolic proteins in lysosomes. CMA declines with age because of a decrease in the levels of lysosome-associated membrane protein (LAMP) type 2A, a lysosomal receptor for this pathway. We have selectively blocked the expression of LAMP-2A in mouse fibroblasts in culture and analyzed the cellular consequences of reduced CMA activity. CMA-defective cells maintain normal rates of long-lived protein degradation by up-regulating macroautophagy, the major form of autophagy. Constitutive up-regulation of macroautophagy is unable, however, to compensate for all CMA functions. Thus, CMA-defective cells are more sensitive to stressors, suggesting that, although protein turnover is maintained, the selectivity of CMA is necessary as part of the cellular response to stress. Our results also denote the existence of cross-talk among different forms of autophagy. PMID:16585521

  4. Regulation of organismal proteostasis by trans-cellular chaperone signaling

    PubMed Central

    van Oosten-Hawle, Patricija; Porter, Robert S.; Morimoto, Richard I.

    2013-01-01

    Summary A major challenge for metazoans is to ensure that different tissues each expressing distinctive proteomes are, nevertheless, well protected at an organismal level from proteotoxic stress. We have examined this and show that expression of endogenous metastable protein sensors in muscle cells induces a systemic stress response throughout multiple tissues of C. elegans. Suppression of misfolding in muscle cells can be achieved not only by enhanced expression of HSP90 in muscle cells, but as effective by elevated expression of HSP90 in intestine or neuronal cells. This cell-non-autonomous control of HSP90 expression relies upon transcriptional feedback between somatic tissues that is regulated by the FoxA transcription factor PHA-4. This trans-cellular chaperone signaling response maintains organismal proteostasis when challenged by a local tissue imbalance in folding and provides the basis for a novel form of organismal stress sensing surveillance. PMID:23746847

  5. Flagellin Polymerisation Control by a Cytosolic Export Chaperone

    PubMed Central

    Auvray, Frédéric; Thomas, Joanne; Fraser, Gillian M.; Hughes, Colin

    2008-01-01

    Assembly of the long helical filament of the bacterial flagellum requires polymerisation of ca 20,000 flagellin (FliC) monomeric subunits into the growing structure extending from the cell surface. Here, we show that export of Salmonella flagellin is facilitated specifically by a cytosolic protein, FliS, and that FliS binds to the FliC C-terminal helical domain, which contributes to stabilisation of flagellin subunit interactions during polymerisation. Stable complexes of FliS with flagellin were assembled efficiently in vitro, apparently by FliS homodimers binding to FliC monomers. The data suggest that FliS acts as a substrate-specific chaperone, preventing premature interaction of newly synthesised flagellin subunits in the cytosol. Compatible with this view, FliS was able to prevent in vitro polymerisation of FliC into filaments. PMID:11327763

  6. Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy

    PubMed Central

    Pedrozo, Zully; Torrealba, Natalia; Fernández, Carolina; Gatica, Damian; Toro, Barbra; Quiroga, Clara; Rodriguez, Andrea E.; Sanchez, Gina; Gillette, Thomas G.; Hill, Joseph A.; Donoso, Paulina; Lavandero, Sergio

    2013-01-01

    Time for primary review: 15 days Aims Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation–contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. Methods and results To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. Conclusion These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels. PMID:23404999

  7. Epiplakin attenuates experimental mouse liver injury by chaperoning keratin reorganization

    PubMed Central

    Szabo, Sandra; Wögenstein, Karl L.; Österreicher, Christoph H.; Guldiken, Nurdan; Chen, Yu; Doler, Carina; Wiche, Gerhard; Boor, Peter; Haybaeck, Johannes; Strnad, Pavel; Fuchs, Peter

    2015-01-01

    Background & Aims Epiplakin is a member of the plakin protein family and exclusively expressed in epithelial tissues where it binds to keratins. Epiplakin-deficient (Eppk1−/−) mice displayed no obvious spontaneous phenotype, but their keratinocytes showed a faster keratin network breakdown in response to stress. The role of epiplakin in the stressed liver remained to be elucidated. Methods Wild-type (WT) and Eppk1−/− mice were subjected to common bile duct ligation (CBDL) or fed with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing diet. The importance of epiplakin during keratin reorganization was assessed in primary hepatocytes. Results Our experiments revealed that epiplakin is expressed in hepatocytes and cholangiocytes, and binds to keratin 8 (K8) and K18 via multiple domains. In several liver stress models epiplakin and K8 genes displayed identical expression patterns and transgenic K8 overexpression resulted in elevated hepatic epiplakin levels. After CBDL and DDC treatment, Eppk1−/− mice developed a more pronounced liver injury and their livers contained larger amounts of hepatocellular keratin granules, indicating impaired disease-induced keratin network reorganization. In line with these findings, primary Eppk1−/− hepatocytes showed increased formation of keratin aggregates after treatment with the phosphatase inhibitor okadaic acid, a phenotype which was rescued by the chemical chaperone trimethylamine N-oxide (TMAO). Finally, transfection experiments revealed that Eppk1−/− primary hepatocytes were less able to tolerate forced K8 overexpression and that TMAO treatment rescued this phenotype. Conclusion Our data indicate that epiplakin plays a protective role during experimental liver injuries by chaperoning disease-induced keratin reorganization. PMID:25617501

  8. Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide

    PubMed Central

    Macri, Christophe; Wang, Fengjuan; Tasset, Inmaculada; Schall, Nicolas; Page, Nicolas; Briand, Jean-Paul; Cuervo, Ana Maria; Muller, Sylviane

    2015-01-01

    The P140 peptide, a 21-mer linear peptide (sequence 131–151) generated from the spliceosomal SNRNP70/U1–70K protein, contains a phosphoserine residue at position 140. It significantly ameliorates clinical manifestations in autoimmune patients with systemic lupus erythematosus and enhances survival in MRL/lpr lupus-prone mice. Previous studies showed that after P140 treatment, there is an accumulation of autophagy markers sequestosome 1/p62 and MAP1LC3-II in MRL/lpr B cells, consistent with a downregulation of autophagic flux. We now identify chaperone-mediated autophagy (CMA) as a target of P140 and demonstrate that its inhibitory effect on CMA is likely tied to its ability to alter the composition of HSPA8/HSC70 heterocomplexes. As in the case of HSPA8, expression of the limiting CMA component LAMP2A, which is increased in MRL/lpr B cells, is downregulated after P140 treatment. We also show that P140, but not the unphosphorylated peptide, uses the clathrin-dependent endo-lysosomal pathway to enter into MRL/lpr B lymphocytes and accumulates in the lysosomal lumen where it may directly hamper lysosomal HSPA8 chaperoning functions, and also destabilize LAMP2A in lysosomes as a result of its effect on HSP90AA1. This dual effect may interfere with the endogenous autoantigen processing and loading to major histocompatibility complex class II molecules and as a consequence, lead to lower activation of autoreactive T cells. These results shed light on mechanisms by which P140 can modulate lupus disease and exert its tolerogenic activity in patients. The unique selective inhibitory effect of the P140 peptide on CMA may be harnessed in other pathological conditions in which reduction of CMA activity would be desired. PMID:25719862

  9. Antarctic Krill 454 Pyrosequencing Reveals Chaperone and Stress Transcriptome

    PubMed Central

    Clark, Melody S.; Thorne, Michael A. S.; Toullec, Jean-Yves; Meng, Yan; Guan, Le Luo; Peck, Lloyd S.; Moore, Stephen

    2011-01-01

    Background The Antarctic krill Euphausia superba is a keystone species in the Antarctic food chain. Not only is it a significant grazer of phytoplankton, but it is also a major food item for charismatic megafauna such as whales and seals and an important Southern Ocean fisheries crop. Ecological data suggest that this species is being affected by climate change and this will have considerable consequences for the balance of the Southern Ocean ecosystem. Hence, understanding how this organism functions is a priority area and will provide fundamental data for life history studies, energy budget calculations and food web models. Methodology/Principal Findings The assembly of the 454 transcriptome of E. superba resulted in 22,177 contigs with an average size of 492bp (ranging between 137 and 8515bp). In depth analysis of the data revealed an extensive catalogue of the cellular chaperone systems and the major antioxidant proteins. Full length sequences were characterised for the chaperones HSP70, HSP90 and the super-oxide dismutase antioxidants, with the discovery of potentially novel duplications of these genes. The sequence data contained 41,470 microsatellites and 17,776 Single Nucleotide Polymorphisms (SNPs/INDELS), providing a resource for population and also gene function studies. Conclusions This paper details the first 454 generated data for a pelagic Antarctic species or any pelagic crustacean globally. The classical “stress proteins”, such as HSP70, HSP90, ferritin and GST were all highly expressed. These genes were shown to be over expressed in the transcriptomes of Antarctic notothenioid fish and hypothesized as adaptations to living in the cold, with the associated problems of decreased protein folding efficiency and increased vulnerability to damage by reactive oxygen species. Hence, these data will provide a major resource for future physiological work on krill, but in particular a suite of “stress” genes for studies understanding marine

  10. Macrophages in spinal cord injury: phenotypic and functional change from exposure to myelin debris.

    PubMed

    Wang, Xi; Cao, Kai; Sun, Xin; Chen, Yongxiong; Duan, Zhaoxia; Sun, Li; Guo, Lei; Bai, Paul; Sun, Dongming; Fan, Jianqing; He, Xijing; Young, Wise; Ren, Yi

    2015-04-01

    Macrophage activation and persistent inflammation contribute to the pathological process of spinal cord injury (SCI). It was reported that M2 macrophages were induced at 3-7 days after SCI but M2 markers were reduced or eliminated after 1 week. By contrast, M1 macrophage response is rapidly induced and then maintained at injured spinal cord. However, factors that modulate macrophage phenotype and function are poorly understood. We developed a model to distinguish bone-marrow derived macrophages (BMDMs) from residential microglia and explored how BMDMs change their phenotype and functions in response to the lesion-related factors in injured spinal cord. Infiltrating BMDMs expressing higher Mac-2 and lower CX3CR1 migrate to the epicenter of injury, while microglia expressing lower Mac-2 but higher CX3CR1 distribute to the edges of lesion. Myelin debris at the lesion site switches BMDMs from M2 phenotype towards M1-like phenotype. Myelin debris activates ATP-binding cassette transporter A1 (ABCA1) for cholesterol efflux in response to myelin debris loading in vitro. However, this homeostatic mechanism in injured site is overwhelmed, leading to the development of foamy macrophages and lipid plaque in the lesion site. The persistence of these cells indicates a pro-inflammatory environment, associated with enhanced neurotoxicity and impaired wound healing. These foamy macrophages have poor capacity to phagocytose apoptotic neutrophils resulting in uningested neutrophils releasing their toxic contents and further tissue damage. In conclusion, these data demonstrate for the first time that myelin debris generated in injured spinal cord modulates macrophage activation. Lipid accumulation following macrophage phenotype switch contributes to SCI pathology.

  11. Macrophages in spinal cord injury: phenotypic and functional change from exposure to myelin debris

    PubMed Central

    Wang, Xi; Cao, Kai; Sun, Xin; Chen, Yongxiong; Duan, Zhaoxia; Sun, Li; Guo, Lei; Bai, Paul; Sun, Dongming; Fan, Jianqing; He, Xijing; Young, Wise; Ren, Yi

    2014-01-01

    Macrophage activation and persistent inflammation contribute to the pathological process of spinal cord injury (SCI). It was reported that M2 macrophages were induced at 3–7 days after SCI but M2 markers were reduced or eliminated after 1 week. By contrast, M1 macrophage response is rapidly induced and then maintained at injured spinal cord. However, factors that modulate macrophage phenotype and function are poorly understood. We developed a model to distinguished bone marrow derived macrophages (BMDMs) from residential microglia and explored how BMDMs change their phenotype and functions in response to the lesion-related factors in injured spinal cord. Infiltrating BMDMs expressing higher Mac-2 and lower CX3CR1 migrate to the epicenter of injury, while microglia expressing lower Mac-2 but higher CX3CR1 distribute to the edges of lesion. Myelin debris at the lesion site switches BMDMs from M2 phenotype towards M1-like phenotype. Myelin debris activate ATP-binding cassette transporter A1 (ABCA1) for cholesterol efflux in response to myelin debris loading in vitro. However, this homeostatic mechanism in injured site is overwhelmed, leading to the development of foamy macrophages and lipid plaque in the lesion site. The persistence of these cells indicates a pro-inflammatory environment, associated with enhanced neurotoxicity and impaired wound healing. These foamy macrophages have poor capacity to phagocytose apoptotic neutrophils resulting in uningested neutrophils releasing their toxic contents and further tissue damage. In conclusion, these data demonstrate for the first time that myelin debris generated in injured spinal cord modulates macrophage activation. Lipid accumulation following macrophage phenotype switch contributes to SCI pathology. PMID:25452166

  12. Endotoxin-induced early gene expression in C3H/HeJ (Lpsd) macrophages.

    PubMed

    Manthey, C L; Perera, P Y; Henricson, B E; Hamilton, T A; Qureshi, N; Vogel, S N

    1994-09-15

    C3H/HeJ (Lpsd) macrophages have been shown to respond to certain LPSs, especially from rough mutant bacteria. C3H/OuJ (Lpsn) macrophages are induced by wild-type LPS, rough LPS, or lipid A to express many genes, including TNF-alpha, TNFR-2, IL-1 beta, IP-10, D3, and D8. C3H/HeJ macrophages failed to induce any of these genes when cultured with wild-type LPS or synthetic lipid A, even when pretreated with IFN-gamma. However, rough mutant Salmonella minnesota Ra, Rc, and Rd LPS, and Escherichia coli D31 m3 Rd LPS induced Lpsd macrophages to express a subset of genes within the gene panel. Because bioactive preparations contained trace quantities of endotoxin protein(s), a deoxycholate-modified, phenol-water method was used to repurify rough LPS into an aqueous phase, and extract endotoxin proteins into a phenol phase. Repurified LPS failed to stimulate Lpsd macrophages; however, phenol fractions were approximately 10% as potent in Lpsd macrophages as crude rough LPS. Full potency was restored in C3H/HeJ macrophages when aqueous phase LPS and phenol-phase proteins were co-precipitated, suggesting that LPS and endotoxin proteins interact synergistically. Endotoxin proteins alone induced TNF-alpha, TNFR-2, and IL-1 beta, but not IP-10, D3, and D8 genes in both Lpsd and Lpsn macrophages. Tyrosine phosphorylation of three 41- to 47-kDa proteins was induced by endotoxin proteins, but not by LPS, in Lpsd macrophages. Thus, endotoxin proteins seem to activate a signaling pathway(s) that converges (distal to the Lps gene product) with a subset of LPS-signaling pathways.

  13. Chaperoning to the metabolic party: The emerging therapeutic role of heat-shock proteins in obesity and type 2 diabetes

    PubMed Central

    Henstridge, Darren C.; Whitham, Martin; Febbraio, Mark A.

    2014-01-01

    Background From their initial, accidental discovery 50 years ago, the highly conserved Heat Shock Proteins (HSPs) continue to exhibit fundamental roles in the protection of cell integrity. Meanwhile, in the midst of an obesity epidemic, research demonstrates a key involvement of low grade inflammation, and mitochondrial dysfunction amongst other mechanisms, in the pathology of insulin resistance and type 2 diabetes mellitus (T2DM). In particular, tumor necrosis factor alpha (TNFα), endoplasmic reticulum (ER) and oxidative stress all appear to be associated with obesity and stimulate inflammatory kinases such as c jun amino terminal kinase (JNK), inhibitor of NF-κβ kinase (IKK) and protein kinase C (PKC) which in turn, inhibit insulin signaling. Mitochondrial dysfunction in skeletal muscle has also been proposed to be prominent in the pathogenesis of T2DM either by reducing the ability to oxidize fatty acids, leading to the accumulation of deleterious lipid species in peripheral tissues such as skeletal muscle and liver, or by altering the cellular redox state. Since HSPs act as molecular chaperones and demonstrate crucial protective functions in stressed cells, we and others have postulated that the manipulation of HSP expression in metabolically relevant tissues represents a therapeutic avenue for obesity-induced insulin resistance. Scope of Review This review summarizes the literature from both animal and human studies, that has examined how HSPs, particularly the inducible HSP, Heat Shock Protein 72 (Hsp72) alters glucose homeostasis and the possible approaches to modulating Hsp72 expression. A summation of the role of chemical chaperones in metabolic disorders is also included. Major Conclusions Targeted manipulation of Hsp72 or use of chemical chaperiones may have clinical utility in treating metabolic disorders such as insulin resistance and T2DM. PMID:25379403

  14. Biology of Bony Fish Macrophages

    PubMed Central

    Hodgkinson, Jordan W.; Grayfer, Leon; Belosevic, Miodrag

    2015-01-01

    Macrophages are found across all vertebrate species, reside in virtually all animal tissues, and play critical roles in host protection and homeostasis. Various mechanisms determine and regulate the highly plastic functional phenotypes of macrophages, including antimicrobial host defenses (pro-inflammatory, M1-type), and resolution and repair functions (anti-inflammatory/regulatory, M2-type). The study of inflammatory macrophages in immune defense of teleosts has garnered much attention, and antimicrobial mechanisms of these cells have been extensively studied in various fish models. Intriguingly, both similarities and differences have been documented for the regulation of lower vertebrate macrophage antimicrobial defenses, as compared to what has been described in mammals. Advances in our understanding of the teleost macrophage M2 phenotypes likewise suggest functional conservation through similar and distinct regulatory strategies, compared to their mammalian counterparts. In this review, we discuss the current understanding of the molecular mechanisms governing teleost macrophage functional heterogeneity, including monopoetic development, classical macrophage inflammatory and antimicrobial responses as well as alternative macrophage polarization towards tissues repair and resolution of inflammation. PMID:26633534

  15. The Macrophage Phagocytic Receptor CD36 Promotes Fibrogenic Pathways on Removal of Apoptotic Cells during Chronic Kidney Injury

    PubMed Central

    Pennathur, Subramaniam; Pasichnyk, Katie; Bahrami, Nadia M.; Zeng, Lixia; Febbraio, Maria; Yamaguchi, Ikuyo; Okamura, Daryl M.

    2016-01-01

    The removal of apoptotic cells is an innate function of tissue macrophages; however, its role in disease progression is unclear. The present study was designed to investigate the role of macrophage CD36, a recognized receptor of apoptotic cells and oxidized lipids, in two models of kidney injury: unilateral ureteral obstruction (UUO) and ischemia reperfusion. To differentiate the macrophage CD36-specific effects in vivo, we generated CD36 chimeric mice by bone marrow transplantation and evaluated the two models. Fibrosis severity was substantially decreased after UUO with a corresponding decrease in matrix synthesis in macrophage CD36-deficient mice. Despite a reduction in fibrosis severity, a 56% increase in apoptotic cells was found without an increase in apoptotic effectors. In addition, a substantial reduction was observed in tumor necrosis factor-α and transforming growth factor-β1 mRNA levels and intracellular bioactive oxidized lipid levels in CD36-deficient macrophages. To validate the functional role of macrophage CD36, we performed unilateral ischemia reperfusion, followed by contralateral nephrectomy. Similarly, we found that the severity of fibrosis was reduced by 55% with a corresponding improvement in kidney function by 88% in macrophage CD36-deficient mice. Taken together, these data suggest that macrophage CD36 is a critical regulator of oxidative fibrogenic signaling and that CD36-mediated phagocytosis of apoptotic cells may serve as an important pathway in the progression of fibrosis. PMID:26092500

  16. Lipid Profiling Reveals Arachidonate Deficiency in RAW264.7 Cells: Structural and Functional Implications†

    PubMed Central

    Rouzer, Carol A.; Ivanova, Pavlina T.; Byrne, Mark O.; Milne, Stephen B.; Marnett, Lawrence J.; Brown, H. Alex

    2008-01-01

    Glycerophospholipids containing arachidonic acid (20:4) serve as the precursors for an array of biologically active lipid mediators, most of which are produced by macrophages. We have applied mass spectrometry-based lipid profiling technology to evaluate the glycerophospholipid structure and composition of two macrophage populations, resident peritoneal macrophages and RAW264.7 cells, with regard to their potential for 20:4-based lipid mediator biosynthesis. Fatty acid analysis indicated that RAW264.7 cells were deficient in 20:4 (10 ± 1 mole percent) as compared to peritoneal macrophages (26 ± 1 mole percent). Mass spectrometry of total glycerophospholipids demonstrated a marked difference in the distribution of lipid species, including reduced levels of 20:4-containing lipids, in RAW264.7 cells as compared to peritoneal macrophages. Enrichment of RAW264.7 cells with 20:4 increased the fatty acid to 20 ± 1 mole percent. However, the distribution of the incorporated 20:4 remained different from that of peritoneal macrophages. RAW264.7 cells pretreated with granulocyte-macrophage colony stimulating factor followed by lipopolysaccharide and interferon-gamma mobilized similar quantities of 20:4 and produced similar amounts of prostaglandins as peritoneal macrophages treated with LPS alone. LPS treatment resulted in detectable changes in specific 20:4-containing glycerophospholipids in peritoneal cells, but not in RAW264.7 cells. 20:4-enriched RAW264.7 cells lost 88% of the incorporated fatty acid during the LPS incubation without additional prostaglandin synthesis. These results illustrate that large differences in glycerophospholipid composition may exist, even in closely related cell populations, and demonstrate the importance of interpreting the potential for lipid-mediator biosynthesis in the context of overall glycerophospholipid composition. PMID:17144673

  17. The RNA Chaperone Hfq Impacts Growth, Metabolism and Production of Virulence Factors in Yersinia enterocolitica

    PubMed Central

    Kakoschke, Tamara; Kakoschke, Sara; Magistro, Giuseppe; Schubert, Sören; Borath, Marc; Heesemann, Jürgen; Rossier, Ombeline

    2014-01-01

    To adapt to changes in environmental conditions, bacteria regulate their gene expression at the transcriptional but also at the post-transcriptional level, e.g. by small RNAs (sRNAs) which modulate mRNA stability and translation. The conserved RNA chaperone Hfq mediates the interaction of many sRNAs with their target mRNAs, thereby playing a global role in fine-tuning protein production. In this study, we investigated the significance of Hfq for the enteropathogen Yersina enterocolitica serotype O:8. Hfq facilitated optimal growth in complex and minimal media. Our comparative protein analysis of parental and hfq-negative strains suggested that Hfq promotes lipid metabolism and transport, cell redox homeostasis, mRNA translation and ATP synthesis, and negatively affects carbon and nitrogen metabolism, transport of siderophore and peptides and tRNA synthesis. Accordingly, biochemical tests indicated that Hfq represses ornithine decarboxylase activity, indole production and utilization of glucose, mannitol, inositol and 1,2-propanediol. Moreover, Hfq repressed production of the siderophore yersiniabactin and its outer membrane receptor FyuA. In contrast, hfq mutants exhibited reduced urease production. Finally, strains lacking hfq were more susceptible to acidic pH and oxidative stress. Unlike previous reports in other Gram-negative bacteria, Hfq was dispensable for type III secretion encoded by the virulence plasmid. Using a chromosomally encoded FLAG-tagged Hfq, we observed increased production of Hfq-FLAG in late exponential and stationary phases. Overall, Hfq has a profound effect on metabolism, resistance to stress and modulates the production of two virulence factors in Y. enterocolitica, namely urease and yersiniabactin. PMID:24454955

  18. Macrophage Stimulating Protein Enhances Hepatic Inflammation in a NASH Model

    PubMed Central

    van Gorp, Patrick J.; Jeurissen, Mike L. J.; Houben, Tom; Walenbergh, Sofie M. A.; Debets, Jacques; Oligschlaeger, Yvonne; Gijbels, Marion J. J.; Neumann, Dietbert; Shiri-Sverdlov, Ronit

    2016-01-01

    Non-alcoholic steatohepatitis (NASH) is a common liver disease characterized by hepatic lipid accumulation (steatosis) and inflammation. Currently, therapeutic options are poor and the long-term burden to society is constantly increasing. Previously, macrophage stimulating protein (MSP)—a serum protein mainly secreted by liver—was shown to inhibit oxidized low-density lipoprotein (OxLDL)/lipopolysaccharides (LPS)-induced inflammation in mouse macrophages. Additionally, MSP could reduce palmitic acid (PA)-induced lipid accumulation and lipogenesis in the HepG2 cell line. Altogether, these data suggest MSP as a suppressor for metabolic inflammation. However, so far the potential of MSP to be used as a treatment for NASH was not investigated. We hypothesized that MSP reduces lipid accumulation and hepatic inflammation. To investigate the effects of MSP in the early stage of NASH, low-density lipoprotein receptor (Ldlr-/-) mice were fed either a regular chow or a high fat, high cholesterol (HFC) diet for 7 days. Recombinant MSP or saline (control) was administrated to the mice by utilizing subcutaneously-implanted osmotic mini-pumps for the last 4 days. As expected, mice fed an HFC diet showed increased plasma and hepatic lipid accumulation, as well as enhanced hepatic inflammation, compared with chow-fed controls. Upon MSP administration, the rise in cholesterol and triglyceride levels after an HFC diet remained unaltered. Surprisingly, while hepatic macrophage and neutrophil infiltration was similar between the groups, MSP-treated mice showed increased gene expression of pro-inflammatory and pro-apoptotic mediators in the liver, compared with saline-treated controls. Contrary to our expectations, MSP did not ameliorate NASH. Observed changes in inflammatory gene expression suggest that further research is needed to clarify the long-term effects of MSP. PMID:27685150

  19. Macrophage podosomes go 3D.

    PubMed

    Van Goethem, Emeline; Guiet, Romain; Balor, Stéphanie; Charrière, Guillaume M; Poincloux, Renaud; Labrousse, Arnaud; Maridonneau-Parini, Isabelle; Le Cabec, Véronique

    2011-01-01

    Macrophage tissue infiltration is a critical step in the immune response against microorganisms and is also associated with disease progression in chronic inflammation and cancer. Macrophages are constitutively equipped with specialized structures called podosomes dedicated to extracellular matrix (ECM) degradation. We recently reported that these structures play a critical role in trans-matrix mesenchymal migration mode, a protease-dependent mechanism. Podosome molecular components and their ECM-degrading activity have been extensively studied in two dimensions (2D), but yet very little is known about their fate in three-dimensional (3D) environments. Therefore, localization of podosome markers and proteolytic activity were carefully examined in human macrophages performing mesenchymal migration. Using our gelled collagen I 3D matrix model to obligate human macrophages to perform mesenchymal migration, classical podosome markers including talin, paxillin, vinculin, gelsolin, cortactin were found to accumulate at the tip of F-actin-rich cell protrusions together with β1 integrin and CD44 but not β2 integrin. Macrophage proteolytic activity was observed at podosome-like protrusion sites using confocal fluorescence microscopy and electron microscopy. The formation of migration tunnels by macrophages inside the matrix was accomplished by degradation, engulfment and mechanic compaction of the matrix. In addition, videomicroscopy revealed that 3D F-actin-rich protrusions of migrating macrophages were as dynamic as their 2D counterparts. Overall, the specifications of 3D podosomes resembled those of 2D podosome rosettes rather than those of individual podosomes. This observation was further supported by the aspect of 3D podosomes in fibroblasts expressing Hck, a master regulator of podosome rosettes in macrophages. In conclusion, human macrophage podosomes go 3D and take the shape of spherical podosome rosettes when the cells perform mesenchymal migration. This work

  20. Strain specific transcriptional response in Mycobacterium tuberculosis infected macrophages

    PubMed Central

    2012-01-01

    Background Tuberculosis (TB), a bacterial infection caused by Mycobacterium tuberculosis (Mtb) remains a significant health problem worldwide with a third of the world population infected and nearly nine million new cases claiming 1.1 million deaths every year. The outcome following infection by Mtb is determined by a complex and dynamic host-pathogen interaction in which the phenotype of the pathogen and the immune status of the host play a role. However, the molecular mechanism by which Mtb strains induce different responses during intracellular infection of the host macrophage is not fully understood. To explore the early molecular events triggered upon Mtb infection of macrophages, we studied the transcriptional responses of murine bone marrow-derived macrophages (BMM) to infection with two clinical Mtb strains, CDC1551 and HN878. These strains have previously been shown to differ in their virulence/immunogenicity in the mouse and rabbit models of pulmonary TB. Results In spite of similar intracellular growth rates, we observed that compared to HN878, infection by CDC1551 of BMM was associated with an increased global transcriptome, up-regulation of a specific early (6 hours) immune response network and significantly elevated nitric oxide production. In contrast, at 24 hours post-infection of BMM by HN878, more host genes involved in lipid metabolism, including cholesterol metabolism and prostaglandin synthesis were up-regulated, compared to infection with CDC1551. In association with the differences in the macrophage responses to infection with the 2 Mtb strains, intracellular CDC1551 expressed higher levels of stress response genes than did HN878. Conclusions In association with the early and more robust macrophage activation, intracellular CDC1551 cells were exposed to a higher level of stress leading to increased up-regulation of the bacterial stress response genes. In contrast, sub-optimal activation of macrophages and induction of a dysregulated host cell

  1. A macrophage receptor for apolipoprotein B48: Cloning, expression, and atherosclerosis

    PubMed Central

    Brown, Matthew L.; Ramprasad, M. P.; Umeda, Patrick K.; Tanaka, Akira; Kobayashi, Yasushi; Watanabe, Teruo; Shimoyamada, Hiroaki; Kuo, Wen-Lin; Li, Ran; Song, Ruiling; Bradley, William A.; Gianturco, Sandra H.

    2000-01-01

    We have cloned a human macrophage receptor that binds to apolipoprotein (apo)B48 of dietary triglyceride (TG)-rich lipoproteins. TG-rich lipoprotein uptake by the apoB48R rapidly converts macrophages and apoB48R-transfected Chinese hamster ovary cells in vitro into lipid-filled foam cells, as seen in atherosclerotic lesions. The apoB48R cDNA (3,744 bp) encodes a protein with no known homologs. Its ≈3.8-kb mRNA is expressed primarily by reticuloendothelial cells: monocytes, macrophages, and endothelial cells. Immunohistochemistry shows the apoB48R is in human atherosclerotic lesion foam cells. Normally, the apoB48R may provide essential lipids to reticuloendothelial cells. If overwhelmed, foam cell formation, endothelial dysfunction, and atherothrombogenesis may ensue, a mechanism for cardiovascular disease risk of elevated TG. PMID:10852956

  2. The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes

    DOE PAGES

    Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.; ...

    2014-12-08

    Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

  3. The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes

    SciTech Connect

    Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.; Tainer, John A.

    2014-12-08

    Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

  4. A Common Structural Motif in the Binding of Virulence Factors to Bacterial Secretion Chaperones

    SciTech Connect

    Lilic,M.; Vujanac, M.; Stebbins, C.

    2006-01-01

    Salmonella invasion protein A (SipA) is translocated into host cells by a type III secretion system (T3SS) and comprises two regions: one domain binds its cognate type III secretion chaperone, InvB, in the bacterium to facilitate translocation, while a second domain functions in the host cell, contributing to bacterial uptake by polymerizing actin. We present here the crystal structures of the SipA chaperone binding domain (CBD) alone and in complex with InvB. The SipA CBD is found to consist of a nonglobular polypeptide as well as a large globular domain, both of which are necessary for binding to InvB. We also identify a structural motif that may direct virulence factors to their cognate chaperones in a diverse range of pathogenic bacteria. Disruption of this structural motif leads to a destabilization of several chaperone-substrate complexes from different species, as well as an impairment of secretion in Salmonella.

  5. Histone chaperones FACT and Spt6 prevent histone variants from turning into histone deviants.

    PubMed

    Jeronimo, Célia; Robert, François

    2016-05-01

    Histone variants are specialized histones which replace their canonical counterparts in specific nucleosomes. Together with histone post-translational modifications and DNA methylation, they contribute to the epigenome. Histone variants are incorporated at specific locations by the concerted action of histone chaperones and ATP-dependent chromatin remodelers. Recent studies have shown that the histone chaperone FACT plays key roles in preventing pervasive incorporation of two histone variants: H2A.Z and CenH3/CENP-A. In addition, Spt6, another histone chaperone, was also shown to be important for appropriate H2A.Z localization. FACT and Spt6 are both associated with elongating RNA polymerase II. Based on these two examples, we propose that the establishment and maintenance of histone variant genomic distributions depend on a transcription-coupled epigenome editing (or surveillance) function of histone chaperones.

  6. Evolution of the Chaperone/Usher Assembly Pathway: Fimbrial Classification Goes Greek†

    PubMed Central

    Nuccio, Sean-Paul; Bäumler, Andreas J.

    2007-01-01

    Summary: Many Proteobacteria use the chaperone/usher pathway to assemble proteinaceous filaments on the bacterial surface. These filaments can curl into fimbrial or nonfimbrial surface structures (e.g., a capsule or spore coat). This article reviews the phylogeny of operons belonging to the chaperone/usher assembly class to explore the utility of establishing a scheme for subdividing them into clades of phylogenetically related gene clusters. Based on usher amino acid sequence comparisons, our analysis shows that the chaperone/usher assembly class is subdivided into six major phylogenetic clades, which we have termed α-, β-, γ-, κ-, π-, and σ-fimbriae. Members of each clade share related operon structures and encode fimbrial subunits with similar protein domains. The proposed classification system offers a simple and convenient method for assigning newly discovered chaperone/usher systems to one of the six major phylogenetic groups. PMID:18063717

  7. Cooperative Subunit Refolding of a Light-Harvesting Protein through a Self-Chaperone Mechanism.

    PubMed

    Laos, Alistair J; Dean, Jacob C; Toa, Zi S D; Wilk, Krystyna E; Scholes, Gregory D; Curmi, Paul M G; Thordarson, Pall

    2017-01-27

    The fold of a protein is encoded by its amino acid sequence, but how complex multimeric proteins fold and assemble into functional quaternary structures remains unclear. Here we show that two structurally different phycobiliproteins refold and reassemble in a cooperative manner from their unfolded polypeptide subunits, without biological chaperones. Refolding was confirmed by ultrafast broadband transient absorption and two-dimensional electronic spectroscopy to probe internal chromophores as a marker of quaternary structure. Our results demonstrate a cooperative, self-chaperone refolding mechanism, whereby the β-subunits independently refold, thereby templating the folding of the α-subunits, which then chaperone the assembly of the native complex, quantitatively returning all coherences. Our results indicate that subunit self-chaperoning is a robust mechanism for heteromeric protein folding and assembly that could also be applied in self-assembled synthetic hierarchical systems.

  8. FGL chaperone-assembled fimbrial polyadhesins: anti-immune armament of Gram-negative bacterial pathogens.

    PubMed

    Zavialov, Anton; Zav'yalova, Galina; Korpela, Timo; Zav'yalov, Vladimir

    2007-07-01

    This review summarizes the current knowledge on the structure, function, assembly, and biomedical applications of the family of adhesive fimbrial organelles assembled on the surface of Gram-negative pathogens via the FGL chaperone/usher pathway. Recent studies revealed the unique structural and functional properties of these organelles, distinguishing them from a related family, FGS chaperone-assembled adhesive pili. The FGL chaperone-assembled organelles consist of linear polymers of one or two types of protein subunits, each possessing one or two independent adhesive sites specific to different host cell receptors. This structural organization enables these fimbrial organelles to function as polyadhesins. Fimbrial polyadhesins may ensure polyvalent fastening of bacteria to the host cells, aggregating their receptors and triggering subversive signals that allow pathogens to evade immune defense. The FGL chaperone-assembled fimbrial polyadhesins are attractive targets for vaccine and drug design.

  9. Dissecting functional similarities of ribosome-associated chaperones from Saccharomyces cerevisiae and Escherichia coli.

    PubMed

    Rauch, Thomas; Hundley, Heather A; Pfund, Chris; Wegrzyn, Renee D; Walter, William; Kramer, Günter; Kim, So-Young; Craig, Elizabeth A; Deuerling, Elke

    2005-07-01

    Ribosome-tethered chaperones that interact with nascent polypeptide chains have been identified in both prokaryotic and eukaryotic systems. However, these ribosome-associated chaperones share no sequence similarity: bacterial trigger factors (TF) form an independent protein family while the yeast machinery is Hsp70-based. The absence of any component of the yeast machinery results in slow growth at low temperatures and sensitivity to aminoglycoside protein synthesis inhibitors. After establishing that yeast ribosomal protein Rpl25 is able to recruit TF to ribosomes when expressed in place of its Escherichia coli homologue L23, the ribosomal TF tether, we tested whether such divergent ribosome-associated chaperones are functionally interchangeable. E. coli TF was expressed in yeast cells that lacked the endogenous ribosome-bound machinery. TF associated with yeast ribosomes, cross-linked to yeast nascent polypeptides and partially complemented the aminoglycoside sensitivity, demonstrating that ribosome-associated chaperones from divergent organisms share common functions, despite their lack of sequence similarity.

  10. Bovine monocyte-derived macrophage function in induced copper deficiency.

    PubMed

    Cerone, S; Sansinanea, A; Streitenberger, S; García, C; Auza, N

    2000-03-01

    The effect of molybdenum-induced copper deficiency on monocyte-derived macrophage function was examined. Five female calves were given molybdenum (30 ppm) and sulphate (225 ppm) to induce experimental secondary copper deficiency. Oxidant production by bovine macrophages was measured after stimulation with phorbol myristate acetate (PMA) and opsonized zymosan (OpZ). Lipoperoxidative effects inside of macrophage, superoxide dismutase activity, superoxide anion and hydrogen peroxide formation were determined. Copper deficiency was confirmed from decreased serum copper levels, and animals with values less than 5.9 micromol/l were considered deficient. The content of intracellular copper decreased about 40% in deficient cells compared with the controls. The respiratory burst activity determined by nitroblue tetrazolium reduction was significantly impaired with both stimulants used. Superoxide anion formation was less affected than hydrogen peroxide generation. In addition, increased lipid peroxidation was observed. It could be concluded that the effect of these changes may impair the monocyte-derived macrophage function in the immune system.

  11. Metabolic Reprograming in Macrophage Polarization

    PubMed Central

    Galván-Peña, Silvia; O’Neill, Luke A. J.

    2014-01-01

    Studying the metabolism of immune cells in recent years has emphasized the tight link existing between the metabolic state and the phenotype of these cells. Macrophages in particular are a good example of this phenomenon. Whether the macrophage obtains its energy through glycolysis or through oxidative metabolism can give rise to different phenotypes. Classically activated or M1 macrophages are key players of the first line of defense against bacterial infections and are known to obtain energy through glycolysis. Alternatively activated or M2 macrophages on the other hand are involved in tissue repair and wound healing and use oxidative metabolism to fuel their longer-term functions. Metabolic intermediates, however, are not just a source of energy but can be directly implicated in a particular macrophage phenotype. In M1 macrophages, the Krebs cycle intermediate succinate regulates HIF1α, which is responsible for driving the sustained production of the pro-inflammatory cytokine IL1β. In M2 macrophages, the sedoheptulose kinase carbohydrate kinase-like protein is critical for regulating the pentose phosphate pathway. The potential to target these events and impact on disease is an exciting prospect. PMID:25228902

  12. The Role of the Co-Chaperone, CHIP, in Androgen-Independent Prostate Cancer

    DTIC Science & Technology

    2012-02-01

    Award Number: W81XWH-06-1-0285 TITLE: The Role of the Co-Chaperone, CHIP, in Androgen-Independent Prostate Cancer ...AND SUBTITLE 5a. CONTRACT NUMBER The Role of the Co-Chaperone, CHIP, in Androgen Independent Prostate Cancer 5b. GRANT NUMBER W81XWH-06-1...ADT), is the mainstay of treatment for patients with locally advanced or metastatic prostate cancer . This therapy is only temporizing, however

  13. Survey of molecular chaperone requirement for the biosynthesis of hamster polyomavirus VP1 protein in Saccharomyces cerevisiae.

    PubMed

    Valaviciute, Monika; Norkiene, Milda; Goda, Karolis; Slibinskas, Rimantas; Gedvilaite, Alma

    2016-07-01

    A number of viruses utilize molecular chaperones during various stages of their life cycle. It has been shown that members of the heat-shock protein 70 (Hsp70) chaperone family assist polyomavirus capsids during infection. However, the molecular chaperones that assist the formation of recombinant capsid viral protein 1 (VP1)-derived virus-like particles (VLPs) in yeast remain unclear. A panel of yeast strains with single chaperone gene deletions were used to evaluate the chaperones required for biosynthesis of recombinant hamster polyomavirus capsid protein VP1. The impact of deletion or mild overexpression of chaperone genes was determined in live cells by flow cytometry using enhanced green fluorescent protein (EGFP) fused with VP1. Targeted genetic analysis demonstrated that VP1-EGFP fusion protein levels were significantly higher in yeast strains in which the SSZ1 or ZUO1 genes encoding ribosome-associated complex components were deleted. The results confirmed the participation of cytosolic Hsp70 chaperones and suggested the potential involvement of the Ydj1 and Caj1 co-chaperones and the endoplasmic reticulum chaperones in the biosynthesis of VP1 VLPs in yeast. Likewise, the markedly reduced levels of VP1-EGFP in Δhsc82 and Δhsp82 yeast strains indicated that both Hsp70 and Hsp90 chaperones might assist VP1 VLPs during protein biosynthesis.

  14. Monocyte to Macrophage Differentiation Goes along with Modulation of the Plasmalogen Pattern through Transcriptional Regulation

    PubMed Central

    Wallner, Stefan; Grandl, Margot; Konovalova, Tatiana; Sigrüner, Alexander; Kopf, Thomas; Peer, Markus; Orsó, Evelyn; Liebisch, Gerhard; Schmitz, Gerd

    2014-01-01

    Background Dysregulation of monocyte-macrophage differentiation is a hallmark of vascular and metabolic diseases and associated with persistent low grade inflammation. Plasmalogens represent ether lipids that play a role in diabesity and previous data show diminished plasmalogen levels in obese subjects. We therefore analyzed transcriptomic and lipidomic changes during monocyte-macrophage differentiation in vitro using a bioinformatic approach. Methods Elutriated monocytes from 13 healthy donors were differentiated in vitro to macrophages using rhM-CSF under serum-free conditions. Samples were taken on days 0, 1, 4 and 5 and analyzed for their lipidomic and transcriptomic profiles. Results Gene expression analysis showed strong regulation of lipidome-related transcripts. Enzymes involved in fatty acid desaturation and elongation were increasingly expressed, peroxisomal and ER stress related genes were induced. Total plasmalogen levels remained unchanged, while the PE plasmalogen species pattern became more similar to circulating granulocytes, showing decreases in PUFA and increases in MUFA. A partial least squares discriminant analysis (PLS/DA) revealed that PE plasmalogens discriminate the stage of monocyte-derived macrophage differentiation. Partial correlation analysis could predict novel potential key nodes including DOCK1, PDK4, GNPTAB and FAM126A that might be involved in regulating lipid and especially plasmalogen homeostasis during differentiation. An in silico transcription analysis of lipid related regulation revealed known motifs such as PPAR-gamma and KLF4 as well as novel candidates such as NFY, RNF96 and Zinc-finger proteins. Conclusion Monocyte to macrophage differentiation goes along with profound changes in the lipid-related transcriptome. This leads to an induction of fatty-acid desaturation and elongation. In their PE-plasmalogen profile macrophages become more similar to granulocytes than monocytes, indicating terminal phagocytic differentiation

  15. Differential Transcriptional Response in Macrophages Infected with Cell Wall Deficient versus Normal Mycobacterium Tuberculosis

    PubMed Central

    Fu, Yu-Rong; Gao, Kun-Shan; Ji, Rui; Yi, Zheng-Jun

    2015-01-01

    Host-pathogen interactions determine the outcome following infection by mycobacterium tuberculosis (Mtb). Under adverse circumstances, normal Mtb can form cell-wall deficient (CWD) variants within macrophages, which have been considered an adaptive strategy for facilitating bacterial survival inside macrophages. However, the molecular mechanism by which infection of macrophages with different phenotypic Mtb elicits distinct responses of macrophages is not fully understood. To explore the molecular events triggered upon Mtb infection of macrophages, differential transcriptional responses of RAW264.7 cells infected with two forms of Mtb, CWD-Mtb and normal Mtb, were studied by microarray analysis. Some of the differentially regulated genes were confirmed by RT-qPCR in both RAW264.7 cells and primary macrophages. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was used to analyze functions of differentially expressed genes. Distinct gene expression patterns were observed between CWD-Mtb and normal Mtb group. Mapt was up-regulated, while NOS2 and IL-11 were down-regulated in CWD-Mtb infected RAW264.7 cells and primary macrophages compared with normal Mtb infected ones. Many deregulated genes were found to be related to macrophages activation, immune response, phagosome maturation, autophagy and lipid metabolism. KEGG analysis showed that the differentially expressed genes were mainly involved in MAPK signaling pathway, nitrogen metabolism, cytokine-cytokine receptor interaction and focal adhesion. Taken together, the present study showed that differential macrophage responses were induced by intracellular CWD-Mtb an normal Mtb infection, which suggested that interactions between macrophages and different phenotypic Mtb are very complex. The results provide evidence for further understanding of pathogenesis of CWD-Mtb and may help in improving strategies to eliminate intracellular CWD-Mtb. PMID:25552926

  16. Fat-laden macrophages modulate lobular inflammation in nonalcoholic steatohepatitis (NASH).

    PubMed

    Jindal, Aastha; Bruzzì, Stefania; Sutti, Salvatore; Locatelli, Irene; Bozzola, Cristina; Paternostro, Claudia; Parola, Maurizio; Albano, Emanuele

    2015-08-01

    Nonalcoholic steatohepatitis (NASH) is characterized by extensive hepatic monocyte infiltration and monocyte-derived macrophages have an important role in regulating the disease evolution. However, little is known about the functional changes occurring in liver macrophages during NASH progression. In this study, we investigated phenotypic and functional modifications of hepatic macrophages in experimental NASH induced by feeding C57BL/6 mice with a methionine-choline deficient (MCD) diet up to 8weeks. In mice with steatohepatitis liver F4/80-positive macrophages increased in parallel with the disease progression and formed small clusters of enlarged and vacuolated cells. At immunofluorescence these cells contained lipid vesicles positive for the apoptotic cell marker Annexin V suggesting the phagocytosis of apoptotic bodies derived from dead fat-laden hepatocytes. Flow cytometry revealed that these enlarged macrophages expressed inflammatory monocyte (CD11b, Ly6C, TNF-α) markers. However, as compared to regular size macrophages the enlarged sub-set was characterized by an enhanced production of arginase-1 and of the anti-inflammatory mediators IL-10 and annexin A1. Similar vacuolated macrophages producing annexin A1 were also evident in liver biopsies of NASH patients. In mice with NASH, the accumulation of enlarged F4/80(+) cells paralleled with a decline in the expression of the macrophage M1 activation markers iNOS, IL-12 and CXCL10, while the levels of M2 polarization markers arginase-1 and MGL-1 were unchanged. Interestingly, the lowering of IL-12 expression mainly involved the macrophage sub-set with regular size. We conclude that during the progression of NASH fat accumulation within liver macrophages promotes the production of anti-inflammatory mediators that influence hepatic inflammatory responses.

  17. Macrophages and the Viral Dissemination Super Highway

    PubMed Central

    Klepper, Arielle; Branch, Andrea D

    2016-01-01

    Monocytes and macrophages are key components of the innate immune system yet they are often the victims of attack by infectious agents. This review examines the significance of viral infection of macrophages. The central hypothesis is that macrophage tropism enhances viral dissemination and persistence, but these changes may come at the cost of reduced replication in cells other than macrophages. PMID:26949751

  18. A NAP-Family Histone Chaperone Functions in Abiotic Stress Response and Adaptation1[OPEN

    PubMed Central

    Pareek, Ashwani; Singla-Pareek, Sneh Lata

    2016-01-01

    Modulation of gene expression is one of the most significant molecular mechanisms of abiotic stress response in plants. Via altering DNA accessibility, histone chaperones affect the transcriptional competence of genomic loci. However, in contrast to other factors affecting chromatin dynamics, the role of plant histone chaperones in abiotic stress response and adaptation remains elusive. Here, we studied the physiological function of a stress-responsive putative rice (Oryza sativa) histone chaperone of the NAP superfamily: OsNAPL6. We show that OsNAPL6 is a nuclear-localized H3/H4 histone chaperone capable of assembling a nucleosome-like structure. Utilizing overexpression and knockdown approaches, we found a positive correlation between OsNAPL6 expression levels and adaptation to multiple abiotic stresses. Results of comparative transcriptome profiling and promoter-recruitment studies indicate that OsNAPL6 functions during stress response via modulation of expression of various genes involved in diverse functions. For instance, we show that OsNAPL6 is recruited to OsRad51 promoter, activating its expression and leading to more efficient DNA repair and abrogation of programmed cell death under salinity and genotoxic stress conditions. These results suggest that the histone chaperone OsNAPL6 may serve a regulatory role in abiotic stress physiology possibly via modulating nucleosome dynamics at various stress-associated genomic loci. Taken together, our findings establish a hitherto unknown link between histone chaperones and abiotic stress response in plants. PMID:27342307

  19. Heterogeneous binding of the SH3 client protein to the DnaK molecular chaperone

    PubMed Central

    Lee, Jung Ho; Zhang, Dongyu; Hughes, Christopher; Okuno, Yusuke; Sekhar, Ashok; Cavagnero, Silvia

    2015-01-01

    The molecular chaperone heat shock protein 70 (Hsp70) plays a vital role in cellular processes, including protein folding and assembly, and helps prevent aggregation under physiological and stress-related conditions. Although the structural changes undergone by full-length client proteins upon interaction with DnaK (i.e., Escherichia coli Hsp70) are fundamental to understand chaperone-mediated protein folding, these changes are still largely unexplored. Here, we show that multiple conformations of the SRC homology 3 domain (SH3) client protein interact with the ADP-bound form of the DnaK chaperone. Chaperone-bound SH3 is largely unstructured yet distinct from the unfolded state in the absence of DnaK. The bound client protein shares a highly flexible N terminus and multiple slowly interconverting conformations in different parts of the sequence. In all, there is significant structural and dynamical heterogeneity in the DnaK-bound client protein, revealing that proteins may undergo some conformational sampling while chaperone-bound. This result is important because it shows that the surface of the Hsp70 chaperone provides an aggregation-free environment able to support part of the search for the native state. PMID:26195753

  20. Organismal proteostasis: role of cell-nonautonomous regulation and transcellular chaperone signaling

    PubMed Central

    van Oosten-Hawle, Patricija; Morimoto, Richard I.

    2014-01-01

    Protein quality control is essential in all organisms and regulated by the proteostasis network (PN) and cell stress response pathways that maintain a functional proteome to promote cellular health. In this review, we describe how metazoans employ multiple modes of cell-nonautonomous signaling across tissues to integrate and transmit the heat-shock response (HSR) for balanced expression of molecular chaperones. The HSR and other cell stress responses such as the unfolded protein response (UPR) can function autonomously in single-cell eukaryotes and tissue culture cells; however, within the context of a multicellular animal, the PN is regulated by cell-nonautonomous signaling through specific sensory neurons and by the process of transcellular chaperone signaling. These newly identified forms of stress signaling control the PN between neurons and nonneuronal somatic tissues to achieve balanced tissue expression of chaperones in response to environmental stress and to ensure that metastable aggregation-prone proteins expressed within any single tissue do not generate local proteotoxic risk. Transcellular chaperone signaling leads to the compensatory expression of chaperones in other somatic tissues of the animal, perhaps preventing the spread of proteotoxic damage. Thus, communication between subcellular compartments and across different cells and tissues maintains proteostasis when challenged by acute stress and upon chronic expression of metastable proteins. We propose that transcellular chaperone signaling provides a critical control step for the PN to maintain cellular and organismal health span. PMID:25030693

  1. Amyloid-β oligomers are sequestered by both intracellular and extracellular chaperones

    PubMed Central

    Narayan, Priyanka; Meehan, Sarah; Carver, John A.; Wilson, Mark R.; Dobson, Christopher M.; Klenerman, David

    2016-01-01

    The aberrant aggregation of the amyloid-β peptide into β-sheet rich, fibrillar structures proceeds via a heterogeneous ensemble of oligomeric intermediates that have been associated with neurotoxicity in Alzheimer’s disease (AD). Of particular interest in this context are the mechanisms by which molecular chaperones, part of the primary biological defenses against protein misfolding, influence Aβ aggregation. We have used single-molecule fluorescence techniques to compare the interactions between distinct aggregation states (monomers, oligomers, amyloid fibrils) of the AD-associated amyloid-β(1-40) peptide, and two molecular chaperones, both of which are upregulated in the brains of patients with AD and have been found colocalized with Aβ in senile plaques. One of the chaperones, αB-crystallin, is primarily found inside cells while the other, clusterin, is predominantly located in the extracellular environment. We find that both chaperones bind to misfolded oligomeric species and form long-lived complexes thereby preventing both their further growth into fibrils and their dissociation. From these studies, we conclude that these chaperones have a common mechanism of action based on sequestering Aβ oligomers. This conclusion suggests that these chaperones, both of which are ATP-independent, are able to inhibit potentially pathogenic Aβ oligomer-associated processes whether they occur in the extracellular or intracellular environment. PMID:23106396

  2. Conformational dynamics of a membrane protein chaperone enables spatially regulated substrate capture and release

    PubMed Central

    Liang, Fu-Cheng; Kroon, Gerard; McAvoy, Camille Z.; Chi, Chris; Wright, Peter E.; Shan, Shu-ou

    2016-01-01

    Membrane protein biogenesis poses enormous challenges to cellular protein homeostasis and requires effective molecular chaperones. Compared with chaperones that promote soluble protein folding, membrane protein chaperones require tight spatiotemporal coordination of their substrate binding and release cycles. Here we define the chaperone cycle for cpSRP43, which protects the largest family of membrane proteins, the light harvesting chlorophyll a/b-binding proteins (LHCPs), during their delivery. Biochemical and NMR analyses demonstrate that cpSRP43 samples three distinct conformations. The stromal factor cpSRP54 drives cpSRP43 to the active state, allowing it to tightly bind substrate in the aqueous compartment. Bidentate interactions with the Alb3 translocase drive cpSRP43 to a partially inactive state, triggering selective release of LHCP’s transmembrane domains in a productive unloading complex at the membrane. Our work demonstrates how the intrinsic conformational dynamics of a chaperone enables spatially coordinated substrate capture and release, which may be general to other ATP-independent chaperone systems. PMID:26951662

  3. Macrophage-specific de Novo Synthesis of Ceramide Is Dispensable for Inflammasome-driven Inflammation and Insulin Resistance in Obesity*

    PubMed Central

    Camell, Christina D.; Nguyen, Kim Y.; Jurczak, Michael J.; Christian, Brooke E.; Shulman, Gerald I.; Shadel, Gerald S.; Dixit, Vishwa Deep

    2015-01-01

    Dietary lipid overload and calorie excess during obesity is a low grade chronic inflammatory state with diminished ability to appropriately metabolize glucose or lipids. Macrophages are critical in maintaining adipose tissue homeostasis, in part by regulating lipid metabolism, energy homeostasis, and tissue remodeling. During high fat diet-induced obesity, macrophages are activated by lipid derived “danger signals” such as ceramides and palmitate and promote the adipose tissue inflammation in an Nlrp3 inflammasome-dependent manner. Given that the metabolic fate of fatty acids in macrophages is not entirely elucidated, we have hypothesized that de novo synthesis of ceramide, through the rate-limiting enzyme serine palmitoyltransferase long chain (Sptlc)-2, is required for saturated fatty acid-driven Nlrp3 inflammasome activation in macrophages. Here we report that mitochondrial targeted overexpression of catalase, which is established to mitigate oxidative stress, controls ceramide-induced Nlrp3 inflammasome activation but does not affect the ATP-mediated caspase-1 cleavage. Surprisingly, myeloid cell-specific deletion of Sptlc2 is not required for palmitate-driven Nlrp3 inflammasome activation. Furthermore, the ablation of Sptlc2 in macrophages did not impact macrophage polarization or obesity-induced adipose tissue leukocytosis. Consistent with these data, investigation of insulin resistance using hyperinsulinemic-euglycemic clamps revealed no significant differences in obese mice lacking ceramide de novo synthesis machinery in macrophages. These data suggest that alternate metabolic pathways control fatty acid-derived ceramide synthesis in macrophage and the Nlrp3 inflammasome activation in obesity. PMID:26438821

  4. Amphiphilic Nanoparticles Repress Macrophage Atherogenesis: Novel Core/Shell Designs for Scavenger Receptor Targeting and Down-Regulation

    PubMed Central

    2015-01-01

    Atherosclerosis, an inflammatory lipid-rich plaque disease is perpetuated by the unregulated scavenger-receptor-mediated uptake of oxidized lipoproteins (oxLDL) in macrophages. Current treatments lack the ability to directly inhibit oxLDL accumulation and foam cell conversion within diseased arteries. In this work, we harness nanotechnology to design and fabricate a new class of nanoparticles (NPs) based on hydrophobic mucic acid cores and amphiphilic shells with the ability to inhibit the uncontrolled uptake of modified lipids in human macrophages. Our results indicate that tailored NP core and shell formulations repress oxLDL internalization via dual complementary mechanisms. Specifically, the most atheroprotective molecules in the NP cores competitively reduced NP-mediated uptake to scavenger receptor A (SRA) and also down-regulated the surface expression of SRA and CD36. Thus, nanoparticles can be designed to switch activated, lipid-scavenging macrophages to antiatherogenic phenotypes, which could be the basis for future antiatherosclerotic therapeutics. PMID:24972372

  5. The Macrophage Switch in Obesity Development

    PubMed Central

    Castoldi, Angela; Naffah de Souza, Cristiane; Câmara, Niels Olsen Saraiva; Moraes-Vieira, Pedro M.

    2016-01-01

    Immune cell infiltration in (white) adipose tissue (AT) during obesity is associated with the development of insulin resistance. In AT, the main population of leukocytes are macrophages. Macrophages can be classified into two major populations: M1, classically activated macrophages, and M2, alternatively activated macrophages, although recent studies have identified a broad range of macrophage subsets. During obesity, AT M1 macrophage numbers increase and correlate with AT inflammation and insulin resistance. Upon activation, pro-inflammatory M1 macrophages induce aerobic glycolysis. By contrast, in lean humans and mice, the number of M2 macrophages predominates. M2 macrophages secrete anti-inflammatory cytokines and utilize oxidative metabolism to maintain AT homeostasis. Here, we review the immunologic and metabolic functions of AT macrophages and their different facets in obesity and the metabolic syndrome. PMID:26779183

  6. Development and characterization of amphotericin B bearing emulsomes for passive and active macrophage targeting.

    PubMed

    Gupta, Swati; Vyas, Suresh P

    2007-04-01

    The antifungal and antileishmanial agent amphotericin B (AmB) has been complexed with lipids to develop a less toxic formulation of AmB. Because lipid particles are phagocytized by the reticuloendothelial system, lipid associated AmB should be concentrated in infected macrophages of liver and spleen and be very effective against visceral leishmaniasis (VL) and systemic fungal infections. Therefore, AmB was formulated in trilaurin based nanosize lipid particles (emulsomes) stabilized by soya phosphatidylcholine (PC) as a new intravenous drug delivery system for macrophage targeting. Emulsomes were prepared by cast film technique followed by sonication to obtain particles of nanometric size range. Formulations were optimized for AmB to lipid ratio, sonication time and PC to trilaurin ratio. Emulsomes were modified by coating them with macrophage-specific ligand (O-palmitoyl mannan, OPM). The surface modified emulsomes and their plain counterparts were characterised for size, shape, lamellarity and entrapment efficiency. Fluorescence microscopy study showed significant localization of plain and coated emulsomes inside the liver and spleen cells of golden hamsters. In vivo organ distribution studies in albino rats demonstrated that extent of accumulation of emulsome entrapped AmB in macrophage rich organs, particularly liver, spleen and lungs was significantly high when compared against the free drug (AmB-deoxycholate or AmB-Doc). The rate and extent of accumulation were found to increase further on ligand anchoring. Further, a significantly higher (P < 0.05) drug concentration in the liver was estimated over a period of 24 h for OPM coated emulsomes than for plain emulsomes. We concluded that OPM coated emulsomes could fuse with the macrophages of liver and spleen due to ligand-receptor interaction and could target the bioactives inside them. The proposed plain and OPM coated emulsome based systems showed excellent potential for passive and active intramacrophage

  7. Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting M1 macrophage response in melanoma

    PubMed Central

    Mrad, Marguerite; Imbert, Caroline; Garcia, Virginie; Rambow, Florian; Therville, Nicole; Carpentier, Stéphane; Ségui, Bruno; Levade, Thierry; Azar, Rania; Marine, Jean-Christophe; Diab-Assaf, Mona; Colacios, Céline; Andrieu-Abadie, Nathalie

    2016-01-01

    The infiltration of melanoma tumors by macrophages is often correlated with poor prognosis. However, the molecular signals that regulate the dialogue between malignant cells and the inflammatory microenvironment remain poorly understood. We previously reported an increased expression of sphingosine kinase-1 (SK1), which produces the bioactive lipid sphingosine 1-phosphate (S1P), in melanoma. The present study aimed at defining the role of tumor SK1 in the recruitment and differentiation of macrophages in melanoma. Herein, we show that downregulation of SK1 in melanoma cells causes a reduction in the percentage of CD206highMHCIIlow M2 macrophages in favor of an increased proportion of CD206lowMHCIIhigh M1 macrophages into the tumor. This macrophage differentiation orchestrates T lymphocyte recruitment as well as tumor rejection through the expression of Th1 cytokines and chemokines. In vitro experiments indicated that macrophage migration is triggered by the binding of tumor S1P to S1PR1 receptors present on macrophages whereas macrophage differentiation is stimulated by SK1-induced secretion of TGF-β1. Finally, RNA-seq analysis of human melanoma tumors revealed a positive correlation between SK1 and TGF-β1 expression. Altogether, our findings demonstrate that melanoma SK1 plays a key role in the recruitment and phenotypic shift of the tumor macrophages that promote melanoma growth. PMID:27708249

  8. Combined Two-Photon Luminescence Microscopy and OCT for Macrophage Detection in the Hypercholesterolemic Rabbit Aorta Using Plasmonic Gold Nanorose

    PubMed Central

    Wang, Tianyi; Mancuso, J. Jacob; Kazmi, S.M. Shams; Dwelle, Jordan; Sapozhnikova, Veronika; Willsey, Brian; Ma, Li L.; Qiu, Jinze; Li, Xiankai; Dunn, Andrew K.; Johnston, Keith P.; Feldman, Marc D.; Milner, Thomas E.

    2013-01-01

    Background and Objectives The macrophage is an important early cellular marker related to risk of future rupture of atherosclerotic plaques. Two-channel two-photon luminescence (TPL) microscopy combined with optical coherence tomography (OCT) was used to detect, and further characterize the distribution of aorta-based macrophages using plasmonic gold nanorose as an imaging contrast agent. Study Design/Materials and Methods Nanorose uptake by macrophages was identified by TPL microscopy in macrophage cell culture. Ex vivo aorta segments (8 × 8 × 2 mm3) rich in macrophages from a rabbit model of aorta inflammation were imaged by TPL microscopy in combination with OCT. Aorta histological sections (5 µm in thickness) were also imaged by TPL microscopy. Results Merged two-channel TPL images showed the lateral and depth distribution of nanorose-loaded macrophages (confirmed by RAM-11 stain) and other aorta components (e.g., elastin fiber and lipid droplet), suggesting that nanorose-loaded macrophages are diffusively distributed and mostly detected superficially within 20 µm from the luminal surface of the aorta. Moreover, OCT images depicted detailed surface structure of the diseased aorta. Conclusions Results suggest that TPL microscopy combined with OCT can simultaneously reveal macrophage distribution with respect to aorta surface structure, which has the potential to detect vulnerable plaques and monitor plaque-based macrophages overtime during cardiovascular interventions. PMID:22246984

  9. The macrophage: the intersection between HIV infection and atherosclerosis

    PubMed Central

    Crowe, Suzanne M.; Westhorpe, Clare L. V.; Mukhamedova, Nigora; Jaworowski, Anthony; Sviridov, Dmitri; Bukrinsky, Michael

    2010-01-01

    HIV-infected individuals are at increased risk of coronary artery disease (CAD) with underlying mechanisms including chronic immune activation and inflammation secondary to HIV-induced microbial translocation and low-grade endotoxemia; direct effects of HIV and viral proteins on macrophage cholesterol metabolism; and dyslipidemia related to HIV infection and specific antiretroviral therapies. Monocytes are the precursors of the lipid-laden foam cells within the atherosclerotic plaque and produce high levels of proinflammatory cytokines such as IL-6. The minor CD14+/CD16+ “proinflammatory” monocyte subpopulation is preferentially susceptible to HIV infection and may play a critical role in the pathogenesis of HIV-related CAD. In this review, the central role of monocytes/macrophages in HIV-related CAD and the importance of inflammation and cholesterol metabolism are discussed. PMID:19952353

  10. Metabolic reprogramming in macrophages and dendritic cells in innate immunity

    PubMed Central

    Kelly, Beth; O'Neill, Luke AJ

    2015-01-01

    Activation of macrophages and dendritic cells (DCs) by pro-inflammatory stimuli causes them to undergo a metabolic switch towards glycolysis and away from oxidative phosphorylation (OXPHOS), similar to the Warburg effect in tumors. However, it is only recently that the mechanisms responsible for this metabolic reprogramming have been elucidated in more detail. The transcription factor hypoxia-inducible factor-1α (HIF-1α) plays an important role under conditions of both hypoxia and normoxia. The withdrawal of citrate from the tricarboxylic acid (TCA) cycle has been shown to be critical for lipid biosynthesis in both macrophages and DCs. Interference with this process actually abolishes the ability of DCs to activate T cells. Another TCA cycle intermediate, succinate, activates HIF-1α and promotes inflammatory gene expression. These new insights are providing us with a deeper understanding of the role of metabolic reprogramming in innate immunity. PMID:26045163

  11. Celery Seed Extract Blocks Peroxide Injury in Macrophages via Notch1/NF-κB Pathway.

    PubMed

    Si, Yanhong; Guo, Shoudong; Fang, Yongqi; Qin, Shucun; Li, Furong; Zhang, Ying; Jiao, Peng; Zhang, Chunduo; Gao, Linlin

    2015-01-01

    Oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation and injury is one of the major atherogenic factors. This study is aimed to investigate the protective effect of celery seed extract (CSE) on ox-LDL-induced injury of macrophages and the underlying signaling pathway. RAW264.7 macrophages were pre-incubated with CSE for 24 h, followed by stimulation with ox-LDL. Oil red O staining and enzymatic colorimetry indicated CSE significantly lessened lipid droplets and total cholesterol (TC) content in ox-LDL-injured macrophages. ELISA revealed that CSE decreased the secretion of inflammatory cytokine TNF-α and IL-6 by 12-27% and 5-15% respectively. MTT assay showed CSE promoted cell viability by 16-40%. Cell apoptosis was also analyzed by flow cytometry and laser scanning confocal microscope and the data indicated CSE inhibited ox-LDL-induced apoptosis of macrophages. Meanwhile, western blot analysis showed CSE suppressed NF-κBp65 and notch1 protein expressions stimulated by ox-LDL in macrophages. These results suggest that CSE inhibits ox-LDL-induced macrophages injury via notch1/NF-κB pathway.

  12. Macrophages are novel sites of expression and regulation of retinol binding protein-4 (RBP4).

    PubMed

    Broch, M; Ramírez, R; Auguet, M T; Alcaide, M J; Aguilar, C; Garcia-Espana, A; Richart, C

    2010-01-01

    Obesity is linked to a low-level chronic inflammatory state that may contribute to the development of associated metabolic complications. Retinol-binding protein 4 (RBP4) is an adipokine associated with parameters of obesity including insulin resistance indices, body mass index, waist circumference, lipid profile, and recently, with circulating inflammatory factors. Due to the infiltration of adipose tissue in obesity by macrophages derived from circulating monocytes and, on the other hand, the existence of a close genetic relationship between adipocytes and macrophages, we decided to examine if RBP4 is expressed in monocytes and/or primary human macrophages. While we did not detect expression of RBP4 in undifferentiated monocytes, RBP4 expression became evident during the differentiation of monocytes into macrophages and was highest in differentiated macrophages. Once we demonstrated the expression of RBP4 in macrophages, we checked if RBP4 expression could be regulated by inflammatory stimuli such as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), or the endotoxin lipopolysaccharide (LPS). We observed that while RBP4 expression was strongly inhibited by TNF-alpha and LPS, it was not affected by IL-6. Our results highlight the complexity behind the regulation of this adipokine and demonstrate that RBP4 expression in macrophages could be modulated by inflammatory stimuli.

  13. Mycobacterium avium Subspecies paratuberculosis Recombinant Proteins Modulate Antimycobacterial Functions of Bovine Macrophages.

    PubMed

    Bannantine, John P; Stabel, Judith R; Laws, Elizabeth; D Cardieri, Maria Clara; Souza, Cleverson D

    2015-01-01

    It has been shown that Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) activates the Mitogen Activated Protein Kinase (MAPK) p38 pathway, yet it is unclear which components of M. paratuberculosis are involved in the process. Therefore, a set of 42 M. paratuberculosis recombinant proteins expressed from coding sequences annotated as lipoproteins were screened for their ability to induce IL-10 expression, an indicator of MAPKp38 activation, in bovine monocyte-derived macrophages. A recombinant lipoprotein, designated as MAP3837c, was among a group of 6 proteins that strongly induced IL-10 gene transcription in bovine macrophages, averaging a 3.1-fold increase compared to non-stimulated macrophages. However, a parallel increase in expression of IL-12 and TNF-α was only observed in macrophages exposed to a subset of these 6 proteins. Selected recombinant proteins were further analyzed for their ability to enhance survival of M. avium within bovine macrophages as measured by recovered viable bacteria and nitrite production. All 6 IL-10 inducing MAP recombinant proteins along with M. paratuberculosis cells significantly enhanced phosphorylation of MAPK-p38 in bovine macrophages. Although these proteins are likely not post translationally lipidated in E. coli and thus is a limitation in this study, these results form the foundation of how the protein component of the lipoprotein interacts with the immune system. Collectively, these data reveal M. paratuberculosis proteins that might play a role in MAPK-p38 pathway activation and hence in survival of this organism within bovine macrophages.

  14. Resolvin D1 Polarizes Primary Human Macrophages toward a Proresolution Phenotype through GPR32.

    PubMed

    Schmid, Mattia; Gemperle, Claudio; Rimann, Nicole; Hersberger, Martin

    2016-04-15

    Resolvin D1 (RvD1) was shown to be a potent anti-inflammatory and proresolution lipid mediator in several animal models of inflammation, but its mechanism of action in humans is not clear. We show that the RvD1 receptor GPR32 is present on resting, proinflammatory M(LPS) and alternatively activated primary human M(IL-4) macrophages, whereas TGF-β and IL-6 reduce its membrane expression. Accordingly, stimulation of resting primary human macrophages with 10 nM RvD1 for 48 h maximally reduced the secretion of the proinflammatory cytokines IL-1β and IL-8; abolished chemotaxis to several chemoattractants like chemerin, fMLF, and MCP-1; and doubled the phagocytic activity of these macrophages toward microbial particles. In contrast, these functional changes were not accompanied by surface expression of markers specific for alternatively activated M(IL-4) macrophages. Similar proresolution effects of RvD1 were observed when proinflammatory M(LPS) macrophages were treated with RvD1. In addition, we show that these RvD1-mediated effects are GPR32 dependent because reduction of GPR32 expression by small interfering RNA, TGF-β, and IL-6 treatment ablated these proresolution effects in primary human macrophages. Taken together, our results indicate that in humans RvD1 triggers GPR32 to polarize and repolarize macrophages toward a proresolution phenotype, supporting the role of this mediator in the resolution of inflammation in humans.

  15. ROS sets the stage for macrophage differentiation.

    PubMed

    Covarrubias, Anthony; Byles, Vanessa; Horng, Tiffany

    2013-08-01

    While M1 macrophages are highly pro-inflammatory and microbicidal, M2 macrophages and the related tumor associated macrophages (TAMs) regulate tissue remodeling and angiogenesis and can display immunomodulatory activity. In July issue of Cell Research, Zhang et al. show that ROS production, critical for the activation and functions of M1 macrophages, is necessary for the differentiation of M2 macrophages and TAMs, and that antioxidant therapy blocks TAM differentiation and tumorigenesis in mouse models of cancer.

  16. Pulmonary surfactant and macrophages studied at the air/liquid interface revealed by Brewster angle microscopy (BAM)

    NASA Astrophysics Data System (ADS)

    Telesford, Dana-Marie; Allen, Heather; Carlson, Tracy; Schlesinger, Larry

    2012-04-01

    The alveolus is lined with a complex mixture of lipids and proteins called pulmonary surfactant (PS) that lower surface tension at the alveolar air/liquid interface. The surface area of the lung for a 70 kg adult human at total lung capacity is ˜70 m^2. The large surface area and the direct exposure to the environment with every inhalation make this organ more susceptible to invasion by viruses, bacteria, and small particles. The most abundant cell recovered in human lung lavage is the alveolar macrophage which accounts for 85% of the total. The primary function of the alveolar macrophage is to defend the lung against invasion, but also in the clearance of surfactant components in the lung. Quintero and Wright,^1 in an in vitro study observing alveolar macrophage metabolism of two lipid components dipalmitoyl phosphatidylglycerol (DPPG) and dipalmitoyl phosphatidylcholine (DPPC), noted that DPPG was removed at a faster rate. The mechanism by which this process takes place is not fully understood and our aim is to investigate the interactions of macrophages with different lipids using Brewster angle microscopy. Preliminary studies suggest that THP-1 differentiated macrophages do not significantly perturb DPPC and DPPG monolayers and research utilizing alveolar macrophages is underway. The effect of PS SP-A and SP-D is also discussed.

  17. Effect of oxidative stress on plasma membrane fluidity of THP-1 induced macrophages.

    PubMed

    de la Haba, Carlos; Palacio, José R; Martínez, Paz; Morros, Antoni

    2013-02-01

    Plasma membrane is one of the preferential targets of reactive oxygen species which cause lipid peroxidation. This process modifies membrane properties such as membrane fluidity, a very important physical feature known to modulate membrane protein localization and function. The aim of this study is to evaluate the effect of oxidative stress on plasma membrane fluidity regionalization of single living THP-1 macrophages. These cells were oxidized with H(2)O(2) at different concentrations, and plasma membrane fluidity was analyzed by two-photon microscopy in combination with the environment-sensitive probe Laurdan. Results show a significant H(2)O(2) concentration dependent increase in the frequency of rigid lipid regions, mainly attributable to lipid rafts, at the expense of the intermediate fluidity regions. A novel statistical analysis evaluated changes in size and number of lipid raft domains under oxidative stress conditions, as lipid rafts are platforms aiding cell signaling and are thought to have relevant roles in macrophage functions. It is shown that H(2)O(2) causes an increase in the number, but not the size, of raft domains. As macrophages are highly resistant to H(2)O(2), these new raft domains might be involved in cell survival pathways.

  18. Macrophage-Specific Expression of Human Lysosomal Acid Lipase Corrects Inflammation and Pathogenic Phenotypes in lal−/− Mice

    PubMed Central

    Yan, Cong; Lian, Xuemei; Li, Yuan; Dai, Ying; White, Amanda; Qin, Yulin; Li, Huimin; Hume, David A.; Du, Hong

    2006-01-01

    Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in the cell. The downstream metabolites of these compounds serve as hormonal ligands for nuclear receptors and transcription factors. Genetic ablation of the lal gene in the mouse caused malformation of macrophages and inflammation-triggered multiple pathogenic phenotypes in multiple organs. To assess the relationship between macrophages and lal−/− pathogenic phenotypes, a macrophage-specific doxycycline-inducible transgenic system was generated to induce human LAL (hLAL) expression in the lal−/− genetic background under control of the 7.2-kb c-fms promoter/intron2 regulatory sequence. Doxycycline-induced hLAL expression in macrophages significantly ameliorated aberrant gene expression, inflammatory cell (neutrophil) influx, and pathogenesis in multiple organs. These studies strongly support that neutral lipid metabolism in macrophages contributes to organ inflammation and pathogenesis. PMID:16936266

  19. Photoreceptor IFT complexes containing chaperones, guanylyl cyclase 1 and rhodopsin.

    PubMed

    Bhowmick, Reshma; Li, Mei; Sun, Jun; Baker, Sheila A; Insinna, Christine; Besharse, Joseph C

    2009-06-01

    Intraflagellar transport (IFT) provides a mechanism for the transport of cilium-specific proteins, but the mechanisms for linkage of cargo and IFT proteins have not been identified. Using the sensory outer segments (OS) of photoreceptors, which are derived from sensory cilia, we have identified IFT-cargo complexes containing IFT proteins, kinesin 2 family proteins, two photoreceptor-specific membrane proteins, guanylyl cyclase 1 (GC1, Gucy2e) and rhodopsin (RHO), and the chaperones, mammalian relative of DNAJ, DnajB6 (MRJ), and HSC70 (Hspa8). Analysis of these complexes leads to a model in which MRJ through its binding to IFT88 and GC1 plays a critical role in formation or stabilization of the IFT-cargo complexes. Consistent with the function of MRJ in the activation of HSC70 ATPase activity, Mg-ATP enhances the co-IP of GC1, RHO, and MRJ with IFT proteins. Furthermore, RNAi knockdown of MRJ in IMCD3 cells expressing GC1-green fluorescent protein (GFP) reduces cilium membrane targeting of GC1-GFP without apparent effect on cilium elongation.

  20. Activation of a bacterial lipase by its chaperone.

    PubMed Central

    Hobson, A H; Buckley, C M; Aamand, J L; Jørgensen, S T; Diderichsen, B; McConnell, D J

    1993-01-01

    The gene lipA of Pseudomonas cepacia DSM 3959 encodes a prelipase from which a signal peptide is cleaved during secretion, producing a mature extracellular lipase. Expression of lipase in several heterologous hosts depends on the presence of another gene, limA, in cis or in trans. Lipase protein has been overproduced in Escherichia coli in the presence and absence of the lipase modulator gene limA. Therefore, limA is not required for the transcription of lipA or for the translation of the lipA mRNA. However, no lipase activity is observed in the absence of limA. limA has been overexpressed and encodes a 33-kDa protein, Lim. If lipase protein is denatured in 8 M urea and the urea is removed by dialysis, lipase activity is quantitatively recovered provided Lim protein is present during renaturation. Lip and Lim proteins form a complex precipitable either by an anti-lipase or anti-Lim antibody. The Lim protein has therefore the properties of a chaperone. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7685908

  1. A Novel Function of Molecular Chaperone HSP70

    PubMed Central

    Halasi, Marianna; Váraljai, Renáta; Benevolenskaya, Elizaveta; Gartel, Andrei L.

    2016-01-01

    The oncogenic transcription factor FOXM1 is overexpressed in the majority of human cancers, and it is a potential target for anticancer therapy. We identified proteasome inhibitors as the first type of drugs that target FOXM1 in cancer cells. Here we found that HSP90 inhibitor PF-4942847 and heat shock also suppress FOXM1. The common effector, which was induced after treatment with proteasome and HSP90 inhibitors or heat shock, was the molecular chaperone HSP70. We show that HSP70 binds to FOXM1 following proteotoxic stress and that HSP70 inhibits FOXM1 DNA-binding ability. Inhibition of FOXM1 transcriptional autoregulation by HSP70 leads to the suppression of FOXM1 protein expression. In addition, HSP70 suppression elevates FOXM1 expression, and simultaneous inhibition of FOXM1 and HSP70 increases the sensitivity of human cancer cells to anticancer drug-induced apoptosis. Overall, we determined the unique and novel mechanism of FOXM1 suppression by proteasome inhibitors. PMID:26559972

  2. Fab Chaperone-Assisted RNA Crystallography (Fab CARC).

    PubMed

    Sherman, Eileen; Archer, Jennifer; Ye, Jing-Dong

    2016-01-01

    Recent discovery of structured RNAs such as ribozymes and riboswitches shows that there is still much to learn about the structure and function of RNAs. Knowledge learned can be employed in both biochemical research and clinical applications. X-ray crystallography gives unparalleled atomic-level structural detail from which functional inferences can be deduced. However, the difficulty in obtaining high-quality crystals and their phasing information make it a very challenging task. RNA crystallography is particularly arduous due to several factors such as RNA's paucity of surface chemical diversity, lability, repetitive anionic backbone, and flexibility, all of which are counterproductive to crystal packing. Here we describe Fab chaperone assisted RNA crystallography (CARC), a systematic technique to increase RNA crystallography success by facilitating crystal packing as well as expediting phase determination through molecular replacement of conserved Fab domains. Major steps described in this chapter include selection of a synthetic Fab library displayed on M13 phage against a structured RNA crystallization target, ELISA for initial choice of binding Fabs, Fab expression followed by protein A affinity then cation exchange chromatography purification, final choice of Fab by binding specificity and affinity as determined by a dot blot assay, and lastly gel filtration purification of a large quantity of chosen Fabs for crystallization.

  3. Diabetic Peripheral Neuropathy: Should a Chaperone Accompany Our Therapeutic Approach?

    PubMed Central

    Farmer, Kevin L.; Li, Chengyuan

    2012-01-01

    Diabetic peripheral neuropathy (DPN) is a common complication of diabetes that is associated with axonal atrophy, demyelination, blunted regenerative potential, and loss of peripheral nerve fibers. The development and progression of DPN is due in large part to hyperglycemia but is also affected by insulin deficiency and dyslipidemia. Although numerous biochemical mechanisms contribute to DPN, increased oxidative/nitrosative stress and mitochondrial dysfunction seem intimately associated with nerve dysfunction and diminished regenerative capacity. Despite advances in understanding the etiology of DPN, few approved therapies exist for the pharmacological management of painful or insensate DPN. Therefore, identifying novel therapeutic strategies remains paramount. Because DPN does not develop with either temporal or biochemical uniformity, its therapeutic management may benefit from a multifaceted approach that inhibits pathogenic mechanisms, manages inflammation, and increases cytoprotective responses. Finally, exercise has long been recognized as a part of the therapeutic management of diabetes, and exercise can delay and/or prevent the development of painful DPN. This review presents an overview of existing therapies that target both causal and symptomatic features of DPN and discusses the role of up-regulating cytoprotective pathways via modulating molecular chaperones. Overall, it may be unrealistic to expect that a single pharmacologic entity will suffice to ameliorate the multiple symptoms of human DPN. Thus, combinatorial therapies that target causal mechanisms and enhance endogenous reparative capacity may enhance nerve function and improve regeneration in DPN if they converge to decrease oxidative stress, improve mitochondrial bioenergetics, and increase response to trophic factors. PMID:22885705

  4. Bacterial Discrimination by FISH using Molecular Chaperon GroE

    NASA Astrophysics Data System (ADS)

    Nakamura, T.; Maruyama, A.; Kurusu, Y.

    2004-12-01

    FISH(Fluorescence In Situ hybridization) is a powerful method for the analysis of the phylogenetic classification of microorganism in the environment. In many cases, 16s rRNA sequences of microorganisms are employed as target probe. Here we showed that novel probe was used in FISH in order to discriminate among the bacteria including psychrophile, mesophile, and thermophile. Molecular Chaperon GroE is a best characterized protein based on Escherichia coli and essential for bacterial proliferation. In E. coli, the amount of GroEL protein per cell reaches to about 5% of total cellualr protein at heat-shock response. This response occurred at transcription levels, the amount of groEL mRNA increases at about 10-fold per cell, reaches to 0.4% of total synthesized RNA. Therefore, we considered that groEL gene was employed FISH analysis as a target probe. Moreover, we found that Gly-Gly-Met (GGM) repeats in the carboxy-terminal of GroEL strongly conserved among psychrophile and mesophile, but not thermophile. In this report, we attempted to discriminate among the bacteria including psychrophile, mesophile, and thermophile by FISH using the specific sequence of GroEL as a probe. Furthermore, we proposed the novel phylogenetic trees based on the amino acids sequences of carboxy-terminal of GroEL for bacterial evolution by temperature adaptation.

  5. Pharmacological Chaperoning: A Potential Treatment for PMM2-CDG.

    PubMed

    Yuste-Checa, Patricia; Brasil, Sandra; Gámez, Alejandra; Underhaug, Jarl; Desviat, Lourdes R; Ugarte, Magdalena; Pérez-Cerdá, Celia; Martinez, Aurora; Pérez, Belén

    2017-02-01

    The congenital disorder of glycosylation (CDG) due to phosphomannomutase 2 deficiency (PMM2-CDG), the most common N-glycosylation disorder, is a multisystem disease for which no effective treatment is available. The recent functional characterization of disease-causing mutations described in patients with PMM2-CDG led to the idea of a therapeutic strategy involving pharmacological chaperones (PC) to rescue PMM2 loss-of-function mutations. The present work describes the high-throughput screening, by differential scanning fluorimetry, of 10,000 low-molecular-weight compounds from a commercial library, to search for possible PCs for the enzyme PMM2. This exercise identified eight compounds that increased the thermal stability of PMM2. Of these, four compounds functioned as potential PCs that significantly increased the stability of several destabilizing and oligomerization mutants and also increased PMM activity in a disease model of cells overexpressing PMM2 mutations. Structural analysis revealed one of these compounds to provide an excellent starting point for chemical optimization since it passed tests based on a number of pharmacochemical quality filters. The present results provide the first proof-of-concept of a possible treatment for PMM2-CDG and describe a promising chemical structure as a starting point for the development of new therapeutic agents for this severe orphan disease.

  6. Withaferin A Analogs That Target the AAA+ Chaperone p97

    PubMed Central

    Wijeratne, E. M. Kithsiri; Xu, Ya-ming; Kang, MinJin; Wu, Tongde; Lau, Eric C.; Mesa, Celestina; Mason, Damian J.; Brown, Robert V.; Clair, James J. La; Gunatilaka, A. A. Leslie; Zhang, Donna D.; Chapman, Eli

    2015-01-01

    Understanding the mode of action (MOA) of many natural products can be puzzling with mechanistic clues that seem to lack a common thread. One such puzzle lies in the evaluation of the antitumor properties of the natural product withaferin A (WFA). A variety of seemingly unrelated pathways have been identified to explain its activity, suggesting a lack of selectivity. We now show that WFA acts as an inhibitor of the chaperone, p97, both in vitro and in cell models in addition to inhibiting the proteasome in vitro. Through medicinal chemistry, we have refined the activity of WFA toward p97 and away from the proteasome. Subsequent studies indicated that these WFA analogs retained p97 activity and cytostatic activity in cell models, suggesting that the modes of action reported for WFA could be connected by proteostasis modulation. Through this endeavor, we highlight how the parallel integration of medicinal chemistry with chemical biology offers a potent solution to one of natures’ intriguing molecular puzzles. PMID:26006219

  7. A new perspective in Parkinson's disease, chaperone-mediated autophagy.

    PubMed

    Li, Boyu; Zhang, Yun; Yuan, Yuhe; Chen, Naihong

    2011-05-01

    Parkinson's disease (PD) is an age-related neurodegenerative disease characterized by loss of dopaminergic neurons and aggregation of alpha-synuclein. Although the role of alpha-synuclein in the pathology of PD is still unclear, the fact that its aggregation contributes to the loss of dopaminergic neurons has been confirmed. Therefore, controlling the alpha-synuclein protein level may be critical for PD pathogenesis and may provide potential therapeutics. Wild-type alpha-synuclein is physiologically degraded by chaperone-mediated autophagy (CMA), and dysfunction of CMA results in alpha-synuclein aggregation and compensative macroautophagy activation which finally leads to cell death. Therefore, CMA may participate in PD pathogenesis as a very important factor, and up-regulating CMA activity could degrade overloaded alpha-synuclein. In view of potential compensative effects, maintenance of the balance of CMA activity will be another major challenge in the future development of the therapeutic strategy. Herein we review the current knowledge of the role of CMA in PD.

  8. Sulphur shuttling across a chaperone during molybdenum cofactor maturation.

    PubMed

    Arnoux, Pascal; Ruppelt, Christian; Oudouhou, Flore; Lavergne, Jérôme; Siponen, Marina I; Toci, René; Mendel, Ralf R; Bittner, Florian; Pignol, David; Magalon, Axel; Walburger, Anne

    2015-02-04

    Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhD binds Mo-bisPGD in vivo and has submicromolar affinity for GDP-used as a surrogate of the molybdenum cofactor's nucleotide moieties. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.

  9. Macrophage Responses to B. Anthracis

    DTIC Science & Technology

    2006-08-14

    LPS were reflective of a profound rophage responses to close relatives like Bacillus cereus as well change in cellular signaling, and in general these...published (attached) in 2005 [Bergman, et al. Murine Macrophage Transcriptional Responses to Bacillus I Final Report anthracis Infection and Intoxication...Macrophage Transcriptional Responses to Bacillus anthracis Infection and Intoxication. Infection & Immunity. 73:1069-1079. Parallel to the mRNA data

  10. Proteins with RNA Chaperone Activity: A World of Diverse Proteins with a Common Task—Impediment of RNA Misfolding

    PubMed Central

    Semrad, Katharina

    2011-01-01

    Proteins with RNA chaperone activity are ubiquitous proteins that play important roles in cellular mechanisms. They prevent RNA from misfolding by loosening misfolded structures without ATP consumption. RNA chaperone activity is studied in vitro and in vivo using oligonucleotide- or ribozyme-based assays. Due to their functional as well as structural diversity, a common chaperoning mechanism or universal motif has not yet been identified. A growing database of proteins with RNA chaperone activity has been established based on evaluation of chaperone activity via the described assays. Although the exact mechanism is not yet understood, it is more and more believed that disordered regions within proteins play an important role. This possible mechanism and which proteins were found to possess RNA chaperone activity are discussed here. PMID:21234377

  11. Myeloperoxidase-Oxidized LDLs Enhance an Anti-Inflammatory M2 and Antioxidant Phenotype in Murine Macrophages

    PubMed Central

    Sauvage, Aude; Van Steenbrugge, Martine

    2016-01-01

    Macrophages and oxidized LDLs play a key role in atherogenesis but their heterogeneity has been neglected up to now. Macrophages are prone to polarization and subsets of polarized macrophages have been described in atheromas. LDLs can be oxidized not only chemically by copper (Ox-LDLs) but also enzymatically by myeloperoxidase (MpOx-LDLs) resulting in oxidized LDLs poor in lipid peroxides. The effects of physiologically relevant myeloperoxidase-oxidized LDLs on macrophage polarization or on polarized macrophages remain largely unknown. In this study, the effects of LDLs on macrophage polarization were investigated by monitoring the expression of M1 and M2 genes following stimulation with native LDLs, Ox-LDLs, or MpOx-LDLs in RAW 264.7 cells. Except for MRC1, which is induced only by Ox-LDLs, MpOx-LDLs induced an overexpression of most of the selected marker genes at the mRNA level. MpOx-LDLs also modulate marker gene expression in polarized macrophages favoring notably anti-inflammatory Arg1 expression in M2 cells and also in the other phenotypes. Noteworthy, MpOx-LDLs were the most efficient to accumulate lipids intracellularly in (un)polarized macrophages whatever the phenotype. These data were largely confirmed in murine bone marrow-derived macrophages. Our data suggest that MpOx-LDLs were the most efficient to accumulate within cells and to enhance an anti-inflammatory and antioxidant phenotype in M2 cells and also in the other macrophage phenotypes. PMID:27656049

  12. AR-12 Inhibits Multiple Chaperones Concomitant With Stimulating Autophagosome Formation Collectively Preventing Virus Replication.

    PubMed

    Booth, Laurence; Roberts, Jane L; Ecroyd, Heath; Tritsch, Sarah R; Bavari, Sina; Reid, St Patrick; Proniuk, Stefan; Zukiwski, Alexander; Jacob, Abraham; Sepúlveda, Claudia S; Giovannoni, Federico; García, Cybele C; Damonte, Elsa; González-Gallego, Javier; Tuñón, María J; Dent, Paul

    2016-10-01

    We have recently demonstrated that AR-12 (OSU-03012) reduces the function and ATPase activities of multiple HSP90 and HSP70 family chaperones. Combined knock down of chaperones or AR-12 treatment acted to reduce the expression of virus receptors and essential glucosidase proteins. Combined knock down of chaperones or AR-12 treatment inactivated mTOR and elevated ATG13 S318 phosphorylation concomitant with inducing an endoplasmic reticulum stress response that in an eIF2α-dependent fashion increased Beclin1 and LC3 expression and autophagosome formation. Over-expression of chaperones prevented the reduction in receptor/glucosidase expression, mTOR inactivation, the ER stress response, and autophagosome formation. AR-12 reduced the reproduction of viruses including Mumps, Influenza, Measles, Junín, Rubella, HIV (wild type and protease resistant), and Ebola, an effect replicated by knock down of multiple chaperone proteins. AR-12-stimulated the co-localization of Influenza, EBV and HIV virus proteins with LC3 in autophagosomes and reduced viral protein association with the chaperones HSP90, HSP70, and GRP78. Knock down of Beclin1 suppressed drug-induced autophagosome formation and reduced the anti-viral protection afforded by AR-12. In an animal model of hemorrhagic fever virus, a transient exposure of animals to low doses of AR-12 doubled animal survival from ∼30% to ∼60% and suppressed liver damage as measured by ATL, GGT and LDH release. Thus through inhibition of chaperone protein functions; reducing the production, stability and processing of viral proteins; and stimulating autophagosome formation/viral protein degradation, AR-12 acts as a broad-specificity anti-viral drug in vitro and in vivo. We argue future patient studies with AR-12 are warranted. J. Cell. Physiol. 231: 2286-2302, 2016. © 2016 Wiley Periodicals, Inc.

  13. Platelet microparticles reprogram macrophage gene expression and function.

    PubMed

    Laffont, Benoit; Corduan, Aurélie; Rousseau, Matthieu; Duchez, Anne-Claire; Lee, Chan Ho C; Boilard, Eric; Provost, Patrick

    2016-01-01

    Platelet microparticles (MPs) represent the most abundant MPs subtype in the circulation, and can mediate intercellular communication through delivery of bioactives molecules, such as cytokines, proteins, lipids and RNAs. Here, we show that platelet MPs can be internalised by primary human macrophages and deliver functional miR-126-3p. The increase in macrophage miR-126-3p levels was not prevented by actinomycin D, suggesting that it was not due to de novo gene transcription. Platelet MPs dose-dependently downregulated expression of four predicted mRNA targets of miR-126-3p, two of which were confirmed also at the protein level. The mRNA downregulatory effects of platelet MPs were abrogated by expression of a neutralising miR-126-3p sponge, implying the involvement of miR-126-3p. Transcriptome-wide, microarray analyses revealed that as many as 66 microRNAs and 653 additional RNAs were significantly and differentially expressed in macrophages upon exposure to platelet MPs. More specifically, platelet MPs induced an upregulation of 34 microRNAs and a concomitant downregulation of 367 RNAs, including mRNAs encoding for cytokines/chemokines CCL4, CSF1 and TNF. These changes were associated with reduced CCL4, CSF1 and TNF cytokine/chemokine release by macrophages, and accompanied by a marked increase in their phagocytic capacity. These findings demonstrate that platelet MPs can modify the transcriptome of macrophages, and reprogram their function towards a phagocytic phenotype.

  14. Maresin conjugates in tissue regeneration biosynthesis enzymes in human macrophages

    PubMed Central

    Dalli, Jesmond; Vlasakov, Iliyan; Riley, Ian R.; Rodriguez, Ana R.; Spur, Bernd W.; Chiang, Nan; Serhan, Charles N.

    2016-01-01

    Macrophages are central in coordinating immune responses, tissue repair, and regeneration, with different subtypes being associated with inflammation-initiating and proresolving actions. We recently identified a family of macrophage-derived proresolving and tissue regenerative molecules coined maresin conjugates in tissue regeneration (MCTR). Herein, using lipid mediator profiling we identified MCTR in human serum, lymph nodes, and plasma and investigated MCTR biosynthetic pathways in human macrophages. With human recombinant enzymes, primary cells, and enantiomerically pure compounds we found that the synthetic maresin epoxide intermediate 13S,14S-eMaR (13S,14S-epoxy- 4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid) was converted to MCTR1 (13R-glutathionyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) by LTC4S and GSTM4. Incubation of human macrophages with LTC4S inhibitors blocked LTC4 and increased resolvins and lipoxins. The conversion of MCTR1 to MCTR2 (13R-cysteinylglycinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) was catalyzed by γ-glutamyl transferase (GGT) in human macrophages. Biosynthesis of MCTR3 was mediated by dipeptidases that cleaved the cysteinyl-glycinyl bond of MCTR2 to give 13R-cysteinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid. Of note, both GSTM4 and GGT enzymes displayed higher affinity to 13S,14S-eMaR and MCTR1 compared with their classic substrates in the cysteinyl leukotriene metabolome. Together these results establish the MCTR biosynthetic pathway and provide mechanisms in tissue repair and regeneration. PMID:27791009

  15. Rspo2 suppresses CD36-mediated apoptosis in oxidized low density lipoprotein-induced macrophages

    PubMed Central

    Yan, Hui; Wang, Shuai; Li, Zhenwei; Sun, Zewei; Zan, Jie; Zhao, Wenting; Pan, Yanyun; Wang, Zhen; Wu, Mingjie; Zhu, Jianhua

    2016-01-01

    Oxidized low density lipoprotein (oxLDL)-induced apoptosis of macrophages contributes to the formation of atherosclerotic plaques. R-spondin 2 (Rspo2), a member of the cysteine-rich secreted proteins, has been shown to be involved in the oncogenesis of several types of cancer. It has also been found to be abundantly expressed among the four R-spondin members in macrophages. The present study was performed to determine whether Rspo2 is involved in the ox-LDL-induced apoptosis of macrophages. It was identified that Rspo2 inhibited oxLDL-induced apoptosis in the presence of endoplasmic reticulum (ER) stress activator using flow cytometry. In addition, Rspo2 was observed to suppress oxLDL-induced ER stress and reactive oxygen species production as demonstrated by western blotting. Furthermore, analysis of the role of Rspo2 in macrophage lipid uptake identified that Rspo2 negatively regulated the Dil-oxLDL uptake by inhibiting the expression of cluster of differentiation (CD)36, through the transcription factor, peroxisome proliferator-activated receptor (PPAR)-γ. The manipulation of Rspo2 had a direct effect on PPAR-γ nuclear translocation. In addition, chromatin immunoprecipitation analysis revealed that Rspo2 manipulation led to regulation of the direct binding between PPAR-γ and CD36. In conclusion, Rspo2 was found to have a negative regulatory effect during oxLDL-induced macrophage apoptosis by regulating lipid uptake. PMID:27571704

  16. Increased inflammatory properties of adipose tissue macrophages recruited during diet-induced obesity.

    PubMed

    Lumeng, Carey N; Deyoung, Stephanie M; Bodzin, Jennifer L; Saltiel, Alan R

    2007-01-01

    Although recent studies show that adipose tissue macrophages (ATMs) participate in the inflammatory changes in obesity and contribute to insulin resistance, the properties of these cells are not well understood. We hypothesized that ATMs recruited to adipose tissue during a high-fat diet have unique inflammatory properties compared with resident tissue ATMs. Using a dye (PKH26) to pulse label ATMs in vivo, we purified macrophages recruited to white adipose tissue during a high-fat diet. Comparison of gene expression in recruited and resident ATMs using real-time RT-PCR and cDNA microarrays showed that recruited ATMs overexpress genes important in macrophage migration and phagocytosis, including interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and C-C chemokine receptor 2 (CCR2). Many of these genes were not induced in ATMs from high-fat diet-fed CCR2 knockout mice, supporting the importance of CCR2 in regulating recruitment of inflammatory ATMs during obesity. Additionally, expression of Apoe was decreased, whereas genes important in lipid metabolism, such as Pparg, Adfp, Srepf1, and Apob48r, were increased in the recruited macrophages. In agreement with this, ATMs from obese mice had increased lipid content compared with those from lean mice. These studies demonstrate that recruited ATMs in obese animals represent a subclass of macrophages with unique properties.

  17. Comparative Evaluation of Nephrotoxicity and Management by Macrophages of Intravenous Pharmaceutical Iron Formulations

    PubMed Central

    Connor, James R.; Zhang, Xuesheng; Nixon, Anne M.; Webb, Becky; Perno, Joseph R.

    2015-01-01

    Background There is a significant clinical need for effective treatment of iron deficiency. A number of compounds that can be administered intravenously have been developed. This study examines how the compounds are handled by macrophages and their relative potential to provoke oxidative stress. Methods Human kidney (HK-2) cells, rat peritoneal macrophages and renal cortical homogenates were exposed to pharmaceutical iron preparations. Analyses were performed for indices of oxidative stress and cell integrity. In addition, in macrophages, iron uptake and release and cytokine secretion was monitored. Results HK-2 cell viability was decreased by iron isomaltoside and ferumoxytol and all compounds induced lipid peroxidation. In the renal cortical homogenates, lipid peroxidation occurred at lowest concentrations with ferric carboxymaltose, iron dextran, iron sucrose and sodium ferric gluconate. In the macrophages, iron sucrose caused loss of cell viability. Iron uptake was highest for ferumoxytol and iron isomaltoside and lowest for iron sucrose and sodium ferric gluconate. Iron was released as secretion of ferritin or as ferrous iron via ferroportin. The latter was blocked by hepcidin. Exposure to ferric carboxymaltose and iron dextran resulted in release of tumor necrosis factor α. Conclusions Exposure to iron compounds increased cell stress but was tissue and dose dependent. There was a clear difference in the handling of iron from the different compounds by macrophages that suggests in vivo responses may differ. PMID:25973894

  18. Ribosomal crystallography: peptide bond formation, chaperone assistance and antibiotics activity.

    PubMed

    Yonath, Ada

    2005-08-31

    The peptidyl transferase center (PTC) is located in a protein free environment, thus confirming that the ribosome is a ribozyme. This arched void has dimensions suitable for accommodating the 3' ends of the A-and the P-site tRNAs, and is situated within a universal sizable symmetry-related region that connects all ribosomal functional centers involved in amino-acid polymerization. The linkage between the elaborate PTC architecture and the A-site tRNA position revealed that the A- to P-site passage of the tRNA 3' end is performed by a rotatory motion, which leads to stereochemistry suitable for peptide bond formation and for substrate mediated catalysis, thus suggesting that the PTC evolved by gene-fusion. Adjacent to the PTC is the entrance of the protein exit tunnel, shown to play active roles in sequence-specific gating of nascent chains and in responding to cellular signals. This tunnel also provides a site that may be exploited for local co-translational folding and seems to assist in nascent chain trafficking into the hydrophobic space formed by the first bacterial chaperone, the trigger factor. Many antibiotics target ribosomes. Although the ribosome is highly conserved, subtle sequence and/or conformational variations enable drug selectivity, thus facilitating clinical usage. Comparisons of high-resolution structures of complexes of antibiotics bound to ribosomes from eubacteria resembling pathogens, to an archaeon that shares properties with eukaryotes and to its mutant that allows antibiotics binding, demonstrated the unambiguous difference between mere binding and therapeutical effectiveness. The observed variability in antibiotics inhibitory modes, accompanied by the elucidation of the structural basis to antibiotics mechanism justifies expectations for structural based improved properties of existing compounds as well as for the development of novel drugs.

  19. The expanding proteome of the molecular chaperone HSP90

    PubMed Central

    Samant, Rahul S; Clarke, Paul A

    2012-01-01

    The molecular chaperone HSP90 maintains the activity and stability of a diverse set of “client” proteins that play key roles in normal and disease biology. Around 20 HSP90 inhibitors that deplete the oncogenic clientele have entered clinical trials for cancer. However, the full extent of the HSP90-dependent proteome, which encompasses not only clients but also proteins modulated by downstream transcriptional responses, is still incompletely characterized and poorly understood. Earlier large-scale efforts to define the HSP90 proteome have been valuable but are incomplete because of limited technical sensitivity. Here, we discuss previous large-scale surveys of proteome perturbations induced by HSP90 inhibitors in light of a significant new study using state-of-the-art stable isotope labeling by amino acids (SILAC) technology combined with more sensitive high-resolution mass spectrometry (MS) that extends the catalog of proteomic changes in inhibitor-treated cancer cells. Among wide-ranging changes, major functional responses include downregulation of protein kinase activity and the DNA damage response alongside upregulation of the protein degradation machinery. Despite this improved proteomic coverage, there was surprisingly little overlap with previous studies. This may be due in part to technical issues but is likely also due to the variability of the HSP90 proteome with the inhibitor conditions used, the cancer cell type and the genetic status of client proteins. We suggest future proteomic studies to address these factors, to help distinguish client protein components from indirect transcriptional components and to address other key questions in fundamental and translational HSP90 research. Such studies should also reveal new biomarkers for patient selection and novel targets for therapeutic intervention. PMID:22421145

  20. Trypanosoma cruzi Calreticulin Topographical Variations in Parasites Infecting Murine Macrophages.

    PubMed

    González, Andrea; Valck, Carolina; Sánchez, Gittith; Härtel, Steffen; Mansilla, Jorge; Ramírez, Galia; Fernández, María Soledad; Arias, José Luis; Galanti, Norbel; Ferreira, Arturo

    2015-05-01

    Trypanosoma cruzi calreticulin (TcCRT), a 47-kDa chaperone, translocates from the endoplasmic reticulum to the area of flagellum emergence. There, it binds to complement components C1 and mannan-binding lectin (MBL), thus acting as a main virulence factor, and inhibits the classical and lectin pathways. The localization and functions of TcCRT, once the parasite is inside the host cell, are unknown. In parasites infecting murine macrophages, polyclonal anti-TcCRT antibodies detected TcCRT mainly in the parasite nucleus and kinetoplast. However, with a monoclonal antibody (E2G7), the resolution and specificity of the label markedly improved, and TcCRT was detected mainly in the parasite kinetoplast. Gold particles, bound to the respective antibodies, were used as probes in electron microscopy. This organelle may represent a stopover and accumulation site for TcCRT, previous its translocation to the area of flagellum emergence. Finally, early during T. cruzi infection and by unknown mechanisms, an important decrease in the number of MHC-I positive host cells was observed.

  1. An integrated signal transduction network of macrophage migration inhibitory factor.

    PubMed

    Subbannayya, Tejaswini; Variar, Prathyaksha; Advani, Jayshree; Nair, Bipin; Shankar, Subramanian; Gowda, Harsha; Saussez, Sven; Chatterjee, Aditi; Prasad, T S Keshava

    2016-06-01

    Macrophage migration inhibitory factor (MIF) is a glycosylated multi-functional protein that acts as an enzyme as well as a cytokine. MIF mediates its actions through a cell surface class II major histocompatibility chaperone, CD74 and co-receptors such as CD44, CXCR2, CXCR4 or CXCR7. MIF has been implicated in the pathogenesis of several acute and chronic inflammatory diseases. Although MIF is a molecule of biomedical importance, a public resource of MIF signaling pathway is currently lacking. In view of this, we carried out detailed data mining and documentation of the signaling events pertaining to MIF from published literature and developed an integrated reaction map of MIF signaling. This resulted in the cataloguing of 68 molecules belonging to MIF signaling pathway, which includes 24 protein-protein interactions, 44 post-translational modifications, 11 protein translocation events and 8 activation/inhibition events. In addition, 65 gene regulation events at the mRNA levels induced by MIF signaling have also been catalogued. This signaling pathway has been integrated into NetPath ( http://www.netpath.org ), a freely available human signaling pathway resource developed previously by our group. The MIF pathway data is freely available online in various community standard data exchange formats. We expect that data on signaling events and a detailed signaling map of MIF will provide the scientific community with an improved platform to facilitate further molecular as well as biomedical investigations on MIF.

  2. Trypanosoma cruzi Calreticulin Topographical Variations in Parasites Infecting Murine Macrophages

    PubMed Central

    González, Andrea; Valck, Carolina; Sánchez, Gittith; Härtel, Steffen; Mansilla, Jorge; Ramírez, Galia; Fernández, María Soledad; Arias, José Luis; Galanti, Norbel; Ferreira, Arturo

    2015-01-01

    Trypanosoma cruzi calreticulin (TcCRT), a 47-kDa chaperone, translocates from the endoplasmic reticulum to the area of flagellum emergence. There, it binds to complement components C1 and mannan-binding lectin (MBL), thus acting as a main virulence factor, and inhibits the classical and lectin pathways. The localization and functions of TcCRT, once the parasite is inside the host cell, are unknown. In parasites infecting murine macrophages, polyclonal anti-TcCRT antibodies detected TcCRT mainly in the parasite nucleus and kinetoplast. However, with a monoclonal antibody (E2G7), the resolution and specificity of the label markedly improved, and TcCRT was detected mainly in the parasite kinetoplast. Gold particles, bound to the respective antibodies, were used as probes in electron microscopy. This organelle may represent a stopover and accumulation site for TcCRT, previous its translocation to the area of flagellum emergence. Finally, early during T. cruzi infection and by unknown mechanisms, an important decrease in the number of MHC-I positive host cells was observed. PMID:25758653

  3. Biologic activities of molecular chaperones and pharmacologic chaperone imidazole-containing dipeptide-based compounds: natural skin care help and the ultimate challenge: implication for adaptive responses in the skin.

    PubMed

    Babizhayev, Mark A; Nikolayev, Gennady M; Nikolayeva, Juliana G; Yegorov, Yegor E

    2012-03-01

    Accumulation of molecular damage and increased molecular heterogeneity are hallmarks of photoaged skin and pathogenesis of human cutaneous disease. Growing evidence demonstrates the ability of molecular chaperone proteins and of pharmacologic chaperones to decrease the environmental stress and ameliorate the oxidation stress-related and glycation disease phenotypes, suggesting that the field of chaperone therapy might hold novel treatments for skin diseases and aging. In this review, we examine the evidence suggesting a role for molecular chaperone proteins in the skin and their inducer and protecting agents: pharmacologic chaperone imidazole dipeptide-based agents (carcinine and related compounds) in cosmetics and dermatology. Furthermore, we discuss the use of chaperone therapy for the treatment of skin photoaging diseases and other skin pathologies that have a component of increased glycation and/or free radical-induced oxidation in their genesis. We examine biologic activities of molecular and pharmacologic chaperones, including strategies for identifying potential chaperone compounds and for experimentally demonstrating chaperone activity in in vitro and in vivo models of human skin disease. This allows the protein to function and traffic to the appropriate location in the skin, thereby increasing protein activity and cellular function and reducing stress on skin cells. The benefits of imidazole dipeptide antioxidants with transglycating activity (such as carcinine) in skin care are that they help protect and repair cell membrane damage and help retain youthful, younger-looking skin. All skin types will benefit from daily, topical application of pharmacologic chaperone antioxidants, anti-irritants, in combination with water-binding protein agents that work to mimic the structure and function of healthy skin. General strategies are presented addressing ground techniques to improve absorption of usually active chaperone proteins and dipeptide compounds, include

  4. Resolvins attenuate inflammation and promote resolution in cigarette smoke-exposed human macrophages.

    PubMed

    Croasdell, Amanda; Thatcher, Thomas H; Kottmann, R Matthew; Colas, Romain A; Dalli, Jesmond; Serhan, Charles N; Sime, Patricia J; Phipps, Richard P

    2015-10-15

    Inflammation is a protective response to injury, but it can become chronic, leading to tissue damage and disease. Cigarette smoke causes multiple inflammatory diseases, which account for thousands of deaths and cost billions of dollars annually. Cigarette smoke disrupts the function of immune cells, such as macrophages, by prolonging inflammatory signaling, promoting oxidative stress, and impairing phagocytosis, contributing to increased incidence of infections. Recently, new families of lipid-derived mediators, "specialized proresolving mediators" (SPMs), were identified. SPMs play a critical role in the active resolution of inflammation by counterregulating proinflammatory signaling and promoting resolution pathways. We have identified dysregulated concentrations of lipid mediators in exhaled breath condensate, bronchoalveolar lavage fluid, and serum from patients with chronic obstructive pulmonary disease (COPD). In human alveolar macrophages from COPD and non-COPD patients, D-series resolvins decreased inflammatory cytokines and enhanced phagocytosis. To further investigate the actions of resolvins on human cells, macrophages were differentiated from human blood monocytes and treated with D-series resolvins and then exposed to cigarette smoke extract. Resolvins significantly suppressed macrophage production of proinflammatory cytokines, enzymes, and lipid mediators. Resolvins also increased anti-inflammatory cytokines, promoted an M2 macrophage phenotype, and restored cigarette smoke-induced defects in phagocytosis, highlighting the proresolving functions of these molecules. These actions were receptor-dependent and involved modulation of canonical and noncanonical NF-κB expression, with the first evidence for SPM action on alternative NF-κB signaling. These data show that resolvins act on human macrophages to attenuate cigarette smoke-induced inflammatory effects through proresolving mechanisms and provide new evidence of the therapeutic potential of SPMs.

  5. Hsp72 chaperone function is dispensable for protection against stress-induced apoptosis.

    PubMed

    Chow, Ari M; Steel, Rohan; Anderson, Robin L

    2009-05-01

    In addition to its role as a molecular chaperone, heat shock protein 72 (Hsp72) protects cells against a wide range of apoptosis inducing stresses. However, it is unclear if these two roles are functionally related or whether Hsp72 inhibits apoptosis by a mechanism independent of chaperone activity. The N-terminal adenosine triphosphatase domain, substrate-binding domain and the C-terminal EEVD regulatory motif of Hsp72 are all essential for chaperone activity. In this study, we show that Hsp72 mutants with a functional substrate-binding domain but lacking chaperone activity retain their ability to protect cells against apoptosis induced by heat and tumor necrosis factor alpha. In contrast, a deletion mutant lacking a functional substrate-binding domain has no protective capacity. The ability of the Hsp72 substrate-binding domain to inhibit apoptosis independent of the regulatory effects of the adenosine triphosphate-binding domain indicates that the inhibition of apoptosis may involve a stable binding interaction with a regulatory substrate rather than Hsp72 chaperone activity.

  6. A Quantitative Characterization of Nucleoplasmin/Histone Complexes Reveals Chaperone Versatility

    PubMed Central

    Fernández-Rivero, Noelia; Franco, Aitor; Velázquez-Campoy, Adrian; Alonso, Edurne; Muga, Arturo; Prado, Adelina

    2016-01-01

    Nucleoplasmin (NP) is an abundant histone chaperone in vertebrate oocytes and embryos involved in storing and releasing maternal histones to establish and maintain the zygotic epigenome. NP has been considered a H2A–H2B histone chaperone, and recently it has been shown that it can also interact with H3-H4. However, its interaction with different types of histones has not been quantitatively studied so far. We show here that NP binds H2A–H2B, H3-H4 and linker histones with Kd values in the subnanomolar range, forming different complexes. Post-translational modifications of NP regulate exposure of the polyGlu tract at the disordered distal face of the protein and induce an increase in chaperone affinity for all histones. The relative affinity of NP for H2A–H2B and linker histones and the fact that they interact with the distal face of the chaperone could explain their competition for chaperone binding, a relevant process in NP-mediated sperm chromatin remodelling during fertilization. Our data show that NP binds H3-H4 tetramers in a nucleosomal conformation and dimers, transferring them to DNA to form disomes and tetrasomes. This finding might be relevant to elucidate the role of NP in chromatin disassembly and assembly during replication and transcription. PMID:27558753

  7. mTORC1 links protein quality and quantity control by sensing chaperone availability.

    PubMed

    Qian, Shu-Bing; Zhang, Xingqian; Sun, Jun; Bennink, Jack R; Yewdell, Jonathan W; Patterson, Cam

    2010-08-27

    Balanced protein synthesis and degradation are crucial for proper cellular function. Protein synthesis is tightly coupled to energy status and nutrient levels by the mammalian target of rapamycin complex 1 (mTORC1). Quality of newly synthesized polypeptides is maintained by the molecular chaperone and ubiquitin-proteasome systems. Little is known about how cells integrate information about the quantity and quality of translational products simultaneously. We demonstrate that cells distinguish moderate reductions in protein quality from severe protein misfolding using molecular chaperones to differentially regulate mTORC1 signaling. Moderate reduction of chaperone availability enhances mTORC1 signaling, whereas stress-induced complete depletion of chaperoning capacity suppresses mTORC1 signaling. Molecular chaperones regulate mTORC1 assembly in coordination with nutrient availability. This mechanism enables mTORC1 to rapidly detect and respond to environmental cues while also sensing intracellular protein misfolding. The tight linkage between protein quality and quantity control provides a plausible mechanism coupling protein misfolding with metabolic dyshomeostasis.

  8. Molecular Chaperones of Leishmania: Central Players in Many Stress-Related and -Unrelated Physiological Processes

    PubMed Central

    Requena, Jose M.; Montalvo, Ana M.; Fraga, Jorge

    2015-01-01

    Molecular chaperones are key components in the maintenance of cellular homeostasis and survival, not only during stress but also under optimal growth conditions. Folding of nascent polypeptides is supported by molecular chaperones, which avoid the formation of aggregates by preventing nonspecific interactions and aid, when necessary, the translocation of proteins to their correct intracellular localization. Furthermore, when proteins are damaged, molecular chaperones may also facilitate their refolding or, in the case of irreparable proteins, their removal by the protein degradation machinery of the cell. During their digenetic lifestyle, Leishmania parasites encounter and adapt to harsh environmental conditions, such as nutrient deficiency, hypoxia, oxidative stress, changing pH, and shifts in temperature; all these factors are potential triggers of cellular stress. We summarize here our current knowledge on the main types of molecular chaperones in Leishmania and their functions. Among them, heat shock proteins play important roles in adaptation and survival of this parasite against temperature changes associated with its passage from the poikilothermic insect vector to the warm-blooded vertebrate host. The study of structural features and the function of chaperones in Leishmania biology is providing opportunities (and challenges) for drug discovery and improving of current treatments against leishmaniasis. PMID:26167482

  9. Evolution of a plant-specific copper chaperone family for chloroplast copper homeostasis

    SciTech Connect

    Blaby-Haas, Crysten E.; Padilla-Benavides, Teresita; Stübe, Roland; Argüello, José M.; Merchant, Sabeeha S.

    2014-12-02

    Metallochaperones traffic copper (Cu+) from its point of entry at the plasma membrane to its destination. In plants, one destination is the chloroplast, which houses plastocyanin, a Cu-dependent electron transfer protein involved in photosynthesis. In this paper, we present a previously unidentified Cu+ chaperone that evolved early in the plant lineage by an alternative-splicing event of the pre-mRNA encoding the chloroplast P-type ATPase in Arabidopsis 1 (PAA1). In several land plants, recent duplication events created a separate chaperone-encoding gene coincident with loss of alternative splicing. The plant-specific Cu+ chaperone delivers Cu+ with specificity for PAA1, which is flipped in the envelope relative to prototypical bacterial ATPases, compatible with a role in Cu+ import into the stroma and consistent with the canonical catalytic mechanism of these enzymes. The ubiquity of the chaperone suggests conservation of this Cu+-delivery mechanism and provides a unique snapshot into the evolution of a Cu+ distribution pathway. Finally, we also provide evidence for an interaction between PAA2, the Cu+-ATPase in thylakoids, and the Cu+-chaperone for Cu/Zn superoxide dismutase (CCS), uncovering a Cu+ network that has evolved to fine-tune Cu+ distribution.

  10. Yeast prions are useful for studying protein chaperones and protein quality control.

    PubMed

    Masison, Daniel C; Reidy, Michael

    2015-01-01

    Protein chaperones help proteins adopt and maintain native conformations and play vital roles in cellular processes where proteins are partially folded. They comprise a major part of the cellular protein quality control system that protects the integrity of the proteome. Many disorders are caused when proteins misfold despite this protection. Yeast prions are fibrous amyloid aggregates of misfolded proteins. The normal action of chaperones on yeast prions breaks the fibers into pieces, which results in prion replication. Because this process is necessary for propagation of yeast prions, even small differences in activity of many chaperones noticeably affect prion phenotypes. Several other factors involved in protein processing also influence formation, propagation or elimination of prions in yeast. Thus, in much the same way that the dependency of viruses on cellular functions has allowed us to learn much about cell biology, the dependency of yeast prions on chaperones presents a unique and sensitive way to monitor the functions and interactions of many components of the cell's protein quality control system. Our recent work illustrates the utility of this system for identifying and defining chaperone machinery interactions.

  11. Evolution of a plant-specific copper chaperone family for chloroplast copper homeostasis

    DOE PAGES

    Blaby-Haas, Crysten E.; Padilla-Benavides, Teresita; Stübe, Roland; ...

    2014-12-02

    Metallochaperones traffic copper (Cu+) from its point of entry at the plasma membrane to its destination. In plants, one destination is the chloroplast, which houses plastocyanin, a Cu-dependent electron transfer protein involved in photosynthesis. In this paper, we present a previously unidentified Cu+ chaperone that evolved early in the plant lineage by an alternative-splicing event of the pre-mRNA encoding the chloroplast P-type ATPase in Arabidopsis 1 (PAA1). In several land plants, recent duplication events created a separate chaperone-encoding gene coincident with loss of alternative splicing. The plant-specific Cu+ chaperone delivers Cu+ with specificity for PAA1, which is flipped in themore » envelope relative to prototypical bacterial ATPases, compatible with a role in Cu+ import into the stroma and consistent with the canonical catalytic mechanism of these enzymes. The ubiquity of the chaperone suggests conservation of this Cu+-delivery mechanism and provides a unique snapshot into the evolution of a Cu+ distribution pathway. Finally, we also provide evidence for an interaction between PAA2, the Cu+-ATPase in thylakoids, and the Cu+-chaperone for Cu/Zn superoxide dismutase (CCS), uncovering a Cu+ network that has evolved to fine-tune Cu+ distribution.« less

  12. Calcyclin Binding Protein/Siah-1 Interacting Protein Is a Hsp90 Binding Chaperone

    PubMed Central

    Góral, Agnieszka; Bieganowski, Paweł; Prus, Wiktor; Krzemień-Ojak, Łucja; Kądziołka, Beata; Fabczak, Hanna; Filipek, Anna

    2016-01-01

    The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery. PMID:27249023

  13. Characteristics analysis of the luzA gene encoding chaperone from Photobacterium leiognathi related to bioluminescence.

    PubMed

    Lin, J W; Lin, B J; Chen, H Y; Weng, S F

    1998-03-27

    Nucleotide sequence of the luzA gene (GenBank accession No. AF039303) from Photobacterium leiognathi ATCC 25521 (NCIMB 2193) has been determined, and the chaperone encoded by the luzA gene was deduced. The LuzA chaperone has a calculated M(r) 26,295 and comprises 230 amino acid residues; the hydrophobic alpha-helix N-terminal 21 amino acid residues MKKTIFALLFMSVFI SYPSFA is the leader peptide, therefore the matured LuzA chaperone has a calculated M(r) 23,871 and comprises 209 amino acid residues only. The periplasmic LuzA chaperone is the protein concerned with the protein folding, assembly and stability. The luzA gene and the related genes are closely linked to the sod gene, that encoding Cu/Zn superoxide dismutase enables to enhance bioluminescence of the lux operon; the gene order of the luzA gene and related genes is -ufo'-luzA-ufoI-ufoII-ter->-R&R'-sod-ufo-- >. In trans complementation bioluminoassays in vivo elicit that the LuzA chaperone might be not directly concerned with bioluminescence of the lux operon from P. leiognathi in E. coli, but might enable to stabilize the proteins related to bioluminescence. The unidentified ufoII gene closely linked to the luzA gene is able to enhance bioluminescence.

  14. Is Catalytic Activity of Chaperones a Selectable Trait for the Emergence of Heat Shock Response?

    PubMed Central

    Çetinbaş, Murat; Shakhnovich, Eugene I.

    2015-01-01

    Although heat shock response is ubiquitous in bacterial cells, the underlying physical chemistry behind heat shock response remains poorly understood. To study the response of cell populations to heat shock we employ a physics-based ab initio model of living cells where protein biophysics (i.e., folding and protein-protein interactions in crowded cellular environments) and important aspects of proteins homeostasis are coupled with realistic population dynamics simulations. By postulating a genotype-phenotype relationship we define a cell division rate in terms of functional concentrations of proteins and protein complexes, whose Boltzmann stabilities of folding and strengths of their functional interactions are exactly evaluated from their sequence information. We compare and contrast evolutionary dynamics for two models of chaperon action. In the active model, foldase chaperones function as nonequilibrium machines to accelerate the rate of protein folding. In the passive model, holdase chaperones form reversible complexes with proteins in their misfolded conformations to maintain their solubility. We find that only cells expressing foldase chaperones are capable of genuine heat shock response to the increase in the amount of unfolded proteins at elevated temperatures. In response to heat shock, cells’ limited resources are redistributed differently for active and passive models. For the active model, foldase chaperones are overexpressed at the expense of downregulation of high abundance proteins, whereas for the passive model; cells react to heat shock by downregulating their high abundance proteins, as their low abundance proteins are upregulated. PMID:25606691

  15. Molecular Chaperones of Leishmania: Central Players in Many Stress-Related and -Unrelated Physiological Processes.

    PubMed

    Requena, Jose M; Montalvo, Ana M; Fraga, Jorge

    2015-01-01

    Molecular chaperones are key components in the maintenance of cellular homeostasis and survival, not only during stress but also under optimal growth conditions. Folding of nascent polypeptides is supported by molecular chaperones, which avoid the formation of aggregates by preventing nonspecific interactions and aid, when necessary, the translocation of proteins to their correct intracellular localization. Furthermore, when proteins are damaged, molecular chaperones may also facilitate their refolding or, in the case of irreparable proteins, their removal by the protein degradation machinery of the cell. During their digenetic lifestyle, Leishmania parasites encounter and adapt to harsh environmental conditions, such as nutrient deficiency, hypoxia, oxidative stress, changing pH, and shifts in temperature; all these factors are potential triggers of cellular stress. We summarize here our current knowledge on the main types of molecular chaperones in Leishmania and their functions. Among them, heat shock proteins play important roles in adaptation and survival of this parasite against temperature changes associated with its passage from the poikilothermic insect vector to the warm-blooded vertebrate host. The study of structural features and the function of chaperones in Leishmania biology is providing opportunities (and challenges) for drug discovery and improving of current treatments against leishmaniasis.

  16. Molecular chaperones in lactic acid bacteria: physiological consequences and biochemical properties.

    PubMed

    Sugimoto, Shinya; Abdullah-Al-Mahin; Sonomoto, Kenji

    2008-10-01

    Recently, lactic acid bacteria (LAB) have attracted much attention because of their potential application to probiotics and industrial applications as starters for dairy products or lactic acid fermentation. Additional emphasis is also being paid to them as commensal bacteria in gastrointestinal tract. Since LAB exhibit a stress response, insight into the relationship between stress proteins such as molecular chaperones and stress tolerance or adaptation is increasing gradually along with current research examining these important bacteria. Similar to other bacteria, one of the major stress-response systems in LAB is the expression of molecular chaperones. The recently completed genome sequencing of various LAB strains, combined with the development of advanced molecular techniques, have enabled us to identify molecular chaperones and to understand their regulation systems in response to various stresses. Furthermore, recent biochemical studies provided novel insight into the molecular mechanisms of LAB chaperone systems. This review highlights the physiological consequences and biochemical properties of molecular chaperones (especially sHsps, Hsp70, and Hsp100) in LAB and their use in biotechnological applications.

  17. Recognition and targeting mechanisms by chaperones in flagellum assembly and operation.

    PubMed

    Khanra, Nandish; Rossi, Paolo; Economou, Anastassios; Kalodimos, Charalampos G

    2016-08-30

    The flagellum is a complex bacterial nanomachine that requires the proper assembly of several different proteins for its function. Dedicated chaperones are central in preventing aggregation or undesired interactions of flagellar proteins, including their targeting to the export gate. FliT is a key flagellar chaperone that binds to several flagellar proteins in the cytoplasm, including its cognate filament-capping protein FliD. We have determined the solution structure of the FliT chaperone in the free state and in complex with FliD and the flagellar ATPase FliI. FliT adopts a four-helix bundle and uses a hydrophobic surface formed by the first three helices to recognize its substrate proteins. We show that the fourth helix constitutes the binding site for FlhA, a membrane protein at the export gate. In the absence of a substrate protein FliT adopts an autoinhibited structure wherein both the binding sites for substrates and FlhA are occluded. Substrate binding to FliT activates the complex for FlhA binding and thus targeting of the chaperone-substrate complex to the export gate. The activation and targeting mechanisms reported for FliT appear to be shared among the other flagellar chaperones.

  18. Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin.

    PubMed

    Marchenko, N Yu; Sikorskaya, E V; Marchenkov, V V; Kashparov, I A; Semisotnov, G V

    2016-03-01

    Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme.

  19. Effect of hesperetin on chaperone activity in selenite-induced cataract

    PubMed Central

    Oka, Mikako; Tamura, Hiroomi; Takehana, Makoto

    2016-01-01

    Abstract Background. Chaperone activity of α-crystallin in the lens works to prevent protein aggregation and is important to maintain the lens transparency. This study evaluated the effect of hesperetin on lens chaperone activity in selenite-induced cataracts. Methodology. Thirteen-day-old rats were divided into four groups. Animals were given hesperetin (groups G2 and G4) or vehicle (G1 and G3) on Days 0, 1, and 2. Rats in G3 and G4 were administered selenite subcutaneously 4 hours after the first hesperetin injection. On Days 2, 4, and 6, cataract grades were evaluated using slit-lamp biomicroscopy. The amount of a-crystallin and chaperone activity in water-soluble fraction were measured after animals sacrificed. Results. G3 on day 4 had developed significant cataract, as an average cataract grading of 4.6 ± 0.2. In contrast, G4 had less severe central opacities and lower stage cataracts than G3, as an average cataract grading of 2.4 ± 0.4. The a-crystallin levels in G3 lenses were lower than in G1, but the same as G4. Additionally, chaperone activity was weaker in G3 lenses than G1, but the same as in G4. Conclusions. Our results suggest that hesperetin can prevent the decreasing lens chaperone activity and a-crystallin water solubility by administered of selenite. PMID:28352791

  20. Functional adaptations of the bacterial chaperone trigger factor to extreme environmental temperatures.

    PubMed

    Godin-Roulling, Amandine; Schmidpeter, Philipp A M; Schmid, Franz X; Feller, Georges

    2015-07-01

    Trigger factor (TF) is the first molecular chaperone interacting cotranslationally with virtually all nascent polypeptides synthesized by the ribosome in bacteria. Thermal adaptation of chaperone function was investigated in TFs from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima. This series covers nearly all temperatures encountered by bacteria. Although structurally homologous, these TFs display strikingly distinct properties that are related to the bacterial environmental temperature. The hyperthermophilic TF strongly binds model proteins during their folding and protects them from heat-induced misfolding and aggregation. It decreases the folding rate and counteracts the fast folding rate imposed by high temperature. It also functions as a carrier of partially folded proteins for delivery to downstream chaperones ensuring final maturation. By contrast, the psychrophilic TF displays weak chaperone activities, showing that these functions are less important in cold conditions because protein folding, misfolding and aggregation are slowed down at low temperature. It efficiently catalyses prolyl isomerization at low temperature as a result of its increased cellular concentration rather than from an improved activity. Some chaperone properties of the mesophilic TF possibly reflect its function as a cold shock protein in E. coli.

  1. Hsp72 chaperone function is dispensable for protection against stress-induced apoptosis

    PubMed Central

    Chow, Ari M.; Steel, Rohan

    2008-01-01

    In addition to its role as a molecular chaperone, heat shock protein 72 (Hsp72) protects cells against a wide range of apoptosis inducing stresses. However, it is unclear if these two roles are functionally related or whether Hsp72 inhibits apoptosis by a mechanism independent of chaperone activity. The N-terminal adenosine triphosphatase domain, substrate-binding domain and the C-terminal EEVD regulatory motif of Hsp72 are all essential for chaperone activity. In this study, we show that Hsp72 mutants with a functional substrate-binding domain but lacking chaperone activity retain their ability to protect cells against apoptosis induced by heat and tumor necrosis factor alpha. In contrast, a deletion mutant lacking a functional substrate-binding domain has no protective capacity. The ability of the Hsp72 substrate-binding domain to inhibit apoptosis independent of the regulatory effects of the adenosine triphosphate-binding domain indicates that the inhibition of apoptosis may involve a stable binding interaction with a regulatory substrate rather than Hsp72 chaperone activity. PMID:18819021

  2. Engineering and Evolution of Molecular Chaperones and Protein Disaggregases with Enhanced Activity

    PubMed Central

    Mack, Korrie L.; Shorter, James

    2016-01-01

    Cells have evolved a sophisticated proteostasis network to ensure that proteins acquire and retain their native structure and function. Critical components of this network include molecular chaperones and protein disaggregases, which function to prevent and reverse deleterious protein misfolding. Nevertheless, proteostasis networks have limits, which when exceeded can have fatal consequences as in various neurodegenerative disorders, including Parkinson's disease and amyotrophic lateral sclerosis. A promising strategy is to engineer proteostasis networks to counter challenges presented by specific diseases or specific proteins. Here, we review efforts to enhance the activity of individual molecular chaperones or protein disaggregases via engineering and directed evolution. Remarkably, enhanced global activity or altered substrate specificity of various molecular chaperones, including GroEL, Hsp70, ClpX, and Spy, can be achieved by minor changes in primary sequence and often a single missense mutation. Likewise, small changes in the primary sequence of Hsp104 yield potentiated protein disaggregases that reverse the aggregation and buffer toxicity of various neurodegenerative disease proteins, including α-synuclein, TDP-43, and FUS. Collectively, these advances have revealed key mechanistic and functional insights into chaperone and disaggregase biology. They also suggest that enhanced chaperones and disaggregases could have important applications in treating human disease as well as in the purification of valuable proteins in the pharmaceutical sector. PMID:27014702

  3. Is catalytic activity of chaperones a selectable trait for the emergence of heat shock response?

    PubMed

    Çetinbaş, Murat; Shakhnovich, Eugene I

    2015-01-20

    Although heat shock response is ubiquitous in bacterial cells, the underlying physical chemistry behind heat shock response remains poorly understood. To study the response of cell populations to heat shock we employ a physics-based ab initio model of living cells where protein biophysics (i.e., folding and protein-protein interactions in crowded cellular environments) and important aspects of proteins homeostasis are coupled with realistic population dynamics simulations. By postulating a genotype-phenotype relationship we define a cell division rate in terms of functional concentrations of proteins and protein complexes, whose Boltzmann stabilities of folding and strengths of their functional interactions are exactly evaluated from their sequence information. We compare and contrast evolutionary dynamics for two models of chaperon action. In the active model, foldase chaperones function as nonequilibrium machines to accelerate the rate of protein folding. In the passive model, holdase chaperones form reversible complexes with proteins in their misfolded conformations to maintain their solubility. We find that only cells expressing foldase chaperones are capable of genuine heat shock response to the increase in the amount of unfolded proteins at elevated temperatures. In response to heat shock, cells' limited resources are redistributed differently for active and passive models. For the active model, foldase chaperones are overexpressed at the expense of downregulation of high abundance proteins, whereas for the passive model; cells react to heat shock by downregulating their high abundance proteins, as their low abundance proteins are upregulated.

  4. Interferon-β promotes macrophage foam cell formation by altering both cholesterol influx and efflux mechanisms.

    PubMed

    Boshuizen, Marieke C S; Hoeksema, Marten A; Neele, Annette E; van der Velden, Saskia; Hamers, Anouk A J; Van den Bossche, Jan; Lutgens, Esther; de Winther, Menno P J

    2016-01-01

    Foam cell formation is a crucial event in atherogenesis. While interferon-β (IFNβ) is known to promote atherosclerosis in mice, studies on the role of IFNβ on foam cell formation are minimal and conflicting. We therefore extended these studies using both in vitro and in vivo approaches and examined IFNβ's function in macrophage foam cell formation. To do so, murine bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages were loaded with acLDL overnight, followed by 6h IFNβ co-treatment. This increased lipid content as measured by Oil red O staining. We next analyzed the lipid uptake pathways of IFNβ-stimulated BMDMs and observed increased endocytosis of DiI-acLDL as compared to controls. These effects were mediated via SR-A, as its gene expression was increased and inhibition of SR-A with Poly(I) blocked the IFNβ-induced increase in Oil red O staining and DiI-acLDL endocytosis. The IFNβ-induced increase in lipid content was also associated with decreased ApoA1-mediated cholesterol efflux, in response to decreased ABCA1 protein and gene expression. To validate our findings in vivo, LDLR(-/-) mice were put on chow or a high cholesterol diet for 10weeks. 24 and 8h before sacrifice mice were injected with IFNβ or PBS, after which thioglycollate-elicited peritoneal macrophages were collected and analyzed. In accordance with the in vitro data, IFNβ increased lipid accumulation. In conclusion, our experimental data support the pro-atherogenic role of IFNβ, as we show that IFNβ promotes macrophage foam cell formation by increasing SR-A-mediated cholesterol influx and decreasing ABCA1-mediated efflux mechanisms.

  5. Nicotine potentiates proatherogenic effects of oxLDL by stimulating and upregulating macrophage CD36 signaling.

    PubMed

    Zhou, Ming-Sheng; Chadipiralla, Kiranmai; Mendez, Armando J; Jaimes, Edgar A; Silverstein, Roy L; Webster, Keith; Raij, Leopoldo

    2013-08-15

    Cigarette smoking is a major risk factor for atherosclerosis and cardiovascular disease. CD36 mediates oxidized LDL (oxLDL) uptake and contributes to macrophage foam cell formation. We investigated a role for the CD36 pathway in nicotine-induced activation of macrophages and foam cell formation in vitro and in vivo. Nicotine in the same plasma concentration range found in smokers increased the CD36(+)/CD14(+) cell population in human peripheral blood mononuclear cells, increased CD36 expression of human THP1 macrophages, and increased macrophage production of reactive oxygen species, PKCδ phosphorylation, and peroxisome proliferator-activated receptor-γ (PPARγ) expression. Nicotine-induced CD36 expression was suppressed by antioxidants and by specific PKCδ and PPARγ inhibitors, implicating mechanistic roles for these intermediates. Nicotine synergized with oxLDL to increase macrophage expression of CD36 and cytokines TNF-α, monocyte chemoattractant protein-1, IL-6, and CXCL9, all of which were prevented by CD36 small interfering (si)RNA. Incubation with oxLDL (50 μg/ml) for 72 h resulted in lipid deposition in macrophages and foam cell formation. Preincubation with nicotine further increased oxLDL-induced lipid accumulation and foam cell formation, which was also prevented by CD36 siRNA. Treatment of apoE-/- mice with nicotine markedly exacerbated inflammatory monocyte levels and atherosclerotic plaque accumulation, effects that were not seen in CD36-/- apoE-/- mice. Our results show that physiological levels of nicotine increase CD36 expression in macrophages, a pathway that may account at least in part for the known proinflammatory and proatherogenic properties of nicotine. These results identify such enhanced CD36 expression as a novel nicotine-mediated pathway that may constitute an independent risk factor for atherosclerosis in smokers. The results also suggest that exacerbated atherogenesis by this pathway may be an adverse side effect of extended use

  6. Polyoxygenated Cholesterol Ester Hydroperoxide Activates TLR4 and SYK Dependent Signaling in Macrophages

    PubMed Central

    Choi, Soo-Ho; Yin, Huiyong; Ravandi, Amir; Armando, Aaron; Dumlao, Darren; Kim, Jungsu; Almazan, Felicidad; Taylor, Angela M.; McNamara, Coleen A.; Tsimikas, Sotirios; Dennis, Edward A.; Witztum, Joseph L.; Miller, Yury I.

    2013-01-01

    Oxidation of low-density lipoprotein (LDL) is one of the major causative mechanisms in the development of atherosclerosis. In previous studies, we showed that minimally oxidized LDL (mmLDL) induced inflammatory responses in macrophages, macropinocytosis and intracellular lipid accumulation and that oxidized cholesterol esters (OxCEs) were biologically active components of mmLDL. Here we identified a specific OxCE molecule responsible for the biological activity of mmLDL and characterized signaling pathways in macrophages in response to this OxCE. Using liquid chromatography – tandem mass spectrometry and biological assays, we identified an oxidized cholesteryl arachidonate with bicyclic endoperoxide and hydroperoxide groups (BEP-CE) as a specific OxCE that activates macrophages in a TLR4/MD-2-dependent manner. BEP-CE induced TLR4/MD-2 binding and TLR4 dimerization, phosphorylation of SYK, ERK1/2, JNK and c-Jun, cell spreading and uptake of dextran and native LDL by macrophages. The enhanced macropinocytosis resulted in intracellular lipid accumulation and macrophage foam cell formation. Bone marrow-derived macrophages isolated from TLR4 and SYK knockout mice did not respond to BEP-CE. The presence of BEP-CE was demonstrated in human plasma and in the human plaque material captured in distal protection devices during percutaneous intervention. Our results suggest that BEP-CE is an endogenous ligand that activates the TLR4/SYK signaling pathway. Because BEP-CE is present in human plasma and human atherosclerotic lesions, BEP-CE-induced and TLR4/SYK-mediated macrophage responses may contribute to chronic inflammation in human atherosclerosis. PMID:24376657

  7. Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

    NASA Technical Reports Server (NTRS)

    Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

    2003-01-01

    Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

  8. Induction of heme oxygenase-1 with dietary quercetin reduces obesity-induced hepatic inflammation through macrophage phenotype switching

    PubMed Central

    Kim, Chu-Sook; Choi, Hye-Seon; Joe, Yeonsoo; Chung, Hun Taeg

    2016-01-01

    BACKGROUND/OBJECTIVES Obesity-induced steatohepatitis accompanied by activated hepatic macrophages/Kupffer cells facilitates the progression of hepatic fibrinogenesis and exacerbates metabolic derangements such as insulin resistance. Heme oxyganase-1 (HO-1) modulates tissue macrophage phenotypes and thus is implicated in protection against inflammatory diseases. Here, we show that the flavonoid quercetin reduces obesity-induced hepatic inflammation by inducing HO-1, which promotes hepatic macrophage polarization in favor of the M2 phenotype. MATERIALS/METHODS Male C57BL/6 mice were fed a regular diet (RD), high-fat diet (HFD), or HFD supplemented with quercetin (HF+Que, 0.5g/kg diet) for nine weeks. Inflammatory cytokines and macrophage markers were measured by ELISA and RT-PCR, respectively. HO-1 protein was measured by Western blotting. RESULTS Quercetin supplementation decreased levels of inflammatory cytokines (TNFα, IL-6) and increased that of the anti-inflammatory cytokine (IL-10) in the livers of HFD-fed mice. This was accompanied by upregulation of M2 macrophage marker genes (Arg-1, Mrc1) and downregulation of M1 macrophage marker genes (TNFα, NOS2). In co-cultures of lipid-laden hepatocytes and macrophages, treatment with quercetin induced HO-1 in the macrophages, markedly suppressed expression of M1 macrophage marker genes, and reduced release of MCP-1. Moreover, these effects of quercetin were blunted by an HO-1 inhibitor and deficiency of nuclear factor E2-related factor 2 (Nrf2) in macrophages. CONCLUSIONS Quercetin reduces obesity-induced hepatic inflammation by promoting macrophage phenotype switching. The beneficial effect of quercetin is associated with Nrf2-mediated HO-1 induction. Quercetin may be a useful dietary factor for protecting against obesity-induced steatohepatitis. PMID:27909560

  9. Foamy macrophages from tuberculous patients' granulomas constitute a nutrient-rich reservoir for M. tuberculosis persistence.

    PubMed

    Peyron, Pascale; Vaubourgeix, Julien; Poquet, Yannick; Levillain, Florence; Botanch, Catherine; Bardou, Fabienne; Daffé, Mamadou; Emile, Jean-François; Marchou, Bruno; Cardona, Pere-Joan; de Chastellier, Chantal; Altare, Frédéric

    2008-11-01

    Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis-infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria.

  10. Foamy Macrophages from Tuberculous Patients' Granulomas Constitute a Nutrient-Rich Reservoir for M. tuberculosis Persistence

    PubMed Central

    Poquet, Yannick; Levillain, Florence; Botanch, Catherine; Bardou, Fabienne; Daffé, Mamadou; Emile, Jean-François; Marchou, Bruno; Cardona, Pere-Joan; de Chastellier, Chantal; Altare, Frédéric

    2008-01-01

    Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis–infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria. PMID:19002241

  11. Macrophage Heterogeneity in Respiratory Diseases

    PubMed Central

    Boorsma, Carian E.; Draijer, Christina; Melgert, Barbro N.

    2013-01-01

    Macrophages are among the most abundant cells in the respiratory tract, and they can have strikingly different phenotypes within this environment. Our knowledge of the different phenotypes and their functions in the lung is sketchy at best, but they appear to be linked to the protection of gas exchange against microbial threats and excessive tissue responses. Phenotypical changes of macrophages within the lung are found in many respiratory diseases including asthma, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis. This paper will give an overview of what macrophage phenotypes have been described, what their known functions are, what is known about their presence in the different obstructive and restrictive respiratory diseases (asthma, COPD, pulmonary fibrosis), and how they are thought to contribute to the etiology and resolution of these diseases. PMID:23533311

  12. Restored mutant receptor:Corticoid binding in chaperone complexes by trimethylamine N-oxide

    PubMed Central

    Miller, Aaron L.; Elam, W. Austin; Johnson, Betty H.; Khan, Shagufta H.; Kumar, Raj; Thompson, E. Brad

    2017-01-01

    Without a glucocorticoid (GC) ligand, the transcription factor glucocorticoid receptor (GR) is largely cytoplasmic, with its GC-binding domain held in high affinity conformation by a cluster of chaperones. Binding a GC causes serial dis- and re-associations with chaperones, translocation of the GR to the nucleus, where it binds to DNA sites and associates with coregulatory proteins and basic transcription complexes. Herein, we describe the effects of a potent protective osmolyte, trimethylamine N-oxide (TMAO), on a conditions-dependent “activation-labile” mutant GR (GRact/l), which under GR-activating conditions cannot bind GCs in cells or in cell cytosols. In both cells and cytosols, TMAO restores binding to GRact/l by stabilizing it in complex with chaperones. Cells bathed in much lower concentrations of TMAO than those required in vitro show restoration of GC binding, presumably due to intracellular molecular crowding effects. PMID:28301576

  13. Structural insights on two hypothetical secretion chaperones from Xanthomonas axonopodis pv. citri.

    PubMed

    Fattori, Juliana; Prando, Alessandra; Assis, Leandro H P; Aparicio, Ricardo; Tasic, Ljubica

    2011-06-01

    Several Gram-negative bacterial pathogens have developed type III secretion systems (T3SSs) to deliver virulence proteins directly into eukaryotic cells in a process essential for many diseases. The type III secretion processes require customized chaperones with high specificity for binding partners, thus providing the secretion to occur. Due to the very low sequence similarities among secretion chaperones, annotation and discrimination of a great majority of them is extremely difficult and a task with low scores even if genes are encountered that codify for small (<20 kDa) proteins with low pI and a tendency to dimerise. Concerning about this, herein, we present structural features on two hypothetical T3SSs chaperones belonging to plant pathogen Xanthomonas axonopodis pv. citri and suggest how low resolution models based on Small Angle X-ray Scattering patterns can provide new structural insights that could be very helpful in their analysis and posterior classification.

  14. Chaperone Proteins in the Central Nervous System and Peripheral Nervous System after Nerve Injury.

    PubMed

    Ousman, Shalina S; Frederick, Ariana; Lim, Erin-Mai F

    2017-01-01

    Injury to axons of the central nervous system (CNS) and the peripheral nervous system (PNS) is accompanied by the upregulation and downregulation of numerous molecules that are involved in mediating nerve repair, or in augmentation of the original damage. Promoting the functions of beneficial factors while reducing the properties of injurious agents determines whether regeneration and functional recovery ensues. A number of chaperone proteins display reduced or increased expression following CNS and PNS damage (crush, transection, contusion) where their roles have generally been found to be protective. For example, chaperones are involved in mediating survival of damaged neurons, promoting axon regeneration and remyelination and, improving behavioral outcomes. We review here the various chaperone proteins that are involved after nervous system axonal damage, the functions that they impact in the CNS and PNS, and the possible mechanisms by which they act.

  15. Crystal structure of archaeal homolog of proteasome-assembly chaperone PbaA.

    PubMed

    Sikdar, Arunima; Satoh, Tadashi; Kawasaki, Masato; Kato, Koichi

    2014-10-24

    Formation of the eukaryotic proteasome is not a spontaneous process but a highly ordered process assisted by several assembly chaperones. In contrast, archaeal proteasome subunits can spontaneously assemble into an active form. Recent bioinformatic analysis identified the proteasome-assembly chaperone-like proteins, PbaA and PbaB, in archaea. Our previous study showed that the PbaB homotetramer functions as a proteasome activator through its tentacle-like C-terminal segments. However, a functional role of the other homolog PbaA has remained elusive. Here we determined the 2.25-Å resolution structure of PbaA, illustrating its disparate tertiary and quaternary structures compared with PbaB. PbaA forms a homopentamer in which the C-terminal segments, with a putative proteasome-activating motif, are packed against the core. These findings offer deeper insights into the molecular evolution relationships between the proteasome-assembly chaperones and the proteasome activators.

  16. Structural Basis for Protein anti-Aggregation Activity of the Trigger Factor Chaperone*

    PubMed Central

    Saio, Tomohide; Guan, Xiao; Rossi, Paolo; Economou, Anastassios; Kalodimos, Charalampos G.

    2014-01-01

    Molecular chaperones prevent aggregation and misfolding of proteins but scarcity of structural data has impeded an understanding of the recognition and anti-aggregation mechanisms. Here we report the solution structure, dynamics and energetics of three Trigger Factor (TF) chaperone molecules in complex with alkaline phosphatase (PhoA) captured in the unfolded state. Our data show that TF uses multiple sites to bind to several regions of the PhoA substrate protein primarily through hydrophobic contacts. NMR relaxation experiments show that TF interacts with PhoA in a highly dynamic fashion but as the number and length of the PhoA regions engaged by TF increases, a more stable complex gradually emerges. Multivalent binding keeps the substrate protein in an extended, unfolded conformation. The results show how molecular chaperones recognize unfolded polypeptides and how by acting as unfoldases and holdases prevent the aggregation and premature (mis)folding of unfolded proteins. PMID:24812405

  17. Chaperone Proteins in the Central Nervous System and Peripheral Nervous System after Nerve Injury

    PubMed Central

    Ousman, Shalina S.; Frederick, Ariana; Lim, Erin-Mai F.

    2017-01-01

    Injury to axons of the central nervous system (CNS) and the peripheral nervous system (PNS) is accompanied by the upregulation and downregulation of numerous molecules that are involved in mediating nerve repair, or in augmentation of the original damage. Promoting the functions of beneficial factors while reducing the properties of injurious agents determines whether regeneration and functional recovery ensues. A number of chaperone proteins display reduced or increased expression following CNS and PNS damage (crush, transection, contusion) where their roles have generally been found to be protective. For example, chaperones are involved in mediating survival of damaged neurons, promoting axon regeneration and remyelination and, improving behavioral outcomes. We review here the various chaperone proteins that are involved after nervous system axonal damage, the functions that they impact in the CNS and PNS, and the possible mechanisms by which they act. PMID:28270745

  18. Inhibition of autophagy ameliorates atherogenic inflammation by augmenting apigenin-induced macrophage apoptosis.

    PubMed

    Wang, Qun; Zeng, Ping; Liu, Yuanliang; Wen, Ge; Fu, Xiuqiong; Sun, Xuegang

    2015-07-01

    Increasing evidences showed that the survival of macrophages promotes atherogenesis. Macrophage apoptosis in the early phase of atherosclerotic process negatively regulates the progression of atherosclerotic lesions. We demonstrated that a natural anti-oxidant apigenin could ameliorate atherogenesis in ApoE(-/-) mice. It reduced the number of foam cells and decreased the serum levels of tumor necrosis factor α, interleukin 1β (IL-1β) and IL-6. Our results showed that oxidized low-density lipoprotein (oxLDL) led to the secretion of pro-inflammatory cytokines. Apigenin-induced apoptosis and downregulated the secretion of TNF-α, IL-6 and IL-1β. It is further supported by the use of zVAD, a pan-caspase inhibitor, demonstrating that apigenin lowered cytokine profile through induction of macrophage apoptosis. Moreover, apigenin-induced Atg5/Atg7-dependent autophagy in macrophages pretreated with oxLDL. Results illustrated that autophagy inhibition increased apigenin-induced apoptosis through activation of Bax. The present findings suggest that oxLDL maintained the survival of macrophages and activated the secretion of pro-inflammatory cytokines to initiate atherosclerosis. Apigenin-induced apoptosis of lipid-laden macrophages and resolved inflammation to ameliorate atherosclerosis. In conclusion, combination of apigenin with autophagy inhibition may be a promising strategy to induce foam cell apoptosis and subdue atherogenic cytokines.

  19. Identification of atypical ether-linked glycerophospholipid species in macrophages by mass spectrometry

    PubMed Central

    Ivanova, Pavlina T.; Milne, Stephen B.; Brown, H. Alex

    2010-01-01

    A large scale profiling and analysis of glycerophospholipid species in macrophages has facilitated the identification of several rare and atypical glycerophospholipid species. By using liquid chromatography tandem mass spectrometry and comparison of the elution and fragmentation properties of the rare lipids to synthetic standards, we were able to identify an array of ether-linked phosphatidylinositols (PIs), phosphatidic acids, phosphatidylserines (PSs), very long chain phosphatidylethanolamines (PEs), and phosphatidylcholines (PCs) as well as phosphatidylthreonines (PTs) and a wide collection of odd carbon fatty acid-containing phospholipids in macrophages. A comprehensive qualitative analysis of glycerophospholipids from different macrophage cells was conducted. During the phospholipid profiling of the macrophage-like RAW 264.7 cells, we identified dozens of rare or previously uncharacterized phospholipids, including ether-linked PIs, PSs, and glycerophosphatidic acids, PTs, and PCs and PTs containing very long polyunsaturated fatty acids. Additionally, large numbers of phospholipids containing at least one odd carbon fatty acid were identified. Using the same methodology, we also identified many of the same species of glycerophospholipids in resident peritoneal macrophages, foam cells, and murine bone marrow derived macrophages. PMID:19965583

  20. Role of caveolae in Leishmania chagasi phagocytosis and intracellular survival in macrophages.

    PubMed

    Rodríguez, Nilda E; Gaur, Upasna; Wilson, Mary E

    2006-07-01

    Caveolae are membrane microdomains enriched in cholesterol, ganglioside M1 (GM1) and caveolin-1. We explored whether caveolae facilitate the entry of Leishmania chagasi into murine macrophages. Transient depletion of macrophage membrane cholesterol by 1 h exposure to methyl-beta-cyclodextrin (MbetaCD) impaired the phagocytosis of non-opsonized and serum-opsonized virulent L. chagasi. In contrast, MbetaCD did not affect the phagocytosis of opsonized attenuated L. chagasi. As early as 5 min after phagocytosis, virulent L. chagasi colocalized with the caveolae markers GM1 and caveolin-1, and colocalization continued for over 48 h. We explored the kinetics of lysosome fusion. Whereas fluorescent-labelled dextran entered macrophage lysosomes by 30 min after addition, localization of L. chagasi in lysosomes was delayed for 24-48 h after phagocytosis. However, after transient depletion of cholesterol from macrophage membrane with MbetaCD, the proportion of L. chagasi-containing phagosomes that fused with lysosomes increased significantly. Furthermore, intracellular replication was impaired in parasites entering after transient cholesterol depletion, even though lipid microdomains were restored by 4 h after treatment. These observations suggest that virulent L. chagasi localize in caveolae during phagocytosis by host macrophages, and that cholesterol-containing macrophage membrane domains, such as caveolae, target parasites to a pathway that promotes delay of lysosome fusion and intracellular survival.

  1. Macrophage Independent Regulation of Reverse Cholesterol Transport by Liver X Receptors

    PubMed Central

    Breevoort, Sarah R.; Angdisen, Jerry; Schulman, Ira G.

    2014-01-01

    Objective The ability of high density lipoprotein (HDL) particles to accept cholesterol from peripheral cells such as lipid-laden macrophages and to transport cholesterol to the liver for catabolism and excretion in a process termed reverse cholesterol transport (RCT) is believed to underlie the beneficial cardiovascular effects of elevated HDL. The liver X receptors (LXRα and LXRβ) regulate RCT by controlling the efflux of cholesterol from macrophages to HDL and the excretion, catabolism and absorption of cholesterol in the liver and intestine. Importantly, treatment with LXR agonists increases RCT and decreases atherosclerosis in animal models. Nevertheless, LXRs are expressed in multiple tissues involved in RCT and their tissue specific contributions to RCT are still not well defined. Approach and Results Utilizing tissue-specific LXR deletions together with in vitro and in vivo assays of cholesterol efflux and fecal cholesterol excretion we demonstrate that macrophage LXR activity is neither necessary nor sufficient for LXR agonist-stimulated RCT. In contrast, the ability of LXR agonists primarily acting in the intestine to increase HDL mass and HDL function appears to underlie the ability of LXR agonists to stimulate RCT in vivo. Conclusions We demonstrate that activation of LXR in macrophages makes little or no contribution to LXR agonist-stimulated RCT. Unexpectedly our studies suggest that the ability of macrophages to efflux cholesterol to HDL in vivo is not regulated by macrophage activity but is primarily determined by the quantity and functional activity of HDL. PMID:24947527

  2. Lipid14: The Amber Lipid Force Field

    PubMed Central

    2015-01-01

    The AMBER lipid force field has been updated to create Lipid14, allowing tensionless simulation of a number of lipid types with the AMBER MD package. The modular nature of this force field allows numerous combinations of head and tail groups to create different lipid types, enabling the easy insertion of new lipid species. The Lennard-Jones and torsion parameters of both the head and tail groups have been revised and updated partial charges calculated. The force field has been validated by simulating bilayers of six different lipid types for a total of 0.5 μs each without applying a surface tension; with favorable comparison to experiment for properties such as area per lipid, volume per lipid, bilayer thickness, NMR order parameters, scattering data, and lipid lateral diffusion. As the derivation of this force field is consistent with the AMBER development philosophy, Lipid14 is compatible with the AMBER protein, nucleic acid, carbohydrate, and small molecule force fields. PMID:24803855

  3. Acetylation of αA-crystallin in the human lens: Effects on structure and chaperone function

    PubMed Central

    Nagaraj, Ram H.; Nahomi, Rooban B.; Shanthakumar, Shilpa; Linetsky, Mikhail; Padmanabha, Smitha; Pasupuleti, Nagarekha; Wang, Benlian; Santhoshkumar, Puttur; Panda, Alok Kumar; Biswas, Ashis

    2011-01-01

    α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens.To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a Nε-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it could affect the chaperone function of αA-crystallin. PMID:22120592

  4. Role of Streptococcus intermedius DnaK chaperone system in stress tolerance and pathogenicity.

    PubMed

    Tomoyasu, Toshifumi; Tabata, Atsushi; Imaki, Hidenori; Tsuruno, Keigo; Miyazaki, Aya; Sonomoto, Kenji; Whiley, Robert Alan; Nagamune, Hideaki

    2012-01-01

    Streptococcus intermedius is a facultatively anaerobic, opportunistic pathogen that causes purulent infections and abscess formation. The DnaK chaperone system has been characterized in several pathogenic bacteria and seems to have important functions in stress resistance and pathogenicity. However, the role of DnaK in S. intermedius remains unclear. Therefore, we constructed a dnaK knockout mutant that exhibited slow growth, thermosensitivity, accumulation of GroEL in the cell, and reduced cytotoxicity to HepG2 cells. The level of secretion of a major pathogenic factor, intermedilysin, was not affected by dnaK mutation. We further examined the function and property of the S. intermedius DnaK chaperone system by using Escherichia coli ΔdnaK and ΔrpoH mutant strains. S. intermedius DnaK could not complement the thermosensitivity of E. coli ΔdnaK mutant. However, the intact S. intermedius DnaK chaperone system could complement the thermosensitivity and acid sensitivity of E. coli ΔdnaK mutant. The S. intermedius DnaK chaperone system could regulate the activity and stability of the heat shock transcription factor σ(32) in E. coli, although S. intermedius does not utilize σ(32) for heat shock transcription. The S. intermedius DnaK chaperone system was also able to efficiently eliminate the aggregated proteins from ΔrpoH mutant cells. Overall, our data showed that the S. intermedius DnaK chaperone system has important functions in quality control of cellular proteins but has less participation in the modulation of expression of pathogenic factors.

  5. Quantification of interaction strengths between chaperones and tetratricopeptide repeat domain-containing membrane proteins.

    PubMed

    Schweiger, Regina; Soll, Jürgen; Jung, Kirsten; Heermann, Ralf; Schwenkert, Serena

    2013-10-18

    The three tetratricopeptide repeat domain-containing docking proteins Toc64, OM64, and AtTPR7 reside in the chloroplast, mitochondrion, and endoplasmic reticulum of Arabidopsis thaliana, respectively. They are suggested to act during post-translational protein import by association with chaperone-bound preprotein complexes. Here, we performed a detailed biochemical, biophysical, and computational analysis of the interaction between Toc64, OM64, and AtTPR7 and the five cytosolic chaperones HSP70.1, HSP90.1, HSP90.2, HSP90.3, and HSP90.4. We used surface plasmon resonance spectroscopy in combination with Interaction Map® analysis to distinguish between chaperone oligomerization and docking protein-chaperone interactions and to calculate binding affinities for all tested interactions. Complementary to this, we applied pulldown assays as well as microscale thermophoresis as surface immobilization independent techniques. The data revealed that OM64 prefers HSP70 over HSP90, whereas Toc64 binds all chaperones with comparable affinities. We could further show that AtTPR7 is able to bind HSP90 in addition to HSP70. Moreover, differences between the HSP90 isoforms were detected and revealed a weaker binding for HSP90.1 to AtTPR7 and OM64, showing that slight differences in the amino acid composition or structure of the chaperones influence binding to the tetratricopeptide repeat domain. The combinatory approach of several methods provided a powerful toolkit to determine binding affinities of similar interaction partners in a highly quantitative manner.

  6. Analysis of the potency of various low molecular weight chemical chaperones to prevent protein aggregation.

    PubMed

    Upagupta, Chandak; Carlisle, Rachel E; Dickhout, Jeffrey G

    2017-04-22

    Newly translated proteins must undergo proper folding to ensure their function. To enter a low energy state, misfolded proteins form aggregates, which are associated with many degenerative diseases, such as Huntington's disease and chronic kidney disease (CKD). Recent studies have shown the use of low molecular weight chemical chaperones to be an effective method of reducing protein aggregation in various cell types. This study demonstrates a novel non-biased assay to assess the molecular efficacy of these compounds at preventing protein misfolding and/or aggregation. This assay utilizes a thioflavin T fluorescent stain to provide a qualitative and quantitative measure of protein misfolding within cells. The functionality of this method was first assessed in renal proximal tubule epithelial cells treated with various endoplasmic reticulum (ER) stress inducers. Once established in the renal model system, we analyzed the ability of some known chemical chaperones to reduce ER stress. A total of five different compounds were selected: 4-phenylbutyrate (4-PBA), docosahexaenoic acid (DHA), tauroursodeoxycholic acid, trehalose, and glycerol. The dose-dependent effects of these compounds at reducing thapsigargin-induced ER stress was then analyzed, and used to determine their EC50 values. Of the chaperones, 4-PBA and DHA provided the greatest reduction of ER stress and did so at relatively low concentrations. Upon analyzing the efficiency of these compounds and their corresponding structures, it was determined that chaperones with a localized hydrophilic, polar end followed by a long hydrophobic chain, such as 4-PBA and DHA, were most effective at reducing ER stress. This study provides some insight into the use of low molecular weight chemical chaperones and may serve as the first step towards developing new chaperones of greater potency thereby providing potential treatments for diseases caused by protein aggregation.

  7. Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes.

    PubMed

    Cano, Victoria; March, Catalina; Insua, Jose Luis; Aguiló, Nacho; Llobet, Enrique; Moranta, David; Regueiro, Verónica; Brennan, Gerard P; Millán-Lou, Maria Isabel; Martín, Carlos; Garmendia, Junkal; Bengoechea, José A

    2015-11-01

    Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K. pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of phosphoinositide 3-kinase (PI3K). Microscopy studies revealed that K. pneumoniae resides within a vacuolar compartment, the Klebsiella-containing vacuole (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV-killed bacteria, the majority of live bacteria did not co-localize with markers of the lysosomal compartment. Our data suggest that K. pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K. pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K-Akt-Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the down-regulation of the expression of cps. Altogether, this study proves evidence that K. pneumoniae survives killing by macrophages by manipulating phagosome maturation that may contribute to Klebsiella pathogenesis.

  8. Dysferlin-mediated phosphatidylserine sorting engages macrophages in sarcolemma repair

    PubMed Central

    Middel, Volker; Zhou, Lu; Takamiya, Masanari; Beil, Tanja; Shahid, Maryam; Roostalu, Urmas; Grabher, Clemens; Rastegar, Sepand; Reischl, Markus; Nienhaus, Gerd Ulrich; Strähle, Uwe

    2016-01-01

    Failure to repair the sarcolemma leads to muscle cell death, depletion of stem cells and myopathy. Hence, membrane lesions are instantly sealed by a repair patch consisting of lipids and proteins. It has remained elusive how this patch is removed to restore cell membrane integrity. Here we examine sarcolemmal repair in live zebrafish embryos by real-time imaging. Macrophages remove the patch. Phosphatidylserine (PS), an ‘eat-me' signal for macrophages, is rapidly sorted from adjacent sarcolemma to the repair patch in a Dysferlin (Dysf) dependent process in zebrafish and human cells. A previously unrecognized arginine-rich motif in Dysf is crucial for PS accumulation. It carries mutations in patients presenting with limb-girdle muscular dystrophy 2B. This underscores the relevance of this sequence and uncovers a novel pathophysiological mechanism underlying this class of myopathies. Our data show that membrane repair is a multi-tiered process involving immediate, cell-intrinsic mechanisms as well as myofiber/macrophage interactions. PMID:27641898

  9. HDL-Mimetic PLGA Nanoparticle To Target Atherosclerosis Plaque Macrophages

    PubMed Central

    Sanchez-Gaytan, Brenda L.; Fay, Francois; Lobatto, Mark E.; Tang, Jun; Ouimet, Mireille; Kim, YongTae; van der Staay, Susanne E. M.; van Rijs, Sarian M.; Priem, Bram; Zhang, Liangfang; Fisher, Edward A; Moore, Kathryn J.; Langer, Robert; Fayad, Zahi A.; Mulder, Willem J M

    2015-01-01

    High-density lipoprotein (HDL) is a natural nanoparticle that exhibits an intrinsic affinity for atherosclerotic plaque macrophages. Its natural targeting capability as well as the option to incorporate lipophilic payloads, e.g., imaging or therapeutic components, in both the hydrophobic core and the phospholipid corona make the HDL platform an attractive nanocarrier. To realize controlled release properties, we developed a hybrid polymer/HDL nanoparticle composed of a lipid/apolipoprotein coating that encapsulates a poly(lactic-co-glycolic acid) (PLGA) core. This novel HDL-like nanoparticle (PLGA–HDL) displayed natural HDL characteristics, including preferential uptake by macrophages and a good cholesterol efflux capacity, combined with a typical PLGA nanoparticle slow release profile. In vivo studies carried out with an ApoE knockout mouse model of atherosclerosis showed clear accumulation of PLGA–HDL nanoparticles in atherosclerotic plaques, which colocalized with plaque macrophages. This biomimetic platform integrates the targeting capacity of HDL biomimetic nanoparticles with the characteristic versatility of PLGA-based nanocarriers. PMID:25650634

  10. HDL-mimetic PLGA nanoparticle to target atherosclerosis plaque macrophages.

    PubMed

    Sanchez-Gaytan, Brenda L; Fay, Francois; Lobatto, Mark E; Tang, Jun; Ouimet, Mireille; Kim, YongTae; van der Staay, Susanne E M; van Rijs, Sarian M; Priem, Bram; Zhang, Liangfang; Fisher, Edward A; Moore, Kathryn J; Langer, Robert; Fayad, Zahi A; Mulder, Willem J M

    2015-03-18

    High-density lipoprotein (HDL) is a natural nanoparticle that exhibits an intrinsic affinity for atherosclerotic plaque macrophages. Its natural targeting capability as well as the option to incorporate lipophilic payloads, e.g., imaging or therapeutic components, in both the hydrophobic core and the phospholipid corona make the HDL platform an attractive nanocarrier. To realize controlled release properties, we developed a hybrid polymer/HDL nanoparticle composed of a lipid/apolipoprotein coating that encapsulates a poly(lactic-co-glycolic acid) (PLGA) core. This novel HDL-like nanoparticle (PLGA-HDL) displayed natural HDL characteristics, including preferential uptake by macrophages and a good cholesterol efflux capacity, combined with a typical PLGA nanoparticle slow release profile. In vivo studies carried out with an ApoE knockout mouse model of atherosclerosis showed clear accumulation of PLGA-HDL nanoparticles in atherosclerotic plaques, which colocalized with plaque macrophages. This biomimetic platform integrates the targeting capacity of HDL biomimetic nanoparticles with the characteristic versatility of PLGA-based nanocarriers.

  11. Signaling events in pathogen-induced macrophage foam cell formation.

    PubMed

    Shaik-Dasthagirisaheb, Yazdani B; Mekasha, Samrawit; He, Xianbao; Gibson, Frank C; Ingalls, Robin R

    2016-08-01

    Macrophage foam cell formation is a key event in atherosclerosis. Several triggers induce low-density lipoprotein (LDL) uptake by macrophages to create foam cells, including infections with Porphyromonas gingivalis and Chlamydia pneumoniae, two pathogens that have been linked to atherosclerosis. While gene regulation during foam cell formation has been examined, comparative investigations to identify shared and specific pathogen-elicited molecular events relevant to foam cell formation are not well documented. We infected mouse bone marrow-derived macrophages with P. gingivalis or C. pneumoniae in the presence of LDL to induce foam cell formation, and examined gene expression using an atherosclerosis pathway targeted plate array. We found over 30 genes were significantly induced in response to both pathogens, including PPAR family members that are broadly important in atherosclerosis and matrix remodeling genes that may play a role in plaque development and stability. Six genes mainly involved in lipid transport were significantly downregulated. The response overall was remarkably similar and few genes were regulated in a pathogen-specific manner. Despite very divergent lifestyles, P. gingivalis and C. pneumoniae activate similar gene expression profiles during foam cell formation that may ultimately serve as targets for modulating infection-elicited foam cell burden, and progression of atherosclerosis.

  12. Macrophages and HIV-1: An Unhealthy Constellation.

    PubMed

    Sattentau, Quentin J; Stevenson, Mario

    2016-03-09

    Lentiviruses have a long-documented association with macrophages. Abundant evidence exists for in vitro and, in a tissue-specific manner, in vivo infection of macrophages by the primate lentiviruses HIV-1 and SIV. However, macrophage contribution to aspects of HIV-1 and SIV pathogenesis, and their role in viral persistence in individuals on suppressive antiretroviral therapy, remains unclear. Here we discuss recent evidence implicating macrophages in HIV-1-mediated disease and highlight directions for further investigation.

  13. Macrophage Heterogeneity and Plasticity: Impact of Macrophage Biomarkers on Atherosclerosis

    PubMed Central

    Rojas, Joselyn; Salazar, Juan; Martínez, María Sofía; Palmar, Jim; Bautista, Jordan; Chávez-Castillo, Mervin; Gómez, Alexis; Bermúdez, Valmore

    2015-01-01

    Cardiovascular disease (CVD) is a global epidemic, currently representing the worldwide leading cause of morbidity and mortality. Atherosclerosis is the fundamental pathophysiologic component of CVD, where the immune system plays an essential role. Monocytes and macrophages are key mediators in this aspect: due to their heterogeneity and plasticity, these cells may act as either pro- or anti-inflammatory mediators. Indeed, monocytes may develop heterogeneous functional phenotypes depending on the predominating pro- or anti-inflammatory microenvironment within the lesion, resulting in classic, intermediate, and non-classic monocytes, each with strikingly differing features. Similarly, macrophages may also adopt heterogeneous profiles being mainly M1 and M2, the former showing a proinflammatory profile while the latter demonstrates anti-inflammatory traits; they are further subdivided in several subtypes with more specialized functions. Furthermore, macrophages may display plasticity by dynamically shifting between phenotypes in response to specific signals. Each of these distinct cell profiles is associated with diverse biomarkers which may be exploited for therapeutic intervention, including IL-10, IL-13, PPAR-γ, LXR, NLRP3 inflammasomes, and microRNAs. Direct modulation of the molecular pathways concerning these potential macrophage-related targets represents a promising field for new therapeutic alternatives in atherosclerosis and CVD. PMID:26491604

  14. Copy-choice recombination by reverse transcriptases: Reshuffling of genetic markers mediated by RNA chaperones

    PubMed Central

    Negroni, Matteo; Buc, Henri

    2000-01-01

    Copy-choice recombination efficiently reshuffles genetic markers in retroviruses. In vivo, the folding of the genomic RNA is controlled by the nucleocapsid protein (NC). We show that binding of NC onto the acceptor RNA molecule is sufficient to enhance recombination, providing evidence for a mechanism where the structure of the acceptor template determines the template switch. NC as well as another RNA chaperone (StpA) converts recombination into a widespread process no longer restricted to rare hot spots, an effect maximized when both the NC and the reverse transcriptase come from HIV-1. These data suggest that RNA chaperones confer a higher genetic flexibility to retroviruses. PMID:10829081

  15. Quantifying the role of chaperones in protein translocation by computational modeling

    PubMed Central

    Assenza, Salvatore; De Los Rios, Paolo; Barducci, Alessandro

    2015-01-01

    The molecular chaperone Hsp70 plays a central role in the import of cytoplasmic proteins into organelles, driving their translocation by binding them from the organellar interior. Starting from the experimentally-determined structure of the E. coli Hsp70, we computed, by means of molecular simulations, the effective free-energy profile for substrate translocation upon chaperone binding. We then used the resulting free energy to quantitatively characterize the kinetics of the import process, whose comparison with unassisted translocation highlights the essential role played by Hsp70 in importing cytoplasmic proteins. PMID:25988176

  16. Plasma lipids in beta-thalassemia minor.

    PubMed

    Maioli, M; Pettinato, S; Cherchi, G M; Giraudi, D; Pacifico, A; Pupita, G; Tidore, M G

    1989-02-01

    Because total cholesterol levels have been found to be lower in patients affected by thalassemia major and intermedia, we examined the plasma lipid pattern of 628 beta-thalassemia trait carriers and 4552 controls in order to evaluate whether the plasma lipid impairment is also present in the heterozygous state. Total cholesterol and low density lipoprotein (LDL)-cholesterol levels were significantly lower in beta-thalassemia trait carriers when compared to controls, whereas plasma triglycerides and high density lipoprotein (HDL)-cholesterol levels did not differ between the two groups. We suggest that accelerated erythropoiesis and increased uptake of LDL by macrophages and histiocytes of the reticuloendothelial system are the main determinants of low plasma cholesterol levels in heterozygous thalassemia.

  17. Exploring lipids with nonlinear optical microscopy in multiple biological systems

    NASA Astrophysics Data System (ADS)

    Alfonso-Garcia, Alba

    Lipids are crucial biomolecules for the well being of humans. Altered lipid metabolism may give rise to a variety of diseases that affect organs from the cardiovascular to the central nervous system. A deeper understanding of lipid metabolic processes would spur medical research towards developing precise diagnostic tools, treatment methods, and preventive strategies for reducing the impact of lipid diseases. Lipid visualization remains a complex task because of the perturbative effect exerted by traditional biochemical assays and most fluorescence markers. Coherent Raman scattering (CRS) microscopy enables interrogation of biological samples with minimum disturbance, and is particularly well suited for label-free visualization of lipids, providing chemical specificity without compromising on spatial resolution. Hyperspectral imaging yields large datasets that benefit from tailored multivariate analysis. In this thesis, CRS microscopy was combined with Raman spectroscopy and other label-free nonlinear optical techniques to analyze lipid metabolism in multiple biological systems. We used nonlinear Raman techniques to characterize Meibum secretions in the progression of dry eye disease, where the lipid and protein contributions change in ratio and phase segregation. We employed similar tools to examine lipid droplets in mice livers aboard a spaceflight mission, which lose their retinol content contributing to the onset of nonalcoholic fatty-liver disease. We also focused on atherosclerosis, a disease that revolves around lipid-rich plaques in arterial walls. We examined the lipid content of macrophages, whose variable phenotype gives rise to contrasting healing and inflammatory activities. We also proposed new label-free markers, based on lifetime imaging, for macrophage phenotype, and to detect products of lipid oxidation. Cholesterol was also detected in hepatitis C virus infected cells, and in specific strains of age-related macular degeneration diseased cells by

  18. Hold on to your friends: Dedicated chaperones of ribosomal proteins: Dedicated chaperones mediate the safe transfer of ribosomal proteins to their site of pre-ribosome incorporation.

    PubMed

    Pillet, Benjamin; Mitterer, Valentin; Kressler, Dieter; Pertschy, Brigitte

    2017-01-01

    Eukaryotic ribosomes are assembled from their components, the ribosomal RNAs and ribosomal proteins, in a tremendously complex, multi-step process, which primarily takes place in the nuclear compartment. Therefore, most ribosomal proteins have to travel from the cytoplasm to their incorporation site on pre-ribosomes within the nucleus. However, due to their particular characteristics, such as a highly basic amino acid composition and the presence of unstructured extensions, ribosomal proteins are especially prone to aggregation and degradation in their unassembled state, hence specific mechanisms must operate to ensure their safe delivery. Recent studies have uncovered a group of proteins, termed dedicated chaperones, specialized in accompanying and guarding individual ribosomal proteins. In this essay, we review how these dedicated chaperones utilize different folds to interact with their ribosomal protein clients and how they ensure their soluble expression and interconnect their intracellular transport with their efficient assembly into pre-ribosomes.

  19. The histone chaperones Nap1 and Vps75 bind histones H3 and H4 in a tetrameric conformation.

    PubMed

    Bowman, Andrew; Ward, Richard; Wiechens, Nicola; Singh, Vijender; El-Mkami, Hassane; Norman, David George; Owen-Hughes, Tom

    2011-02-18

    Histone chaperones physically interact with histones to direct proper assembly and disassembly of nucleosomes regulating diverse nuclear processes such as DNA replication, promoter remodeling, transcription elongation, DNA damage, and histone variant exchange. Currently, the best-characterized chaperone-histone interaction is that between the ubiquitous chaperone Asf1 and a dimer of H3 and H4. Nucleosome assembly proteins (Nap proteins) represent a distinct class of histone chaperone. Using pulsed electron double resonance (PELDOR) measurements and protein crosslinking, we show that two members of this class, Nap1 and Vps75, bind histones in the tetrameric conformation also observed when they are sequestered within the nucleosome. Furthermore, H3 and H4 trapped in their tetrameric state can be used as substrates in nucleosome assembly and chaperone-mediated lysine acetylation. This alternate mode of histone interaction provides a potential means of maintaining the integrity of the histone tetramer during cycles of nucleosome reassembly.

  20. Mammalian Fe-S proteins: definition of a consensus motif recognized by the co-chaperone HSC20

    PubMed Central

    Maio, N.; Rouault, T. A.

    2017-01-01

    Iron-sulfur (Fe-S) clusters are inorganic cofactors that are fundamental to several biological processes in all three kingdoms of life. In most organisms, Fe-S clusters are initially assembled on a scaffold protein, ISCU, and subsequently transferred to target proteins or to intermediate carriers by a dedicated chaperone/co-chaperone system. The delivery of assembled Fe-S clusters to recipient proteins is a crucial step in the biogenesis of Fe-S proteins, and, in mammals, it relies on the activity of a multiprotein transfer complex that contains the chaperone HSPA9, the co-chaperone HSC20 and the scaffold ISCU. How the transfer complex efficiently engages recipient Fe-S target proteins involves specific protein interactions that are not fully understood. This mini review focuses on recent insights into the molecular mechanism of amino acid motif recognition and discrimination by the co-chaperone HSC20, which guides Fe-S cluster delivery. PMID:27714045

  1. Emerging evidence for beneficial macrophage functions in atherosclerosis and obesity-induced insulin resistance.

    PubMed

    Fitzgibbons, Timothy P; Czech, Michael P

    2016-03-01

    The discovery that obesity promotes macrophage accumulation in visceral fat led to the emergence of a new field of inquiry termed "immunometabolism". This broad field of study was founded on the premise that inflammation and the corresponding increase in macrophage number and activity was a pathologic feature of metabolic diseases. There is abundant data in both animal and human studies that supports this assertation. Established adverse effects of inflammation in visceral fat include decreased glucose and fatty acid uptake, inhibition of insulin signaling, and ectopic triglyceride accumulation. Likewise, in the atherosclerotic plaque, macrophage accumulation and activation results in plaque expansion and destabilization. Despite these facts, there is an accumulating body of evidence that macrophages also have beneficial functions in both atherosclerosis and visceral obesity. Potentially beneficial functions that are common to these different contexts include the regulation of efferocytosis, lipid buffering, and anti-inflammatory effects. Autophagy, the process by which cytoplasmic contents are delivered to the lysosome for degradation, is integral to many of these protective biologic functions. The macrophage utilizes autophagy as a molecular tool to maintain tissue integrity and homeostasis at baseline (e.g., bone growth) and in the face of ongoing metabolic insults (e.g., fasting, hypercholesterolemia, obesity). Herein, we highlight recent evidence demonstrating that abrogation of certain macrophage functions, in particular autophagy, exacerbates both atherosclerosis and obesity-induced insulin resistance. Insulin signaling through mammalian target of rapamycin (mTOR) is a crucial regulatory node that links nutrient availability to macrophage autophagic flux. A more precise understanding of the metabolic substrates and triggers for macrophage autophagy may allow therapeutic manipulation of this pathway. These observations underscore the complexity of the field

  2. C/EBP homologous protein-induced macrophage apoptosis protects mice from steatohepatitis.

    PubMed

    Malhi, Harmeet; Kropp, Erin M; Clavo, Vinna F; Kobrossi, Christina R; Han, JaeSeok; Mauer, Amy S; Yong, Jing; Kaufman, Randal J

    2013-06-28

    Nonalcoholic fatty liver disease is a heterogeneous disorder characterized by liver steatosis; inflammation and fibrosis are features of the progressive form nonalcoholic steatohepatitis. The endoplasmic reticulum stress response is postulated to play a role in the pathogenesis of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. In particular, C/EBP homologous protein (CHOP) is undetectable under normal conditions but is induced by cellular stress, including endoplasmic reticulum stress. Chop wild type (Chop(+/+)) and knock-out (Chop(-/-)) mice were used in these studies to elucidate the role of CHOP in the pathogenesis of fatty liver disease. Paradoxically, Chop(-/-) mice developed greater liver injury, inflammation, and fibrosis than Chop(+/+) mice, with greater macrophage activation. Primary, bone marrow-derived, and peritoneal macrophages from Chop(+/+) and Chop(-/-) were challenged with palmitic acid, an abundant saturated free fatty acid in plasma and liver lipids. Where palmitic acid treatment activated Chop(+/+) and Chop(-/-) macrophages, Chop(-/-) macrophages were resistant to its lipotoxicity. Chop(-/-) mice were sensitized to liver injury in a second model of dietary steatohepatitis using the methionine-choline-deficient diet. Analysis of bone marrow chimeras between Chop(-/-) and Chop(+/+) mice demonstrated that Chop in macrophages protects from liver injury and inflammation when fed the methionine-choline-deficient diet. We conclude that Chop deletion has a proinflammatory effect in fatty liver injury apparently due to decreased cell death of activated macrophages, resulting in their net accumulation in the liver. Thus, macrophage CHOP plays a key role in protecting the liver from steatohepatitis likely by limiting macrophage survival during lipotoxicity.

  3. C/EBP Homologous Protein-induced Macrophage Apoptosis Protects Mice from Steatohepatitis*

    PubMed Central

    Malhi, Harmeet; Kropp, Erin M.; Clavo, Vinna F.; Kobrossi, Christina R.; Han, JaeSeok; Mauer, Amy S.; Yong, Jing; Kaufman, Randal J.

    2013-01-01

    Nonalcoholic fatty liver disease is a heterogeneous disorder characterized by liver steatosis; inflammation and fibrosis are features of the progressive form nonalcoholic steatohepatitis. The endoplasmic reticulum stress response is postulated to play a role in the pathogenesis of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. In particular, C/EBP homologous protein (CHOP) is undetectable under normal conditions but is induced by cellular stress, including endoplasmic reticulum stress. Chop wild type (Chop+/+) and knock-out (Chop−/−) mice were used in these studies to elucidate the role of CHOP in the pathogenesis of fatty liver disease. Paradoxically, Chop−/− mice developed greater liver injury, inflammation, and fibrosis than Chop+/+ mice, with greater macrophage activation. Primary, bone marrow-derived, and peritoneal macrophages from Chop+/+ and Chop−/− were challenged with palmitic acid, an abundant saturated free fatty acid in plasma and liver lipids. Where palmitic acid treatment activated Chop+/+ and Chop−/− macrophages, Chop−/− macrophages were resistant to its lipotoxicity. Chop−/− mice were sensitized to liver injury in a second model of dietary steatohepatitis using the methionine-choline-deficient diet. Analysis of bone marrow chimeras between Chop−/− and Chop+/+ mice demonstrated that Chop in macrophages protects from liver injury and inflammation when fed the methionine-choline-deficient diet. We conclude that Chop deletion has a proinflammatory effect in fatty liver injury apparently due to decreased cell death of activated macrophages, resulting in their net accumulation in the liver. Thus, macrophage CHOP plays a key role in protecting the liver from steatohepatitis likely by limiting macrophage survival during lipotoxicity. PMID:23720735

  4. PPAR{gamma} regulates the expression of cholesterol metabolism genes in alveolar macrophages

    SciTech Connect

    Baker, Anna D.; Malur, Anagha; Barna, Barbara P.; Kavuru, Mani S.; Malur, Achut G.; Thomassen, Mary Jane

    2010-03-19

    Peroxisome proliferator-activated receptor-gamma (PPAR{gamma}) is a nuclear transcription factor involved in lipid metabolism that is constitutively expressed in the alveolar macrophages of healthy individuals. PPAR{gamma} has recently been implicated in the catabolism of surfactant by alveolar macrophages, specifically the cholesterol component of surfactant while the mechanism remains unclear. Studies from other tissue macrophages have shown that PPAR{gamma} regulates cholesterol influx, efflux, and metabolism. PPAR{gamma} promotes cholesterol efflux through the liver X receptor-alpha (LXR{alpha}) and ATP-binding cassette G1 (ABCG1). We have recently shown that macrophage-specific PPAR{gamma} knockout (PPAR{gamma} KO) mice accumulate cholesterol-laden alveolar macrophages that exhibit decreased expression of LXR{alpha} and ABCG1 and reduced cholesterol efflux. We hypothesized that in addition to the dysregulation of these cholesterol efflux genes, the expression of genes involved in cholesterol synthesis and influx was also dysregulated and that replacement of PPAR{gamma} would restore regulation of these genes. To investigate this hypothesis, we have utilized a Lentivirus expression system (Lenti-PPAR{gamma}) to restore PPAR{gamma} expression in the alveolar macrophages of PPAR{gamma} KO mice. Our results show that the alveolar macrophages of PPAR{gamma} KO mice have decreased expression of key cholesterol synthesis genes and increased expression of cholesterol receptors CD36 and scavenger receptor A-I (SRA-I). The replacement of PPAR{gamma} (1) induced transcription of LXR{alpha} and ABCG1; (2) corrected suppressed expression of cholesterol synthesis genes; and (3) enhanced the expression of scavenger receptors CD36. These results suggest that PPAR{gamma} regulates cholesterol metabolism in alveolar macrophages.

  5. A comparison of two distinct murine macrophage gene expression profiles in response to Leishmania amazonensis infection

    PubMed Central

    2012-01-01

    Background The experimental murine model of leishmaniasis has been widely used to characterize the immune response against Leishmania. CBA mice develop severe lesions, while C57BL/6 present small chronic lesions under L. amazonensis infection. Employing a transcriptomic approach combined with biological network analysis, the gene expression profiles of C57BL/6 and CBA macrophages, before and after L. amazonensis infection in vitro, were compared. These strains were selected due to their different degrees of susceptibility to this parasite. Results The genes expressed by C57BL/6 and CBA macrophages, before and after infection, differ greatly, both with respect to absolute number as well as cell function. Uninfected C57BL/6 macrophages express genes involved in the deactivation pathway of macrophages at lower levels, while genes related to the activation of the host immune inflammatory response, including apoptosis and phagocytosis, have elevated expression levels. Several genes that participate in the apoptosis process were also observed to be up-regulated in C57BL/6 macrophages infected with L. amazonensis, which is very likely related to the capacity of these cells to control parasite infection. By contrast, genes involved in lipid metabolism were found to be up-regulated in CBA macrophages in response to infection, which supports the notion that L. amazonensis probably modulates parasitophorous vacuoles in order to survive and multiply in host cells. Conclusion The transcriptomic profiles of C57BL/6 macrophages, before and after infection, were shown to be involved in the macrophage pathway of activation, which may aid in the control of L. amazonensis infection, in contrast to the profiles of CBA cells. PMID:22321871

  6. A [Cu]rious Ribosomal Profiling Pattern Leads to the Discovery of Ribosomal Frameshifting in the Synthesis of a Copper Chaperone.

    PubMed

    Atkins, John F; Loughran, Gary; Baranov, Pavel V

    2017-01-19

    In many bacteria, separate genes encode a copper binding chaperone and a copper efflux pump, but in some the chaperone encoding gene has been elusive. In this issue of Molecular Cell, Meydan et al. (2017) report that ribosomes translating the ORF that encodes the copper pump frequently frameshift and terminate to produce the copper chaperone.

  7. Murine macrophages response to iron.

    PubMed

    Polati, Rita; Castagna, Annalisa; Bossi, Alessandra Maria; Alberio, Tiziana; De Domenico, Ivana; Kaplan, Jerry; Timperio, Anna Maria; Zolla, Lello; Gevi, Federica; D'Alessandro, Angelo; Brunch, Ryan; Olivieri, Oliviero; Girelli, Domenico

    2012-12-05

    Macrophages play a critical role at the crossroad between iron metabolism and immunity, being able to store and recycle iron derived from the phagocytosis of senescent erythrocytes. The way by which macrophages manage non-heme iron at physiological concentration is still not fully understood. We investigated protein changes in mouse bone marrow macrophages incubated with ferric ammonium citrate (FAC 10 μM iron). Differentially expressed spots were identified by nano RP-HPLC-ESI-MS/MS. Transcriptomic, metabolomics and western immunoblotting analyses complemented the proteomic approach. Pattern analysis was also used for identifying networks of proteins involved in iron homeostasis. FAC treatment resulted in higher abundance of several proteins including ferritins, cytoskeleton related proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at the membrane level, vimentin, arginase, galectin-3 and macrophage migration inhibitory factor (MIF). Interestingly, GAPDH has been recently proposed to act as an alternative transferrin receptor for iron acquisition through internalization of the GAPDH-transferrin complex into the early endosomes. FAC treatment also induced the up-regulation of oxidative stress-related proteins (PRDX), which was further confirmed at the metabolic level (increase in GSSG, 8-isoprostane and pentose phosphate pathway intermediates) through mass spectrometry-based targeted metabolomics approaches. This study represents an example of the potential usefulness of "integarated omics" in the field of iron biology, especially for the elucidation of the molecular mechanisms controlling iron homeostasis in normal and disease conditions. This article is part of a Special Issue entitled: Integrated omics.

  8. Modulating macrophage response to biomaterials

    NASA Astrophysics Data System (ADS)

    Zaveri, Toral

    Macrophages recruited to the site of biomaterial implantation are the primary mediators of the chronic foreign body response to implanted materials. Since foreign body response limits performance and functional life of numerous implanted biomaterials/medical devices, various approaches have been investigated to modulate macrophage interactions with biomaterial surfaces to mitigate this response. In this work we have explored two independent approaches to modulate the macrophage inflammatory response to biomaterials. The first approach targets surface integrins, cell surface receptors that mediate cell adhesion to biomaterials through adhesive proteins spontaneously adsorbed on biomaterial surfaces. The second approach involves surface modification of biomaterials using nanotopographic features since nanotopography has been reported to modulate cell adhesion and viability in a cell type-dependent manner. More specifically, Zinc Oxide (ZnO) nanorod surface was investigated for its role in modulating macrophage adhesion and survival in vitro and foreign body response in vivo. For the first approach, we have investigated the role of integrin Mac-1 and RGD-binding integrins in the in-vivo osteolysis response and macrophage inflammatory processes of phagocytosis as well as inflammatory cytokine secretion in response to particulate biomaterials. We have also investigated the in vivo foreign body response (FBR) to subcutaneously implanted biomaterials by evaluating the thickness of fibrous capsule formed around the implants after 2 weeks of implantation. The role of Mac-1 integrin was isolated using a Mac-1 KO mouse and comparing it to a WT control. The role of RGD binding integrins in FBR was investigated by coating the implanted biomaterial with ELVAX(TM) polymer loaded with Echistatin which contains the RGD sequence. For the in-vivo osteolysis study and to study the in-vitro macrophage response to particulate biomaterials, we used the RGD peptide encapsulated in ELVAX

  9. Antimicrobial proteins of murine macrophages.

    PubMed Central

    Hiemstra, P S; Eisenhauer, P B; Harwig, S S; van den Barselaar, M T; van Furth, R; Lehrer, R I

    1993-01-01

    Three murine microbicidal proteins (MUMPs) were purified from cells of the murine macrophage cell line RAW264.7 that had been activated by gamma interferon. Similar proteins were also present in nonactivated RAW264.7 cells, in cells of the murine macrophage cell line J774A.1, and in resident and activated murine peritoneal macrophages. MUMP-1, MUMP-2, and MUMP-3 killed Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Mycobacterium fortuitum, and Cryptococcus neoformans in vitro. MUMP-1 resembled an H1 histone but was unusual because its N-terminal residue (serine) was not N acetylated. Although MUMP-2 was N terminally blocked, its high lysine/arginine ratio and its reactivity with an antibody to H1 histones suggested that it also belonged to the H1 histone family. MUMP-3 was identical to histone H2B in 30 of 30 amino-terminal residues. Although the antimicrobial properties of histones have been recognized for decades, this is the first evidence that such proteins may endow the lysosomal apparatus of macrophages with nonoxidative antimicrobial potential. Other MUMPs, including some with a more restricted antimicrobial spectrum and one that appeared to be induced in RAW264.7 cells after gamma interferon stimulation, were noted but remain to be characterized. Images PMID:8514411

  10. Lipid droplet accumulation is associated with an increase in hyperglycemia-induced renal damage: prevention by liver X receptors.

    PubMed

    Kiss, Eva; Kränzlin, Bettina; Wagenblaβ, Katja; Bonrouhi, Mahnaz; Thiery, Joachim; Gröne, Elisabeth; Nordström, Viola; Teupser, Daniel; Gretz, Norbert; Malle, Ernst; Gröne, Hermann-Josef

    2013-03-01

    Dyslipidemia is a frequent component of the metabolic disorder of diabetic patients contributing to organ damage. Herein, in low-density lipoprotein receptor-deficient hyperlipidemic and streptozotozin-induced diabetic mice, hyperglycemia and hyperlipidemia acted reciprocally, accentuating renal injury and altering renal function. In hyperglycemic-hyperlipidemic kidneys, the accumulation of Tip47-positive lipid droplets in glomeruli, tubular epithelia, and macrophages was accompanied by the concomitant presence of the oxidative stress markers xanthine oxidoreductase and nitrotyrosine, findings that could also be evidenced in renal biopsy samples of diabetic patients. As liver X receptors (LXRα,β) regulate genes linked to lipid and carbohydrate homeostasis and inhibit inflammatory gene expression in macrophages, the effects of systemic and macrophage-specific LXR activation were analyzed on renal damage in hyperlipidemic-hyperglycemic mice. LXR stimulation by GW3965 up-regulated genes involved in cholesterol efflux and down-regulated proinflammatory/profibrotic cytokines, inhibiting the pathomorphology of diabetic nephropathy, renal lipid accumulation, and improving renal function. Xanthine oxidoreductase and nitrotyrosine levels were reduced. In macrophages, GW3965 or LXRα overexpression significantly suppressed glycated or acetylated low-density lipoprotein-induced cytokines and reactive oxygen species. Specifically, in mice, transgenic expression of LXRα in macrophages significantly ameliorated hyperlipidemic-hyperglycemic nephropathy. The results demonstrate the presence of lipid droplet-induced oxidative mechanisms and the pathophysiologic role of macrophages in diabetic kidneys and indicate the potent regulatory role of LXRs in preventing renal damage in diabetes.

  11. The journey from stem cell to macrophage

    PubMed Central

    Pittet, Mikael J.; Nahrendorf, Matthias; Swirski, Filip K.

    2014-01-01

    Essential protectors against infection and injury, macrophages can also contribute to many common and fatal diseases. Here we discuss the mechanisms that control different types of macrophage activities in mice. We follow the cells’ maturational pathways over time and space, and elaborate on events that influence the type of macrophage eventually settling a particular destination. The nature of the precursor cells, developmental niches, tissues, environmental cues, and other connecting processes appear to contribute to the identity of macrophage type. Together, the spatial and developmental relationships of macrophages comprise a topo-ontogenic map that can guide our understanding of their biology. PMID:24673186

  12. Macrophage cholesterol homeostasis and metabolic diseases: critical role of cholesteryl ester mobilization.

    PubMed

    Ghosh, Shobha

    2011-03-01

    Atherogenic dyslipidemia, including low HDL levels, is the major contributor of residual risk of cardiovascular disease that remains even after aggressive statin therapy to reduce LDL-cholesterol. Currently, distinction is not made between HDL-cholesterol and HDL, which is a lipoprotein consisting of several proteins and a core containing cholesteryl esters (CEs). The importance of assessing HDL functionality, specifically its role in facilitating cholesterol efflux from foam cells, is relevant to atherogenesis. Since HDLs can only remove unesterified cholesterol from macrophages while cholesterol is stored as CEs within foam cells, intracellular CE hydrolysis by CE hydrolase is vital. Reduction in macrophage lipid burden not only attenuates atherosclerosis but also reduces inflammation and linked pathologies such as Type 2 diabetes and chronic kidney disease. Targeting reduction in macrophage CE levels and focusing on enhancing cholesterol flux from peripheral tissues to liver for final elimination is proposed.

  13. Modulation of macrophage-mediated cytotoxicity by kerosene soot: Possible role of reactive oxygen species

    SciTech Connect

    Arif, J.M.; Khan, S.G.; Ashquin, M.; Rahman, Q. )

    1993-05-01

    The involvement of reactive oxygen species (ROS) in the cytotoxicity of soot on rat alveolar macrophages has been postulated. A single intratracheal injection of soot (5 mg) in corn oil significantly induced the macrophage population, hydrogen peroxide (H[sub 2]O[sub 2]) generation, thiobarbituric acid (TBA)-reactive substanced of lipid peroxidation, and the activities of extracellular acid phosphatase (AP) and lactate dehydrogenase (LDH) at 1, 4, 8, and 16 days of postinoculation. The activities of glutathione peroxidase (GPX) and catalase (CAT) were significantly inhibited at all the stages, while glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD) showed a different pattern. These results show that soot is cytotoxic to alveolar macrophages and suggest that ROS may play a primary role in the cytotoxic process. 28 refs., 4 figs., 1 tab.

  14. Mechanisms of glucocorticoid induced suppression of phagocytosis in murine peritoneal macrophage cultures

    SciTech Connect

    Becker, J.L.

    1986-01-01

    Glucocorticoids suppress phagocytosis of heat killed Saccharomyces cerevisiae in macrophage cultures. In order to determine the mechanisms by which this response occurs, this investigation was initiated to examine whether the suppression of phagocytosis is mediated by a steroid induced phagocytosis inhibitory protein (PIP). Furthermore, it is postulated that these suppressive effects may be associated with alterations in macrophage phospholipid metabolism. To assess the association between phospholipid metabolism and phagocytosis, control and 1 ..mu..M dexamethasone treated macrophages were exposed to the phospholipase inhibitor bromophenacylbromide. The enzyme inhibitor suppressed phagocytosis in a time and dose dependent manner. However, supplying dexamethasone treated cultures with arachidonate did not reverse the steroid induced suppression of phagocytosis, whether the arachidonate was supplied alone or together with indomethacin and nordihydroguaiaretic acid. Control cells, prelabeled with /sup 3/H-arachidonate, exhibited an increased percentage of the radiolabeled fatty acid in neutral lipids following phagocytosis, with a corresponding decrease in the percentage associated with phosphatidylcholine.

  15. Vinpocetine attenuates lipid accumulation and atherosclerosis formation

    SciTech Connect

    Cai, Yujun; Li, Jian-Dong; Yan, Chen

    2013-05-10

    Highlights: •Vinpocetine attenuates hyperlipidemia-induced atherosclerosis in a mouse model. •Vinpocetine antagonizes ox-LDL uptake and accumulation in macrophages. •Vinpocetine blocks the induction of ox-LDL receptor LOX-1 in vitro and in vivo. -- Abstract: Atherosclerosis, the major cause of myocardial infarction and stroke, is a chronic arterial disease characterized by lipid deposition and inflammation in the vessel wall. Cholesterol, in low-density lipoprotein (LDL), plays a critical role in the pathogenesis of atherosclerosis. Vinpocetine, a derivative of the alkaloid vincamine, has long been used as a cerebral blood flow enhancer for treating cognitive impairment. Recent study indicated that vinpocetine is a potent anti-inflammatory agent. However, its role in the pathogenesis of atherosclerosis remains unexplored. In the present study, we show that vinpocetine significantly reduced atherosclerotic lesion formation in ApoE knockout mice fed with a high-fat diet. In cultured murine macrophage RAW264.7 cells, vinpocetine markedly attenuated oxidized LDL (ox-LDL) uptake and foam cell formation. Moreover, vinpocetine greatly blocked the induction of ox-LDL receptor 1 (LOX-1) in cultured macrophages as well as in the LOX-1 level in atherosclerotic lesions. Taken together, our data reveal a novel role of vinpocetine in reduction of pathogenesis of atherosclerosis, at least partially through suppressing LOX-1 signaling pathway. Given the excellent safety profile of vinpocetine, this study suggests vinpocetine may be a therapeutic candidate for treating atherosclerosis.

  16. Identification of polarized macrophage subsets in zebrafish.

    PubMed

    Nguyen-Chi, Mai; Laplace-Builhe, Béryl; Travnickova, Jana; Luz-Crawford, Patricia; Tejedor, Gautier; Phan, Quang Tien; Duroux-Richard, Isabelle; Levraud, Jean-Pierre; Kissa, Karima; Lutfalla, Georges; Jorgensen, Christian; Djouad, Farida

    2015-07-08

    While the mammalian macrophage phenotypes have been intensively studied in vitro, the dynamic of their phenotypic polarization has never been investigated in live vertebrates. We used the zebrafish as a live model to identify and trail macrophage subtypes. We generated a transgenic line whose macrophages expressing tumour necrosis factor alpha (tnfa), a key feature of classically activated (M1) macrophages, express fluorescent proteins Tg(mpeg1:mCherryF/tnfa:eGFP-F). Using 4D-confocal microscopy, we showed that both aseptic wounding and Escherichia coli inoculation triggered macrophage recruitment, some of which started to express tnfa. RT-qPCR on Fluorescence Activated Cell Sorting (FACS)-sorted tnfa(+) and tnfa(-) macrophages showed that they, respectively, expressed M1 and alternatively activated (M2) mammalian markers. Fate tracing of tnfa(+) macrophages during the time-course of inflammation demonstrated that pro-inflammatory macrophages converted into M2-like phenotype during the resolution step. Our results reveal the diversity and plasticity of zebrafish macrophage subsets and underline the similarities with mammalian macrophages proposing a new system to study macrophage functional dynamic.

  17. Identification of polarized macrophage subsets in zebrafish

    PubMed Central

    Nguyen-Chi, Mai; Laplace-Builhe, Béryl; Travnickova, Jana; Luz-Crawford, Patricia; Tejedor, Gautier; Phan, Quang Tien; Duroux-Richard, Isabelle; Levraud, Jean-Pierre; Kissa, Karima; Lutfalla, Georges

    2015-01-01

    While the mammalian macrophage phenotypes have been intensively studied in vitro, the dynamic of their phenotypic polarization has never been investigated in live vertebrates. We used the zebrafish as a live model to identify and trail macrophage subtypes. We generated a transgenic line whose macrophages expressing tumour necrosis factor alpha (tnfa), a key feature of classically activated (M1) macrophages, express fluorescent proteins Tg(mpeg1:mCherryF/tnfa:eGFP-F). Using 4D-confocal microscopy, we showed that both aseptic wounding and Escherichia coli inoculation triggered macrophage recruitment, some of which started to express tnfa. RT-qPCR on Fluorescence Activated Cell Sorting (FACS)-sorted tnfa+ and tnfa− macrophages showed that they, respectively, expressed M1 and alternatively activated (M2) mammalian markers. Fate tracing of tnfa+ macrophages during the time-course of inflammation demonstrated that pro-inflammatory macrophages converted into M2-like phenotype during the resolution step. Our results reveal the diversity and plasticity of zebrafish macrophage subsets and underline the similarities with mammalian macrophages proposing a new system to study macrophage functional dynamic. DOI: http://dx.doi.org/10.7554/eLife.07288.001 PMID:26154973

  18. Inhibiting macrophage proliferation suppresses atherosclerotic plaque inflammation.

    PubMed

    Tang, Jun; Lobatto, Mark E; Hassing, Laurien; van der Staay, Susanne; van Rijs, Sarian M; Calcagno, Claudia; Braza, Mounia S; Baxter, Samantha; Fay, Francois; Sanchez-Gaytan, Brenda L; Duivenvoorden, Raphaël; Sager, Hendrik; Astudillo, Yaritzy M; Leong, Wei; Ramachandran, Sarayu; Storm, Gert; Pérez-Medina, Carlos; Reiner, Thomas; Cormode, David P; Strijkers, Gustav J; Stroes, Erik S G; Swirski, Filip K; Nahrendorf, Matthias; Fisher, Edward A; Fayad, Zahi A; Mulder, Willem J M

    2015-04-01

    Inflammation drives atherosclerotic plaque progression and rupture, and is a compelling therapeutic target. Consequently, attenuating inflammation by reducing local macrophage accumulation is an appealing approach. This can potentially be accomplished by either blocking blood monocyte recruitment to the plaque or increasing macrophage apoptosis and emigration. Because macrophage proliferation was recently shown to dominate macrophage accumulation in advanced plaques, locally inhibiting macrophage proliferation may reduce plaque inflammation and produce long-term therapeutic benefits. To test this hypothesis, we used nanoparticle-based delivery of simvastatin to inhibit plaque macrophage proliferation in apolipoprotein E deficient mice (Apoe(-/-) ) with advanced atherosclerotic plaques. This resulted in rapid reduction of plaque inflammation and favorable phenotype remodeling. We then combined this short-term nanoparticle intervention with an eight-week oral statin treatment, and this regimen rapidly reduced and continuously suppressed plaque inflammation. Our results demonstrate that pharmacologically inhibiting local macrophage proliferation can effectively treat inflammation in atherosclerosis.

  19. Inhibiting macrophage proliferation suppresses atherosclerotic plaque inflammation

    PubMed Central

    Tang, Jun; Lobatto, Mark E.; Hassing, Laurien; van der Staay, Susanne; van Rijs, Sarian M.; Calcagno, Claudia; Braza, Mounia S.; Baxter, Samantha; Fay, Francois; Sanchez-Gaytan, Brenda L.; Duivenvoorden, Raphaël; Sager, Hendrik B.; Astudillo, Yaritzy M.; Leong, Wei; Ramachandran, Sarayu; Storm, Gert; Pérez-Medina, Carlos; Reiner, Thomas; Cormode, David P.; Strijkers, Gustav J.; Stroes, Erik S. G.; Swirski, Filip K.; Nahrendorf, Matthias; Fisher, Edward A.; Fayad, Zahi A.; Mulder, Willem J. M.

    2015-01-01

    Inflammation drives atherosclerotic plaque progression and rupture, and is a compelling therapeutic target. Consequently, attenuating inflammation by reducing local macrophage accumulation is an appealing approach. This can potentially be accomplished by either blocking blood monocyte recruitment to the plaque or increasing macrophage apoptosis and emigration. Because macrophage proliferation was recently shown to dominate macrophage accumulation in advanced plaques, locally inhibiting macrophage proliferation may reduce plaque inflammation and produce long-term therapeutic benefits. To test this hypothesis, we used nanoparticle-based delivery of simvastatin to inhibit plaque macrophage proliferation in apolipoprotein E–deficient mice (Apoe−/−) with advanced atherosclerotic plaques. This resulted in the rapid reduction of plaque inflammation and favorable phenotype remodeling. We then combined this short-term nanoparticle intervention with an 8-week oral statin treatment, and this regimen rapidly reduced and continuously suppressed plaque inflammation. Our results demonstrate that pharmacologically inhibiting local macrophage proliferation can effectively treat inflammation in atherosclerosis. PMID:26295063

  20. Macrophages in tissue repair, regeneration, and fibrosis

    PubMed Central

    Wynn, Thomas A.; Vannella, Kevin M.

    2016-01-01

    Inflammatory monocytes and resident tissue macrophages are key regulators of tissue repair, regeneration, and fibrosis. Following tissue injury, monocytes and macrophages undergo marked phenotypic and functional changes to play critical roles during the initiation, maintenance, and resolution phases of tissue repair. Disturbances in macrophage function can lead to aberrant repair, with uncontrolled inflammatory mediator and growth factor production, deficient generation of anti-inflammatory macrophages, or failed communication between macrophages and epithelial cells, endothelial cells, fibroblasts, and stem or tissue progenitor cells all contributing to a state of persistent injury, which may lead to the development of pathological fibrosis. In this review, we discuss the mechanisms that instruct macrophages to adopt pro-inflammatory, pro-wound healing, pro-fibrotic, anti-inflammatory, anti-fibrotic, pro-resolving, and tissue regenerating phenotypes following injury, and highlight how some of these mechanisms and macrophage activation states could be exploited therapeutically. PMID:26982353

  1. Proliferation of macrophages due to the inhibition of inducible nitric oxide synthesis by oxidized low-density lipoproteins

    PubMed Central

    Brunner, Monika; Gruber, Miriam; Schmid, Diethart; Baran, Halina; Moeslinger, Thomas

    2015-01-01

    Oxidized low-density lipoprotein (ox-LDL) is assumed to be a major causal agent in hypercholesteraemia-induced atherosclerosis. Because the proliferation of lipid-loaden macrophages within atherosclerotic lesions has been described, we investigated the dependence of macrophage proliferation on the inhibition of inducible nitric oxide synthase (iNOS) by hypochlorite oxidized LDL. Ox-LDL induces a dose dependent inhibition of inducible nitric oxide synthesis in lipopolysaccharide-interferon stimulated mouse macrophages (J774.A1) with concomitant macrophage proliferation as assayed by cell counting, tritiated-thymidine incorporation and measurement of cell protein. Native LDL did not influence macrophage proliferation and inducible nitric oxide synthesis. iNOS protein and mRNA was reduced by HOCl-oxidized LDL (0-40 µg/ml) as revealed by immunoblotting and competitive semiquantitative PCR. Macrophage proliferation was increased by the addition of the iNOS inhibitor L-NAME. The addition of ox-LDL to L-NAME containing incubations induced no further statistically significant increase in cell number. Nitric oxide donors decreased ox-LDL induced macrophage proliferation and nitric oxide scavengers restored macrophage proliferation to the initial values achieved by ox-LDL. The decrease of cytosolic DNA fragments in stimulated macrophages incubated with ox-LDL demonstrates that the proliferative actions of ox-LDL are associated with a decrease of NO-induced apoptosis. Our data show that inhibition of iNOS dependent nitric oxide production caused by hypochlorite oxidized LDL enhances macrophage proliferation. This might be a key event in the pathogenesis of atherosclerotic lesions. PMID:26600745

  2. Protein aggregation can inhibit clathrin-mediated endocytosis by chaperone competition

    PubMed Central

    Yu, Anan; Shibata, Yoko; Shah, Bijal; Calamini, Barbara; Lo, Donald C.; Morimoto, Richard I.

    2014-01-01

    Protein conformational diseases exhibit complex pathologies linked to numerous molecular defects. Aggregation of a disease-associated protein causes the misfolding and aggregation of other proteins, but how this interferes with diverse cellular pathways is unclear. Here, we show that aggregation of neurodegenerative disease-related proteins (polyglutamine, huntingtin, ataxin-1, and superoxide dismutase-1) inhibits clathrin-mediated endocytosis (CME) in mammalian cells by aggregate-driven sequestration of the major molecular chaperone heat shock cognate protein 70 (HSC70), which is required to drive multiple steps of CME. CME suppression was also phenocopied by HSC70 RNAi depletion and could be restored by conditionally increasing HSC70 abundance. Aggregation caused dysregulated AMPA receptor internalization and also inhibited CME in primary neurons expressing mutant huntingtin, showing direct relevance of our findings to the pathology in neurodegenerative diseases. We propose that aggregate-associated chaperone competition leads to both gain-of-function and loss-of-function phenotypes as chaperones become functionally depleted from multiple clients, leading to the decline of multiple cellular processes. The inherent properties of chaperones place them at risk, contributing to the complex pathologies of protein conformational diseases. PMID:24706768

  3. Plant Leucine Aminopeptidases Moonlight as Molecular Chaperones to Alleviate Stress-induced Damage*

    PubMed Central

    Scranton, Melissa A.; Yee, Ashley; Park, Sang-Youl; Walling, Linda L.

    2012-01-01

    Leucine aminopeptidases (LAPs) are present in animals, plants, and microbes. In plants, there are two classes of LAPs. The neutral LAPs (LAP-N and its orthologs) are constitutively expressed and detected in all plants, whereas the stress-induced acidic LAPs (LAP-A) are expressed only in a subset of the Solanaceae. LAPs have a role in insect defense and act as a regulator of the late branch of wound signaling in Solanum lycopersicum (tomato). Although the mechanism of LAP-A action is unknown, it has been presumed that LAP peptidase activity is essential for regulating wound signaling. Here we show that plant LAPs are bifunctional. Using three assays to monitor protein protection from heat-induced damage, it was shown that the tomato LAP-A and LAP-N and the Arabidopsis thaliana LAP1 and LAP2 are molecular chaperones. Assays using LAP-A catalytic site mutants demonstrated that LAP-A chaperone activity was independent of its peptidase activity. Furthermore, disruption of the LAP-A hexameric structure increased chaperone activity. Together, these data identify a new class of molecular chaperones and a new function for the plant LAPs as well as suggesting new mechanisms for LAP action in the defense of solanaceous plants against stress. PMID:22493451

  4. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    NASA Astrophysics Data System (ADS)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  5. Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding

    PubMed Central

    Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi

    2015-01-01

    Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spec