Role of Erk1/2 Signaling in the Regulation of Neutrophil versus Monocyte Development in Response to G-CSF and M-CSF [Immunology]

August 20th, 2015 by Hu, N., Qiu, Y., Dong, F.

Lineage specification in the hematopoietic system depends on the expression of lineage specific transcription factors. However, the role of hematopoietic cytokines in this process has been controversial and little is known about the intracellular signaling mechanisms by which cytokines instruct lineage choice. G-CSF and M-CSF are two lineage-specific cytokines that play a dominant role in granulopoiesis and monopoiesis, respectively. We show here that a G-CSFR mutant in which tyrosine 729 had been mutated to phenylalanine (Y729F) promoted monocyte rather than neutrophil development in myeloid precursors, which was associated with prolonged activation of Erk1/2 and augmented activation of downstream targets c-Fos and Egr1. Inhibition of Erk1/2 activation or knockdown of c-Fos or Egr1 largely rescued neutrophil development in cells expressing G-CSFR Y729F. We also show that M-CSF, but not G-CSF, stimulated strong and sustained activation of Erk1/2 in mouse lineage marker negative (Lin-) bone marrow cells. Significantly, inhibition of Erk1/2 signaling in these cells favored neutrophil over monocyte development in response to M-CSF. Thus, prolonged Erk1/2 activation resulted in monocyte development following G-CSF induction whereas inhibition of Erk1/2 signaling promoted neutrophil development at the expense of monocyte formation in response to M-CSF. These results reveal an important mechanism by which G-CSF and M-CSF instruct neutrophil versus monocyte lineage choice, i.e., differential activation of Erk1/2 pathway.

Selective Irreversible Inhibition of Neuronal and Inducible Nitric-Oxide Synthase in the Combined Presence of Hydrogen Sulfide and Nitric Oxide [Enzymology]

August 20th, 2015 by

Citrulline formation by both human neuronal nitric-oxide synthase (nNOS) and mouse macrophage inducible nitric-oxide synthase (iNOS) was inhibited by the hydrogen sulfide (H2S) donor Na2S with IC50-values of ~ 2.4×10−5 M and ~ 7.9×10−5 M, respectively, whereas human endothelial nitric-oxide synthase (eNOS) was hardly affected at all. Inhibition of nNOS was not affected by the concentrations of L-arginine (Arg), NADPH, FAD, FMN, tetrahydrobiopterin (BH4), and calmodulin (CaM), indicating that H2S does not interfere with substrate or cofactor binding. The IC50 decreased to ~ 1.5×10−5 M at pH 6.0 and increased to ~ 8.3×10−5 M at pH 8.0. Preincubation of concentrated nNOS with H2S under turnover conditions decreased activity after dilution by ~ 70 %, suggesting irreversible inhibition. However, when CaM was omitted during preincubation, activity was not affected, suggesting that irreversible inhibition requires both H2S and NO. Likewise, NADPH oxidation was inhibited with IC50 ~ 1.9×10−5 M in the presence of Arg and BH4, but exhibited much higher IC50-values (~ 1.0-6.1×10−4 M) when Arg and/or BH4 were omitted. Moreover, the relatively weak inhibition of nNOS by Na2S in the absence of Arg and/or BH4 was markedly potentiated by the NO-donor PROLI/NO (IC50 ~ 1.3-2.0×10−5 M). These results suggest that nNOS and iNOS, but not eNOS, are irreversibly inhibited by H2S/NO at modest concentrations of H2S in a reaction that may allow feedback inhibition of NO production under conditions of excessive NO/H2S formation.
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Brain Expressed X-Linked 2 Is Pivotal for Hyperactive mTOR-Mediated Tumorigenesis [Signal Transduction]

August 20th, 2015 by

Frequent alteration of upstream proto-oncogenes and tumor suppressor genes activates mechanistic target of rapamycin (mTOR) and causes cancer. However, the downstream effectors of mTOR remain largely elusive. Here we report that brain expressed X-linked 2 (BEX2) is a novel downstream effector of mTOR. Elevated BEX2 in Tsc2-/- MEFs, Pten-/- MEFs, Tsc2-deficient rat uterine leiomyoma cells, and brains of neuronal-specific Tsc1 knockout mice were abolished by mTOR inhibitor rapamycin. Furthermore, BEX2 was also increased in the liver of a hepatic-specific Pten knockout mouse and the kidneys of Tsc2 heterozygous deletion mice, and a patient with tuberous sclerosis complex (TSC). mTOR up-regulation of BEX2 was mediated in parallel by both STAT3 and NF-κB. BEX2 was involved in mTOR up-regulation of VEGF production and angiogenesis. Depletion of BEX2 blunted the tumorigenesis of cells with activated mTOR. Therefore, enhanced STAT3/NF-κB-BEX2-VEGF signaling pathway contributes to hyperactive mTOR-induced tumorigenesis. BEX2 may be targeted for the treatment of the cancers with aberrantly activated mTOR signaling pathway.

Differential recognition preferences of the three Src homology 3 (SH3) domains from the adaptor CD2-associated protein (CD2AP), and direct association with Ras and Rab interactor 3 (RIN3) [Molecular Biophysics]

August 20th, 2015 by

CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three SH3 domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide-array screen based on the recognition motif P-x-P-x-P-R, and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine-nucleotide exchange factor (GEF). CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested, yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry (ITC) data show that the preferred binding motif is P-x-P/A-x-p-R. Two high-resolution crystal structures (1.65 Å and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains to c-CBL, ALIX and RIN3.
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Multiple Surface Regions on the Niemann-Pick C2 Protein Facilitate Intracellular Cholesterol Transport [Lipids]

August 20th, 2015 by

The cholesterol storage disorder Niemann-Pick type C (NPC) disease is caused by defects in either of two late endosomal/lysosomal (LE/LY) proteins, NPC1 and NPC2. NPC2 is a 16 kDa soluble protein that binds cholesterol in a 1:1 stoichiometry and can transfer cholesterol between membranes by a mechanism that involves protein-membrane interactions. To examine the structural basis of NPC2 function in cholesterol trafficking, a series of point mutations were generated across the surface of the protein. Several NPC2 mutants exhibited deficient sterol transport properties in a set of fluorescence-based assays. Notably, these mutants were also unable to promote egress of accumulated intracellular cholesterol from npc2-/- fibroblasts. The mutations mapped to several regions on the protein surface, suggesting that NPC2 can bind to more than one membrane simultaneously. Indeed, we have previously demonstrated that WT NPC2 promotes vesicle-vesicle interactions. These interactions were abrogated, however, by mutations causing defective sterol transfer properties. Molecular modeling shows that NPC2 is highly plastic, with several intense positively charged regions across the surface that could interact favorably with negatively charged membrane phospholipids. The point mutations generated in this study caused changes in NPC2 surface charge distribution with minimal conformational changes. The plasticity, coupled with membrane flexibility, likely allows for multiple cholesterol transfer routes. Thus, we hypothesize that, in part, NPC2 rapidly traffics cholesterol between closely appositioned membranes within the multilamellar interior of LE/LYs, ultimately effecting cholesterol egress from this compartment.

Human Mincle Binds to Cholesterol Crystals and Triggers Innate Immune Responses [Immunology]

August 20th, 2015 by

C-type lectin receptors (CLRs) are an emerging family of pattern-recognition receptors that recognizes pathogens or damaged-tissue to trigger innate immune responses. However, endogenous ligands for CLRs are not fully understood. In this study, we sought to identify an endogenous ligand(s) for human macrophage-inducible C-type lectin (hMincle). A particular fraction of lipid extracts from liver selectively activated reporter cells expressing hMincle. Mass spectrometry (MS) analysis determined the chemical structure of the active component as cholesterol. Purified cholesterol in plate-coated and crystalized forms activates reporter cells expressing hMincle but not murine Mincle (mMincle). Cholesterol crystals are known to activate immune cells and induce inflammatory responses through lysosomal damage. However, direct innate immune receptors for cholesterol crystals have not been identified. Murine macrophages transfected with hMincle responded to cholesterol crystals by producing pro-inflammatory cytokines. Human dendritic cells (DCs) expressed a set of inflammatory genes in response to cholesterol crystals and this was inhibited by anti-human Mincle. Importantly, other related CLRs did not bind cholesterol crystals, while other steroids were not recognized by hMincle. These results suggest that cholesterol crystals are an endogenous ligand for hMincle and activates innate immune responses.

The E3-ubiquitin ligases, HUWE1 and NEDD4-1, are involved in the post-translational regulation of the ABCG1 and ABCG4 lipid transporters [Cell Biology]

August 20th, 2015 by

The ATP-binding cassette transporter ABCG1 has an essential role in cellular cholesterol homeostasis, and dysregulation has been associated with a number of high burden diseases. Previous studies reported that ABCG1 is ubiquitinated and degraded via the ubiquitin proteasome system. However, so far the molecular mechanism, including the identity of any of the rate limiting ubiquitination enzymes, or E3 ligases, is unknown. Using liquid chromatography mass spectrometry, we identified two HECT domain E3 ligases associated with ABCG1, named HUWE1 (HECT, UBA and WWE domain containing 1, E3 ubiquitin protein ligase) and NEDD4-1 (Neural precursor cell-expressed developmentally down regulated gene 4), of which the latter is the founding member of the NEDD4 family of ubiquitin ligases. Silencing both HUWE1 and NEDD4-1 in cells overexpressing human ABCG1 significantly increased levels of the ABCG1 monomeric and dimeric protein forms, however ABCA1 protein expression was unaffected. In addition, ligase silencing increased ABCG1-mediated cholesterol export to HDL in cells overexpressing the transporter as well as in THP-1 macrophages. Reciprocally, overexpression of both ligases resulted in a significant reduction in protein levels of both the ABCG1 monomeric and dimeric forms. Like ABCG1, ABCG4 protein levels and cholesterol export activity were significantly increased after silencing both HUWE1 and NEDD4-1 in cells overexpressing this closely related ABC half-transporter. In summary, we have identified for the first time two E3 ligases that are fundamental enzymes in the post-translational regulation of ABCG1 and ABCG4 protein levels and cellular cholesterol export activity.
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The GH130 family of mannoside phosphorylases contains glycoside hydrolases that target beta-1,2 mannosidic linkages in Candida mannan [Glycobiology and Extracellular Matrices]

August 18th, 2015 by

The depolymerization of complex glycans is an important biological process that is of considerable interest to environmentally relevant industries. beta-mannose is a major component of plant structural polysaccharides and eukaryotic N-glycans. These linkages are primarily cleaved by glycoside hydrolases, although a family of glycoside phosphorylases, GH130, have also been shown to target beta-1,2 and beta-1,4 mannosidic linkages. In these phosphorylases bond cleavage was mediated by a single displacement reaction in which phosphate functions as the catalytic nucleophile. A cohort of GH130 enzymes, however, lack the conserved basic residues that bind the phosphate nucleophile, and it was proposed that these enzymes function as glycoside hydrolases. Here we show that two Bacteroides enzymes, BT3780 and BACOVA03624, which lack the phosphate binding residues are indeed betamannosidases that hydrolyse beta-1,2-mannosidic linkages through an inverting mechanism. As the genes encoding these enzymes are located in genetic loci that orchestrate the depolymerisation of yeast alpha-mannans, it is likely that the two enzymes target the beta-1,2-mannose residues that cap the glycan produced by Candida albicans. The crystal structure of BT3780 in complex with mannose bound in the -1 and +1 subsites showed a pair of glutamates, Glu227 and Glu268 hydrogen bond to O1 of alpha-mannose, and either of these residues may function as the catalytic base. The candidate catalytic acid and the other residues that interact with the active site mannose are conserved in both GH130 mannoside phosphorylases and beta-1,2-mannosidases. Functional phylogeny identified a conserved lysine, Lys199 in BT3780, as a key specificity determinant for beta-1,2-mannosidic linkages.
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Site-Directed Mutagenesis Shows the Significance of Interactions with Phospholipids and the G-protein OsYchF1 on the Physiological Functions of the Rice GTPase-Activating Protein 1 (OsGAP1) [Protein Structure and Folding]

August 18th, 2015 by

The C2 domain is one of the most diverse phospholipid-binding domains mediating cellular signaling. One group of C2-domain proteins are plant-specific and are characterized by their small sizes and simple structures. We have previously reported that a member of this group, OsGAP1, is able to alleviate salt stress and stimulate defense responses, and bind to both phospholipids and an unconventional G-protein, OsYchF1. Here we solved the crystal structure of OsGAP1 to a resolution of 1.63 Å. Using site-directed mutagenesis, we successfully differentiated between the clusters of surface residues that are required for binding to phospholipids versus OsYchF1, which, in turn, is critical for its role in stimulating defense responses. On the other hand, the ability to alleviate salt stress by OsGAP1 is dependent only on its ability to bind OsYchF1 and is independent of its phospholipid-binding activity.
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The capsule polymerase CslB of Neisseria meningitidis serogroup L catalyzes the synthesis of a complex trimeric repeating unit comprising glycosidic and phosphodiester linkages [Microbiology]

August 18th, 2015 by

Neisseria meningitidis (Nm) is a human pathogen causing bacterial meningitis and sepsis. The capsular polysaccharide (CPS) surrounding Nm is a major virulence factor. The CPS consists of poly-hexosamine-phosphates in NmA and NmX. The capsule polymerases (CPs) of these serogroups are members of the stealth protein family comprising D-hexose-1-phosphate-transferases from bacterial and protozoan pathogens. CslA, one out of two putative CPs of the pathophysiologically less relevant NmL, is one of the smallest known stealth proteins and caught our attention for structure-function-analyses. Since the NmL capsule polymer (CPSL) consists of a trimeric repeating unit ([→3)-β-D-GlcNAc-(1→3)-β-D-GlcNAc-(1→3)-α-D-GlcNAc-(1→OPO3→]n), we speculated that the two predicted CPs (CslA and CslB) work together in the polymer production. Consequently, both enzymes were cloned, overexpressed, and purified as recombinant proteins. Against our expectation, enzymatic testing identified CslB to be sufficient to catalyze the synthesis of the complex trimeric CPSL repeating unit. In contrast, no polymerase activity was detected for CslA, albeit the enzyme facilitated the hydrolysis of UDP-GlcNAc. Bioinformatics analyses identified two glycosyltransferase (GT) domains in CslB. The N-terminal domain modeled with 100% confidence onto a number of GT-A folded proteins while the C-terminal domain modelled with 100% confidence onto TagF, a teichoic acid polymerase from S. epidermidis. Amino acid positions known to have critical catalytic functions in the template proteins were conserved in CslB and their point mutation abolished enzyme activity. CslB represents an enzyme of so far unique complexity regarding both, the catalyzed reaction and enzyme architecture.
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