Structure of the Legionella Effector, lpg1496, Suggests a Role in Nucleotide Metabolism [Protein Structure and Folding]

August 20th, 2015 by Wong, K., Kozlov, G., Zhang, Y., Gehring, K.

Pathogenic gram-negative bacteria use specialized secretion systems that translocate bacterial proteins, termed effectors, directly into host cells where they interact with host proteins and biochemical processes for the benefit of the pathogen. Lpg1496 is a previously uncharacterized effector of Legionella pneumophila, the causative agent of Legionnaire's disease. Here, we crystallized three nucleotide-binding domains from lpg1496. The C-terminal domain, which is conserved among the SidE family of effectors, is formed of two largely α-helical lobes with a nucleotide-binding cleft. A structural homology search has shown similarity to phosphodiesterases involved in cleavage of cyclic nucleotides. We have also crystallized a novel domain that occurs twice in the N-terminal half of the protein that we term KLAMP domains due to the presence of homologous domains in bacterial histidine kinase-like ATP-binding region-containing proteins and S-adenosylmethionine-dependent methyltransferase proteins. Both KLAMP structures are very similar but selectively bind 3′,5′-cAMP and ADP. A co-crystal of the KLAMP1 domain with 3′,5′-cAMP reveals the contribution of Tyr61 and Tyr69 that produce π-stacking interactions with the adenine ring of the nucleotide. Our study provides the first structural insights into two novel nucleotide-binding domains associated with bacterial virulence.

The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP [Signal Transduction]

August 20th, 2015 by

Evidence suggests that the plasma membrane Ca2+ ATPase (PMCA), which is critical for maintaining a low intracellular Ca2+ concentration ([Ca2+]i), utilises glycolytically-derived ATP in pancreatic ductal adenocarcinoma (PDAC), and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition and an irreversible [Ca2+]i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype towards mitochondrial metabolism and assessing the effects of mitochondrial vs glycolytic inhibitors on ATP depletion, PMCA inhibition and [Ca2+]i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate (KIC) or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitising cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca2+]i in KIC and galactose-cultured cells, suggesting that the glycolytic dependency of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.

Rapid Fine Conformational Epitope Mapping Using Comprehensive Mutagenesis and Deep Sequencing [Immunology]

August 20th, 2015 by

Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high-throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions, and show the method effectiveness by mapping the fine epitope for different antibodies targeting TNF, Pertussis Toxin, and the cancer target TROP2. In all three cases, the experimentally determined conformational epitope was consistent with previous experimental datasets, confirming the reliability of the experimental pipeline. Once the comprehensive library is generated, fine conformational epitope maps can be prepared at a rate of four per day.

Lipin1 regulates skeletal muscle differentiation through ERK activation and Cyclin D complex regulated cell cycle withdrawal [Molecular Bases of Disease]

August 20th, 2015 by

Lipin1, an intracellular protein, plays critical roles in controlling lipid synthesis and energy metabolism through its enzymatic activity and nuclear transcriptional functions. Several mouse models of skeletal muscle wasting are associated with lipin1 mutation or altered expression. Recent human studies have suggested that children with homozygous null mutations in the LPIN1 gene suffer from rhabdomyolysis. However, the underlying pathophysiologic mechanism is still poorly understood. In the present study, we examined whether lipin1 contributes to regulating muscle regeneration. We characterized the time course of skeletal muscle regeneration in lipin1 deficient fld mice following injury. We found that fld mice exhibited smaller regenerated muscle fiber cross-sectional areas and increased levels of fibrosis compared with wild-type mice in response to injury. Our results from a series of in vitro experiments suggest that lipin1 is upregulated and translocated to the nucleus during myoblast differentiation, and plays a key role in myogenesis by regulating the cytosolic activation of ERK1/2 to form a complex and a downstream effector Cyclin D3-mediated cell cycle withdrawal. Overall, our study reveals a previously unknown role of lipin1 in skeletal muscle regeneration and expands our understanding of the cellular and molecular mechanisms underlying skeletal muscle regeneration.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Lipin1 regulates skeletal muscle differentiation through ERK activation and Cyclin D complex regulated cell cycle withdrawal [Molecular Bases of Disease]

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.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Selective Irreversible Inhibition of Neuronal and Inducible Nitric-Oxide Synthase in the Combined Presence of Hydrogen Sulfide and Nitric Oxide [Enzymology]

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.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on 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]

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.