Untargeted plasma metabolomics identifies endogenous metabolite with drug-like properties in chronic animal model of multiple sclerosis [Immunology]

November 6th, 2015 by

We performed untargeted metabolomics of plasma from B6 mice with experimental autoimmune encephalitis (EAE) at the chronic phase of the disease in search of an altered metabolic pathway(s). Of 324 metabolites measured, 100 metabolites that mapped to various pathways (mainly lipids) linked to mitochondrial function, inflammation and membrane stability were observed to be significantly altered between EAE and healthy control (p < 0.05, false discover rate [< 0.10]. Bioinformatics analysis revealed 6 metabolic pathways being impacted and altered in EAE including alpha linolenic acid and linoleic acid metabolism (PUFA). The metabolites of PUFAs, including omega 3 and omega 6 fatty acids, are commonly decreased in mouse models and in multiple sclerosis patients. Daily oral administration of resolvin D1, a downstream metabolite of omega 3, decreased disease progression by suppressing autoreactive T cells and inducing a M2 phenotype of monocytes/macrophages and resident brain microglial cells. This study provides a proof of principle for the application of a metabolomic approach to identify endogenous metabolite(s) possessing drug-like properties, which is tested for therapy in preclinical mouse models.
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The redox state regulates the conformation of Rv2466c to activate the antitubercular prodrug TP053 [Microbiology]

November 6th, 2015 by

Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin fold with a Cys19-Pro20-Trp21-Cys22 motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle X-ray scattering showed that the fully reduced form of Rv2466c adopts a ′closed′ compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an ′open′ conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily.

Negative feed-forward control of TNF by tristetraprolin (ZFP36) is limited by the mitogen-activated protein kinase phosphatase, DUSP1: implications for regulation by glucocorticoids [Signal Transduction]

November 6th, 2015 by Shah, S., Mostafa, M. M., McWhae, A., Traves, S. L., Newton, R.

Tumor necrosis factor α (TNF) is central to inflammation and may play a role in the pathogenesis of asthma. The 3′-untranslated region of the TNF transcript contains AU-rich elements (AREs) that are targeted by the RNA-binding protein, tristetraprolin (ZFP36), which is itself up-regulated by inflammatory stimuli, to promote mRNA degradation. Using primary human bronchial epithelial (HBE) and pulmonary epithelial A549 cells, we confirm that interleukin-1β (IL1B) induces expression of dual-specificity phosphatase 1 (DUSP1), ZFP36 and TNF. While IL1B-induced DUSP1 is involved in feedback control of MAPK pathways, ZFP36 exerts negative (incoherent) feed-forward control of TNF mRNA and protein expression. DUSP1 silencing increased IL1B-induced ZFP36 expression at 2h and profoundly repressed TNF mRNA at 6h. This was partly due to increased TNF mRNA degradation, an effect that was reduced by ZFP36 silencing. This confirms a regulatory network, whereby DUSP1-dependent negative feedback control reduces feed-forward control by ZFP36. Conversely, while DUSP1 over-expression and inhibition of MAPKs prevented IL1B-induced expression of ZFP36, this was associated with increased TNF mRNA expression at 6h, an effect that was predominantly due to elevated transcription. This points to MAPK-dependent feed-forward control of TNF involving ZFP36-dependent and -independent mechanisms. In terms of repression by dexamethasone, neither silencing of DUSP1, ZFP36, nor both together, prevented the repression of IL1B-induced TNF expression thereby demonstrating the need for further repressive mechanisms by anti-inflammatory glucocorticoids. In summary, these data illustrate why understanding the competing effects of feedback and feed-forward control is relevant to the development of novel anti-inflammatory therapies.
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Sar1 GTPase activity is regulated by membrane curvature [Membrane Biology]

November 6th, 2015 by

The majority of biosynthetic secretory proteins initiate their journey through the endomembrane system from specific subdomains of the endoplasmic reticulum (ER). At these locations, coated transport carriers are generated, with the Sar1 GTPase playing a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation. How these events are appropriately coordinated remains poorly understood. Here, we demonstrate that Sar1 acts as the curvature sensing component of the COPII coat complex and highlight the ability of Sar1 to bind more avidly to membranes of high curvature. Additionally, using an atomic force microscopy-based approach, we further show that the intrinsic GTPase activity of Sar1 is necessary for remodeling lipid bilayers. Consistent with this idea, Sar1-mediated membrane remodeling is dramatically accelerated in the presence of its guanine nucleotide activating protein (GAP), Sec23-Sec24, and blocked upon addition of GMP-PNP, a poorly hydrolysable analog of GTP. Our results also indicate that Sar1 GTPase activity is stimulated by membranes that exhibit elevated curvature, potentially enabling Sar1 membrane scission activity to be spatially restricted to highly bent membranes that are characteristic of a bud neck. Taken together, our data support a stepwise model in which the amino-terminal amphipathic helix of GTP-bound Sar1 stably penetrates the ER membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission.

Cytokine Activation by Antibody Fragments Targeted to Cytokine-Receptor Signaling Complexes [Molecular Biophysics]

November 6th, 2015 by

Exogenous cytokine therapy can induce systemic toxicity, which might be prevented by activating endogenously produced cytokines in local cell niches. Here, we developed antibody-based activators of cytokine signaling (AcCS), which recognize cytokines only when they are bound to their cell surface receptors. AcCS were developed for type I interferons (IFNs), which induce cellular activities by binding to cell surface receptors IFNAR1 and IFNAR2. As a potential alternative to exogenous IFN therapy, AcCS were shown to potentiate the biological activities of natural IFNs by ~100-fold. Biochemical and structural characterization demonstrate the AcCS stabilize the IFN/IFNAR2 binary complex by recognizing an IFN-induced conformational change in IFNAR2. Using IFN mutants that disrupt IFNAR1 binding, AcCS were able to enhance IFN antiviral potency without activating antiproliferative responses. This suggests AcCS can be used to manipulate cytokine signaling for basic science and possibly for therapeutic applications.

Antiviral Cystine Knot {alpha}-Amylase Inhibitors from Alstonia scholaris [Protein Structure and Folding]

November 6th, 2015 by

Cystine knot α-amylase inhibitors are cysteine-rich, proline-rich peptides found in the Amaranthaceae and Apocynaceae plant species. They are characterized by a pseudocyclic backbone with 2-4 prolines and three disulfides arranged in a knotted motif. Similar to other knottins, cystine knot α-amylase inhibitors are highly resistant to degradation by heat and protease treatments. Thus far, only the α-amylase inhibition activity has been described for members of this family. Here, we show that cystine knot α-amylase inhibitors named alstotides discovered from the Alstonia scholaris plant of the Apocynaceae family display antiviral activity. The alstotides (As1-As4) were characterized by both proteomic and genomic methods. All four alsotides are novel, heat- and enzyme-stable and contain 30 residues. NMR determination of As1 and As4 structures reveals their conserved structural fold and the presence of one or more cis proline bonds, characteristics shared by other cystine knot α-amylase inhibitors. Genomic analysis showed that they contain a three-domain precursor, an arrangement common to other knottins. We also showed that alstotides are antiviral and cell-permeable to inhibit the early phase of infectious bronchitis virus and Dengue infection, in addition to their ability to inhibit α-amylase. Taken together, our results expand membership of cystine knot α-amylase inhibitors in the Apocynaceae family and their bioactivity, functional promiscuity which could be exploited as leads in developing therapeutics.

The Inhibitory Mechanism of the {zeta} Subunit of the F1FO-ATPase Nanomotor of Paracoccus denitrificans and Related {alpha}-Proteobacteria. [Enzymology]

November 6th, 2015 by

The ζ subunit is a novel inhibitor of the F1FO-ATPase of Paracoccus denitrificans and related α-proteobacteria, different to bacterial ϵ and mitochondrial IF1 inhibitors. The N-terminus of ζ blocks rotation of the γ subunit of the F1-ATPase of P. denitrificans (Zarco-Zavala, M., Morales-Rios, E., Mendoza-Hernandez, G., Ramirez-Silva, L., Perez-Hernandez, G., and Garcia-Trejo, J.J. (2014) FASEB J. 24, 599-608) by a hitherto unknown quaternary structure that was modeled here by structural homology and protein docking. The F1 and F1-ζ models of Paracoccus denitrificans were supported by cross-linking, limited proteolysis, mass spectrometry, and functional data. The final models show that ζ enters into F1 at the open catalytic αE-βE interface, and two partial γ rotations lock the N-terminus of ζ in an inhibition-general core region, blocking further γ rotation, whilst the ζ globular domain anchors it to the closed αDP-βDP interface. Heterologous inhibition of the F1-ATPase of Paracoccus denitrificans by the mitochondrial IF1 supported both, the modelled ζ binding site at the αDP-βDP-γ interface, as well as the endosymbiotic α-proteobacterial origin of mitochondria. In sum, the ζ subunit blocks the intrinsic rotation of the nanomotor by inserting its N-terminal inhibitory domain at the same rotor-stator interface where the mitochondrial IF1 or the bacterial ϵ bind, with a proposed pawl mechanism coupled to the rotation of the central γ subunit working as a ratchet, but with structural differences that makes it a unique control mechanism of the nanomotor to favour the ATP synthase activity over the ATPase turnover in the α-proteobacteria.
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Specificity of collybistin-phosphoinositide interactions: Impact of the individual protein domains [Neurobiology]

November 6th, 2015 by

The regulatory protein collybistin (CB)2 recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full length CB2 (CB2SH3+) harboring the N-terminal SH3-domain adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A-E262A mutation, which conformationally 'opens' CB2SH3+ and allows the PH domain to properly bind lipids depending on the phosphoinositide species with a preference for PI(3)P and PI(4)P. This type of membrane tethering under the control of the release of the SH3-domain of CB is essential for regulating gephyrin clustering.

Cooperative Binding of Stromal Interaction Molecule 1 (STIM1) to the N and C Termini of Calcium Release-Activated Calcium Modulator 1 (Orai1) [Membrane Biology]

November 6th, 2015 by Palty, R., Isacoff, E. Y.

Calcium flux through store operated calcium entry (SOCE) is a central regulator of intracellular calcium signaling. The two key components of the store operated calcium release activated calcium (CRAC) channel are the Ca2+ sensing protein stromal interaction molecule 1 (STIM1) and the channel pore forming protein Orai1. During SOCE activation calcium depletion from the endoplasmic reticulum triggers a series of conformational changes in STIM1 that unmask a minimal Orai1 activating domain (CAD). In order to gate Orai1 channels the exposed STIM1 activating domain binds to two sites in Orai1, one in the N terminus and one in the C terminus. Whether the two sites operate as distinct binding domains or cooperate in CAD binding is unknown. In this study we show that the N and C terminal domains of Orai1 synergistically contribute to interaction with STIM1 and couple STIM1 binding with channel gating and modulation of ion selectivity.
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In silico prediction of human sulfotransferase 1E1 activity guided by pharmacophores from molecular dynamics simulations [Computational Biology]

November 5th, 2015 by

Acting during phase II metabolism, sulfotransferases (SULTs) serve detoxification by transforming a broad spectrum of compounds from pharmaceutical, nutritional, or environmental sources into more easily excretable metabolites. However, SULT activity has also been shown to promote formation of reactive metabolites that may have genotoxic effects. SULT subtype 1E1 (SULT1E1) was identified as a key player in estrogen homeostasis, which is involved in many physiological processes and the pathogenesis of breast and endometrial cancer. The development of an in silico prediction model for SULT1E1 ligands would therefore support the development of metabolically inert drugs and help to assess health risks related to hormonal imbalances. Here, we report on a novel approach to develop a model that enables prediction of substrates and inhibitors of SULT1E1. Molecular dynamics simulations were performed to investigate enzyme flexibility and sample protein conformations. Pharmacophores were developed that served as a cornerstone of the model and machine learning techniques were applied for prediction refinement. The prediction model was used to screen the DrugBank (a database of experimental and approved drugs): 28 % of the predicted hits were reported in literature as ligands of SULT1E1. From the remaining hits, a selection of nine molecules was subjected to biochemical assay validation and experimental results were in accordance with the in silico prediction of SULT1E1 inhibitors and substrates, thus affirming our prediction hypotheses.
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