Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site [Protein Structure and Folding]

November 12th, 2015 by

The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic BAX is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here, we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by BH3-only protein, tBID, in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen- deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices α1/α6 and the α1-α2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights on BAX inhibition and provide a blueprint for developing inhibitors of BAX activation.
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A Revised Mechanism for Human Cyclooxygenase-2 [Molecular Bases of Disease]

November 12th, 2015 by Liu, Y., Roth, J.

The mechanism of omega-6 polyunsaturated fatty acid oxidation by wild-type COX-2 and the Y334F variant, lacking a conserved tyrosine that hydrogen bond to the catalytic tyrosyl radical/tyrosine, was examined for the first time under physiologically relevant conditions. The enzymes show apparent bimolecular rate constants and deuterium kinetic isotope effects that increase in proportion to co-substrate concentrations before converging to limiting values. The trends exclude multiple dioxygenase mechanisms as well as the proposal that initial hydrogen atom abstraction from the fatty acid is the first irreversible step in catalysis. Temperature-dependent studies reinforce the novel finding that hydrogen transfer from the reduced catalytic tyrosine to a terminal peroxyl radical is likely the first irreversible step that controls regio and stereospecific product formation

Tetrahydrobiopterin Biosynthesis as a Potential Target of the Kynurenine Pathway Metabolite Xanthurenic Acid [Metabolism]

November 12th, 2015 by Haruki, H., Hovius, R., Gronlund Pedersen, M., Johnsson, K.

Tryptophan metabolites in the kynurenine pathway are up-regulated by pro-inflammatory cytokines or glucocorticoids, and are linked to anti-inflammatory and immunosuppressive activities. In addition, they are up-regulated in pathologies such as cancer, autoimmune diseases, and psychiatric disorders. The molecular mechanisms of how kynurenine pathway metabolites cause these effects are incompletely understood. On the other hand, pro-inflammatory cytokines also up-regulate the amounts of tetrahydrobiopterin (BH4), an enzyme cofactor essential for the synthesis of several neurotransmitter and nitric oxide species. Here we show that xanthurenic acid is a potent inhibitor of sepiapterin reductase (SPR), the final enzyme in de novo BH4 synthesis. The crystal structure of xanthurenic acid bound to the active site of SPR reveals why among all kynurenine pathway metabolites xanthurenic acid is the most potent SPR inhibitor. Our findings suggest that increased xanthurenic acid levels resulting from up-regulation of the kynurenine pathway could attenuate BH4 biosynthesis and BH4-dependent enzymatic reactions, linking two major metabolic pathways known to be highly up-regulated in inflammation.

Monitoring Ras Interactions with the Nucleotide Exchange Factor Sos using Site-specific NMR Reporter Signals and Intrinsic Fluorescence [Molecular Biophysics]

November 12th, 2015 by

The activity of Ras is controlled by the inter-conversion between GTP- and GDP-bound forms, partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly [15N]-labeled Ras, as well as [13C-methyl-M,I]-labeled Sos, for observing site-specific details of Ras:Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP, or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized, by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants, as well as their selected functional mutants, was also investigated using intrinsic fluorescence. The data supports a positive feedback activation of Sos by Ras-GTP, with Ras-GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras-GDP, suggesting that Sos should actively promote unidirectional GDP→GTP exchange on Ras, in preference of passive homonucleotide exchange. Ras-GDP weakly binds to the catalytic, but not to the allosteric site of Sos. This confirms that Ras-GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras:Sos interactions.
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Contributions of the Prion Protein Sequence, Strain and Environment to the Species Barrier [Protein Structure and Folding]

November 12th, 2015 by

Amyloid propagation requires high levels of sequence specificity so that only molecules with very high sequence identity can form cross-β-sheet structures of sufficient stringency for incorporation into the amyloid fibril. This sequence specificity presents a barrier to transmission of prions between two species with divergent sequences, termed a species barrier. Here, we study the relative effects of protein sequence, seed conformation and environment on the species barrier strength and specificity for the yeast prion protein, Sup35p, from three closely related species of the Saccharomyces senso stricto group, namely S. cerevisiae, S. bayanus and S. paradoxus. Through in vivo plasmid shuffle experiments we show that the major characteristics of the transmission barrier and conformational fidelity are determined by the protein sequence rather than cellular environment. In vitro data confirm that kinetics and structural preferences of aggregation of the S. paradoxus and S. bayanus proteins are influenced by anions in accordance to their positions in the Hofmeister series, as previously observed for S. cerevisiae. However, the specificity of the species barrier is primarily affected by the sequence and the type of anion present during the formation of the initial seed, while anions present during the seeded aggregation process typically influence kinetics rather than the specificity of prion conversion. Thus, our work shows that the protein sequence and the conformation variant (strain) of prion seed are the primary determinants of cross-species prion specificity both in vivo and in vitro.

Connexin 43 is necessary for salivary gland branching morphogenesis and FGF10-induced ERK1/2 phosphorylation [Molecular Bases of Disease]

November 12th, 2015 by

Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Conenxin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43-/- salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43-/- samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43-/- phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.

The N-terminal region of CHD4 is essential for activity and contains a HMG-box-like-domain that can bind poly(ADP-ribose) [Gene Regulation]

November 12th, 2015 by

Chromodomain Helicase DNA-binding protein 4 (CHD4) is a chromatin-remodeling enzyme that has been reported to regulate DNA damage responses through its N-terminal region in a poly(ADP-ribose) polymerase dependent manner. We have identified and determined the structure of a stable domain (CHD4-N) in this Nterminal region. The fold consists of a four α-helix bundle with structural similarity to the High Mobility Group (HMG) box, a domain that is well known as a DNA-binding module. We show that the CHD4-N domain binds with higher affinity to poly(ADP-ribose) than to DNA. We also show that the N-terminal region of CHD4, although not CHD4-N alone, is essential for full nucleosome remodeling activity and is important for localizing CHD4 to sites of DNA damage. Overall, these data build on our understanding of how CHD4/NuRD acts to regulate gene expression and participates in the DNA-damage response.

A Cytosolic Multiprotein Complex containing p85{alpha} is required for {beta}-catenin Activation in Colitis and Colitis-associated Cancer [Signal Transduction]

November 12th, 2015 by

Wnt/β-catenin signaling is required for crypt structure maintenance. We previously observed nuclear accumulation of Ser552 phosphorylated β-catenin (pβ-CatSer552) in intestinal epithelial cells (IEC) during colitis and colitis associated cancer (CAC). Data here delineate a novel multiprotein cytosolic complex (MCC) involved in β-catenin signaling in the intestine. The MCC contains p85α, the class IA subunit of PI3K, along with β-catenin, 14-3-3ζ, Akt and p110α. MCC levels in IEC increase in colitis and CAC patients. IEC-specific p85α-deficient (p85ΔIEC) mice develop more severe dextran sodium sulfate (DSS) colitis due to delayed ulcer healing and reduced epithelial β-catenin activation. In colonic IEC, p85α deficiency did not alter PI3K signaling. In vitro shRNA depletion of individual complex members disrupts the MCC and reduces β-catenin signaling. Despite worse colitis, p85ΔIEC mice have reduced tumor burden after azoxymethane (AOM)/DSS treatment. Together the data indicate that the β-catenin MCC is needed for mucosal repair and carcinogenesis. This novel MCC may be an attractive therapeutic target in preventing cancer in colitis patients.
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Deletion of Monoglyceride Lipase in Astrocytes Attenuates Lipopolysaccharide-Induced Neuroinflammation [Neurobiology]

November 12th, 2015 by

Monoglyceride lipase (MGL) is required for efficient hydrolysis of the endocannabinoid 2-arachidonoylglyerol (2-AG) in the brain generating arachidonic acid (AA) and glycerol. This metabolic function makes MGL an interesting target for the treatment of neuroinflammation, since 2-AG exhibits anti-inflammatory properties and AA is a precursor for pro-inflammatory prostaglandins. Astrocytes are an important source of AA and 2-AG, and highly express MGL. In the present study, we dissected the distinct contribution of MGL in astrocytes on brain 2-AG and AA metabolism by generating a mouse model with genetic deletion of MGL specifically in astrocytes (MKOGFAP). MKOGFAP mice exhibit moderately increased 2-AG and reduced AA levels in brain. Minor accumulation of 2-AG in the brain of MKOGFAP mice does not cause cannabinoid receptor desensitization as previously observed in mice globally lacking MGL. Importantly, MKOGFAP mice exhibit reduced brain prostaglandin E2 and pro-inflammatory cytokines levels upon peripheral lipopolysaccharide (LPS) administration. These observations indicate that MGL-mediated degradation of 2-AG in astrocytes provides AA for prostaglandin synthesis promoting LPS-induced neuroinflammation. The beneficial effect of astrocyte-specific MGL-deficiency is not fully abrogated by the inverse cannabinoid receptor 1 agonist SR141716 (Rimonabant) suggesting that the anti-inflammatory effects are rather caused by reduced prostaglandin synthesis than by activation of cannabinoid receptors. In conclusion, our data demonstrate that MGL in astrocytes is an important regulator of 2-AG levels, AA availability, and neuroinflammation.

Transforming Growth Factor-{beta}1-induced Apoptosis in Podocytes via Extracellular-signal-regulated Kinase-Mammalian Target of Rapamycin Complex 1-NADPH Oxidase 4 Axis [Cell Biology]

November 12th, 2015 by

Transforming Growth Factor-β (TGF-β) is a pleiotropic cytokine which accumulates during kidney injuries resulting in various renal diseases. We have previously reported that TGF-β1 induces selective upregulation of mitochondrial Nox4 playing critical roles in podocyte apoptosis. Here, we investigated the regulatory mechanism of Nox4 upregulation by mTORC1 activation on TGF-β1-induced apoptosis in immortalized podocytes. TGF-β1 treatment markedly increased phosphorylation of mTOR and its downstream target p70S6K and 4EBP1. Blocking TGF-β receptor-I by SB431542 completely blunted phosphorylation of mTOR, p70S6K and 4EBP1. Transient adenoviral over-expression of mTOR-WT and constitutively active mTORΔ augmented TGF-β1-treated Nox4 expression, ROS generation and apoptosis, while mTOR-KD suppressed above changes. In addition, knock-down of mTOR by simTOR mimicked the effect of mTOR-KD. Inhibition of mTORC1 by low dose of rapamycin or sip70S6K protected podocytes through attenuation of Nox4 expression and subsequent oxidative stress-induced apoptosis by TGF-β1. Pharmacological inhibition of MEK-ERK cascade, but not PI3K-Akt-TSC2 pathway, abolished TGF-β1-induced mTOR activation. Inhibition of neither ERK1/2 nor mTORC1 reduced the TGF-β1-stimulated increase of Nox4 mRNA level, however, significantly inhibited total Nox4 expression, ROS generation and apoptosis induced by TGF-β1. Moreover, double knock-down of Smad2 and 3 or only Smad4 completely suppressed TGF-β1-induced ERK1/2-mTOR activation. Our data suggest that TGF-β1 increases translation of Nox4 through Smad-ERK1/2-mTORC1 axis, which is independent of transcriptional regulation. Activation of this pathway plays a crucial role in ROS generation and mitochondrial dysfunction leading to podocyte apoptosis. Therefore, inhibition of ERK1/2-mTORC1 pathway could be a potential therapeutic and preventive target against proteinuric and chronic kidney diseases.
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