Complement component C5a primes retinal pigment epithelial cells for inflammasome activation by lipofuscin-mediated photooxidative damage [Immunology]

November 12th, 2015 by Brandstetter, C., Holz, F. G., Krohne, T. U.

Complement activation, oxidative damage, and activation of the NLRP3 inflammasome have been implicated in retinal pigment epithelium (RPE) pathology in age-related macular degeneration (AMD). Following priming of RPE cells, the NLRP3 inflammasome can be activated by various stimuli such as lipofuscin-mediated photooxidative damage to lysosomal membranes. We investigated whether products of complement activation are capable of providing the priming signal for the inflammasome in RPE cells. Incubation of primary human RPE cells and ARPE-19 cells with complement-competent human serum resulted in upregulation of C5a receptor, but not C3a receptor. Furthermore, it induced expression of pro-IL-1β and enabled IL-1β secretion in response to lipofuscin phototoxicity, thus indicating inflammasome priming by human serum. Complement heat-inactivation, C5 depletion, and C5a receptor inhibition suppressed the priming effect of human serum whereas recombinant C5a likewise induced priming. Conditioned media of inflammasome-activated RPE cells provided an additional priming effect that was mediated by the IL-1 receptor. These results indicate that complement activation product C5a represents a priming signal for RPE cells that allows for subsequent inflammasome activation by stimuli such as lipofuscin-mediated photooxidative damage. This molecular pathway provides a functional link between key factors of AMD pathogenesis including lipofuscin accumulation, photooxidative damage, complement activation, and RPE degeneration and may provide novel therapeutic targets in this disease.
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MAP1S regulates the phagocytosis of bacteria and TLR signaling [Signal Transduction]

November 12th, 2015 by

Phagocytosis is a critical cellular process for innate immune defense against microbial infection. The regulation of phagocytosis process is complex and has not been well defined. An intracellular molecule might regulate the cell surface initiated phagocytosis; however, the underlying molecular mechanism is poorly understood (1). In this study, we found that the microtubule-associated protein 1S (MAP1S), a recently identified protein involved in autophagy (2), is primarily expressed in macrophages. MAP1S-deficient Macrophages are impaired in phagocytosis of bacteria. Furthermore, we demonstrate that MAP1S directly interacts with MyD88, a key adaptor of TLRs, upon TLR activation and affects the TLR signaling pathway. Intriguingly, we also observe that upon TLR activation, MyD88 participates in autophagy processing, in a MAP1S-dependent manner, by co-localizing with MAP1 light chain-3 (MAP1-LC3, or LC3). Thus, we reveal that an intracellular autophagy-related molecule MAP1S controls bacterial phagocytosis through TLR signaling.

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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