AztD, a Periplasmic Zinc Metallochaperone to an ATP Binding Cassette (ABC) Transporter System in Paracoccus denitrificans [Molecular Biophysics]

October 14th, 2015 by Handali, M., Roychowdhury, H., Neupane, D. P., Yukl, E. T.

Bacterial ATP binding cassette (ABC) transporters of transition metals are essential for acquisition of necessary elements from the environment. A large number of gram-negative bacteria including human pathogens have a fourth conserved gene of unknown function adjacent to the canonical permease, ATPase and solute binding protein (SBP) genes of the AztABC Zn transporter system. To assess the function of this putative accessory factor (AztD) from Paracoccus denitrificans, we have analyzed its transcriptional regulation, metal binding properties and interaction with the SBP (AztC). Transcription of the aztD gene is significantly upregulated under conditions of Zn starvation. Recombinantly expressed AztD purifies with slightly substoichiometric Zn from the periplasm of E. coli and is capable of binding up to 3 Zn ions with high affinity. Size exclusion chromatography and a simple intrinsic fluorescence assay were used to determine that AztD as isolated is able to transfer bound Zn nearly quantitatively to apo-AztC. Transfer occurs through a direct, associative mechanism that prevents loss of metal to the solvent. These results indicate that AztD is a Zn chaperone to AztC and likely functions to maintain Zn homeostasis through interaction with the AztABC system. This work extends our understanding of periplasmic Zn trafficking and the function of chaperones in this process.
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Ca2+ influx through store-operated calcium channels replenishes the functional phosphatidylinositol 4,5-bisphosphate pool used by cysteinyl leukotriene type I receptors [Membrane Biology]

October 14th, 2015 by Alswied, A., Parekh, A. B.

Oscillations in cytoplasmic Ca2+ concentration are a universal mode of signalling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca2+ oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5bisphosphate (PIP2), the source of the Ca2+-releasing second messenger inositol trisphosphate (InsP3). Here we show that cytoplasmic Ca2+ oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pre-treated with Li+, an inhibitor of inositol monophosphatases and which prevents PIP2 resynthesis. In Li+-treated cells, cytoplasmic Ca2+ signals evoked by agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knock down of phosphatidylinositol 4-phosphate 5- kinases (PIP5 kinases) α and γ resulted in rapid loss of the intracellular Ca2+ oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca2+ oscillations and these could not be rescued by inositol or PI4P. In Li+-treated cells, recovery of the cytoplasmic Ca2+ oscillations in the presence of inositol or PI4P was suppressed when Ca2+ influx through store-operated Ca2+ channels was inhibited. After rundown of the Ca2+ signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent InsP3-dependent Ca2+ release. Hence leukotriene and P2Y receptors utilise distinct membrane PIP2 pools. Our findings show that store-operated Ca2+ entry is needed to sustain cytoplasmic Ca2+ signalling following leukotriene receptor activation both by refilling the Ca2+ stores and by helping to replenish the PIP2 pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.
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Metal Ion-Dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix [Protein Structure and Folding]

October 14th, 2015 by

The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a crosslinked network by the cooperative action of three proteins, inter-alpha-inhibitor (IalphaI), pentraxin-3 and TNF-induced protein-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. While it is known that matrix stabilization involves the TSG-6-mediated transfer of IalphaI heavy chains (HC) onto hyaluronan (to form covalent HC-HA complexes that are crosslinked by pentraxin-3), and that this occurs via the formation of covalent HC-TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC-TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of a HC-associated magnesium ion. In addition, we have found that the well-characterised hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with either impaired transferase and/or hyaluronan-binding functions), revealed that while the TSG-6-mediated formation of HC-HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.

A2E Accumulation and the Maintenance of the Visual Cycle are Independent of Atg7-mediated Autophagy in the Retinal Pigmented Epithelium [Cell Biology]

October 14th, 2015 by

Autophagy is an evolutionarily conserved catabolic mechanism that relieves cellular stress by removing/recycling damaged organelles and debris through the action of lysosomes. Compromised autophagy has been implicated in many neurodegenerative diseases, including retinal degeneration. Here we examined retinal phenotypes resulting from RPE-specific deletion of the autophagy regulatory gene Atg7 by generating Atg7flox/flox;VMD2-rtTA-cre+ mice to determine whether autophagy is essential for RPE functions including retinoid recycling. Atg7 deficient RPE displayed abnormal morphology with increased RPE thickness, cellular debris and vacuole formation indicating that autophagy is important in maintaining RPE homeostasis. In contrast, 11-cis-retinal content, ERGs and retinal histology were normal in mice with Atg7 deficient RPE in both fasted and fed states. Because A2E accumulation in the RPE is associated with pathogenesis of both Stargardt disease and age-related macular degeneration (AMD) in humans, deletion of Abca4 was introduced into Atg7flox/flox;VMD2-rtTA-cre+ mice to investigate the role of autophagy during A2E deposition. Comparable A2E concentrations were detected in the eyes of 6-month-old mice with and without Atg7 from both Abca4-/- and Abca4+/+ backgrounds. To identify other autophagy-related molecules involved in A2E accumulation, we performed gene expression array analysis on A2E-treated human RPE cells and found upregulation of four autophagy related genes; DRAM1, NPC1, CASP3, and EIF2AK3/PERK. These observations indicate that Atg7-mediated autophagy is dispensable for retinoid recycling and A2E deposition; however, autophagy plays a role in coping with stress caused by A2E accumulation.
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Characterization of RA839, a non-covalent small-molecule binder to Keap1 and selective activator of Nrf2 signalling [Metabolism]

October 12th, 2015 by

The activation of the transcription factor NF-E2-related factor 2 (Nrf2) maintains cellular homeostasis in response to oxidative stress by the regulation of multiple cytoprotective genes. Without stressors the activity of Nrf2 is inhibited by its interaction with the kelch-like ECH-associated protein 1 (Keap1). Here, we describe RA839, a small molecule that binds non-covalently to the Nrf2-interacting kelch domain of Keap1 with a Kd of approximately 6 μM, as demonstrated by X-ray co-crystallization and isothermal titration calorimetry. Whole-genome DNA arrays showed that at 10 μM RA839 significantly regulated 105 genes in bone marrow-derived macrophages. Canonical pathway mapping of these genes revealed an activation of pathways linked with Nrf2 signalling. These pathways were also activated after the activation of Nrf2 by the silencing of Keap1 expression. RA839 regulated only two genes in Nrf2 knockout macrophages. Similar to the activation of Nrf2 by either silencing of Keap1 expression or by the reactive compound CDDO-Me, RA839 prevented the induction of both inducible nitric oxide synthase expression and nitric oxide release in response to lipopolysaccharides in macrophages. In mice RA839 acutely induced Nrf2-target gene expression in liver. RA839 is a selective inhibitor of the Keap1/Nrf2 interaction and a useful tool compound to study the biology of Nrf2.

The solution structure of the lantibiotic immunity protein NisI and its interactions with nisin [Microbiology]

October 12th, 2015 by

Many Gram-positive bacteria produce lantibiotics - genetically encoded and posttranslationally modified peptide antibiotics, which inhibit the growth of other Gram-positive bacteria. In order to protect themselves against their own lantibiotics these bacteria express a variety of immunity proteins including the LanI lipoproteins. The structural and mechanistic basis for LanI-mediated lantibiotic immunity is not yet understood. Lactococcus lactis produces the lantibiotic nisin, which is widely used as a food preservative. Its LanI protein NisI provides immunity against nisin but not against structurally very similar lantibiotics from other species such as subtilin from B. subtilis. In order to understand the structural basis for LanI mediated immunity and their specificity we investigated the structure of NisI. We found that NisI is a two-domain protein. Surprisingly, each of the two NisI domains has the same structure as the LanI protein from B. subtilis, SpaI, despite the lack of significant sequence homology. The two NisI domains and SpaI differ strongly in their surface properties and function. Additionally, SpaI mediated lantibiotic immunity depends on the presence of a basic unstructured N-terminal region that tethers SpaI to the membrane. Such a region is absent from NisI. Instead, the N-terminal domain of NisI interacts with membranes but not with nisin. In contrast, the C-terminal domain specifically binds nisin and modulates the membrane affinity of the N-terminal domain. Thus, our results reveal an unexpected structural relationship between NisI and SpaI and shed light on the structural basis for LanI mediated lantibiotic immunity.

Uncovering the Mechanism of Aggregation of Human Transthyretin. [Protein Structure and Folding]

October 12th, 2015 by

The tetrameric thyroxine-transport protein transthyretin (TTR) forms amyloid fibrils upon dissociation and monomer unfolding. The aggregation of transthyretin has been reported as the cause of the life-threatening transthyretin amyloidosis. The standard treatment of familial cases of TTR amyloidosis has been liver transplantation. Although aggregation-preventing strategies involving ligands are known, understanding the mechanism of TTR aggregation can lead to additional inhibition approaches. Several models of TTR amyloid fibrils have been proposed, but the segments that drive aggregation of the protein have remained unknown. Here we identify beta-strands F and H as necessary for TTR aggregation. Based on the crystal structures of these segments, we designed two non-natural peptide inhibitors that block aggregation. This work provides the first characterization of peptide inhibitors for TTR aggregation, establishing a novel therapeutic strategy.

Epigenetic Control of the Bone-master Runx2 Gene During Osteoblast-lineage Commitment by the Histone Demethylase JARID1B/KDM5B [Gene Regulation]

October 9th, 2015 by

Transcription factor Runx2 controls bone development and osteoblast differentiation by regulating expression of a significant number of bone-related target genes. Here, we report that transcriptional activation and repression of the Runx2 gene via its osteoblast-specific P1 promoter (encoding mRNA for the Runx2/p57 isoform), is accompanied by selective deposition and elimination of histone marks during differentiation of mesenchymal cells to the osteogenic and myoblastic lineages. These epigenetic profiles are mediated by key components of the Trithorax/COMPASS-like and Polycomb group complexes, together with histone arginine methylases like PRMT5 and lysine demethylases like JARID1B/KDM5B. Importantly, knockdown of the H3K4me2/3 demethylase JARID1B, but not of the demethylases UTX and NO66, prevents repression of the Runx2 P1 promoter during myogenic differentiation of mesenchymal cells. The epigenetically-forced expression of Runx2/p57 and osteocalcin, a classical bone-related target gene, under myoblastic-differentiation is accompanied by enrichment of the H3K4me3 and H3K27ac marks at the Runx2 P1 promoter region. Our results identify JARID1B as a key component of a potent epigenetic switch that controls mesenchymal cell fate into myogenic and osteogenic lineages.
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The organophosphate degradation (opd) island borne esterase induced metabolic diversion in E. coli and its influence on p-nitrophenol degradation [Metabolism]

October 9th, 2015 by

In previous studies of the organophosphate degradation gene cluster we showed that expression of an open reading frame (orf306) present within the cluster in E. coli allowed growth on p-nitrophenol (PNP) as sole carbon source. We have now shown that expression of orf306 in E. coli causes a dramatic up-regulation in genes coding for alternative carbon catabolism. The propionate, glyoxylate and methyl citrate cycle (MCC) pathway-specific enzymes are up regulated, along with hca (phenyl propionate) and mhp (hydroxy phenyl propionate) degradation operons. These hca and mhp operons play a key role in degradation of PNP, enabling E. coli to grow using it as sole carbon source. Supporting growth experiments, PNP degradation products entered central metabolic pathways and got incorporated into the carbon backbone. The protein and RNA samples isolated from E. coli (pSDP10) cells grown in C14 labelled PNP indicated incorporation of C14 carbon suggesting Orf306-dependent assimilation of PNP in E. coli cells.
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Hepatic FOXO1 target genes are co-regulated by thyroid hormone via RICTOR deacetylation and MTORC2-AKT inhibition [Metabolism]

October 9th, 2015 by

MTORC2-AKT is a key regulator of carbohydrate metabolism and insulin signaling due to its effects on FOXO1 phosphorylation. Interestingly, both FOXO1 and thyroid hormone (TH) have similar effects on carbohydrate and energy metabolism as well as overlapping transcriptional regulation of many target genes. Currently, little is known about the regulation of MTORC2-AKT or FOXO1 by TH. Accordingly, we performed hepatic transcriptome profiling in mice after FOXO1 knockdown in the absence or presence of TH, and compared these results with hepatic FOXO1 and THRB1 (TRβ1) ChIP-Seq data. We identified a subset of TH-stimulated FOXO1 target genes that required co-regulation by FOXO1 and TH. TH activation of FOXO1 was directly linked to an increase in SIRT1-MTORC2 interaction and RICTOR deacetylation. This, in turn, led to decreased AKT and FOXO1 phosphorylation. Moreover, TH increased FOXO1 nuclear localization, DNA binding, and target gene transcription by reducing AKT-dependent FOXO1 phosphorylation in a THRB1-dependent manner. These events were associated with TH-mediated oxidative phosphorylation and NAD+ production, and suggested that downstream metabolic effects by TH can post-translationally activate other transcription factors. Finally, TH increased glucose output and inhibited the effect of insulin on AKT phosphorylation in hepatic cells. Our results showed that RICTOR/MTORC2-AKT can integrate convergent hormonal and metabolic signals to provide co-ordinated and sensitive regulation of hepatic FOXO1-target gene expression.