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.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Epigenetic Control of the Bone-master Runx2 Gene During Osteoblast-lineage Commitment by the Histone Demethylase JARID1B/KDM5B [Gene Regulation]

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.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on The organophosphate degradation (opd) island borne esterase induced metabolic diversion in E. coli and its influence on p-nitrophenol degradation [Metabolism]

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.

The Regulation of Synaptic Protein Turnover [Protein Synthesis and Degradation]

October 9th, 2015 by Alvarez-Castelao, B., Schuman, E. M.

Emerging evidence indicates that protein synthesis and degradation are necessary for the remodeling of synapses. These two processes govern cellular protein turnover, are tightly regulated, and neuronal activity modulates them in time and space. The anisotropic anatomy of the neurons presents a challenge for the study of protein turnover, but understanding protein turnover in neurons and its modulation in response to activity can help us to unravel the fine-tuned changes that occur at synapses in response to activity. Here we review the key experimental evidence demonstrating the role of protein synthesis and degradation in synaptic plasticity, and the turnover rates of specific neuronal proteins

TRAF5-mediated K63-linked polyubiquitination play essential role in positive regulation of ROR{gamma}t on promoting IL-17A expression [Cell Biology]

October 9th, 2015 by

Retinoid-related orphan nuclear receptor γt (RORγt) is a key transcription factor for the de-velopment and function of Th17 cells. In this study, we showed that tumor necrosis factor receptor as-sociated factor 5 (TRAF5), known as an E3 ubiq-uitin-protein ligase and signal transducer, interacts with and ubiquitinates RORγt via K63-linked polyubiquitination. TRAF5 stabilizes RORγt pro-tein level depending on its RING-finger domain. And depletion of TRAF5 in Th17 cells destabilizes RORγt protein and down-regulates Th17-related genes, including IL-17A, an inflammatory cytokine involved in pathogenic mechanisms of several au-toimmune diseases, such as systemic lupus erythe-matosus. Moreover, up-regulation of TRAF5 mRNA level was found in SLE patient CD4+ T cells. Our findings reveal a direct link between TRAF5-mediated ubiquitination and RORγt pro-tein regulation, which may aggravate inflamma-tory progress and provide new therapeutic drug targets for autoimmune diseases.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on TRAF5-mediated K63-linked polyubiquitination play essential role in positive regulation of ROR{gamma}t on promoting IL-17A expression [Cell Biology]

Actin Out: Regulation of the Synaptic Cytoskeleton [Cell Biology]

October 9th, 2015 by Spence, E. F., Soderling, S. H.

The small size of dendritic spines belies the elaborate role they play in excitatory synaptic transmission and ultimately complex behaviors. The cytoskeletal architecture of the spine is predominately composed of actin filaments. These filaments, which at first glance might appear simple, are also surprisingly complex. They dynamically assemble into different structures and serve as a platform for orchestrating the elaborate responses of the spine during spinogenesis and experience-dependent plasticity. Multiple mutations associated with human neurodevelopmental and psychiatric disorders involve genes which encode regulators of the synaptic cytoskeleton. A major, unresolved question is how the disruption of specific actin filament structures leads to the onset and progression of complex synaptic and behavioral phenotypes. This review will cover established and emerging mechanisms of actin cytoskeletal remodeling and how this influences specific aspects of spine biology that are implicated in disease.

Neutrophil elastase differentially regulates IL-8 and VEGF production by cigarette smoke extract [Signal Transduction]

October 9th, 2015 by Lee, K.-H., Lee, C.-H., Jeong, J., Jang, A.-H., Yoo, C.-G.

Inflammation by IL-8-induced neutrophil recruitment and apoptosis of epithelial cells by decreased expression of VEGF have been suggested as one of the complicated pathogenic mechanisms of COPD. The role of neutrophil elastase (NE) in the development of COPD is also well-known. However, little is known about how they interact. The objective of this study was to elucidate the effect of NE on the cigarette smoke extract (CSE)-induced IL-8 and VEGF production, and its molecular mechanism in bronchial epithelial cells. CSE increased both IL-8 and VEGF productions in human bronchial epithelial cell (BEAS-2B). While NE significantly enhanced CSE-induced IL-8 production, it suppressed VEGF production. This differential regulation was not CSE-specific. The effect of NE on IL-8 production, but not VEGF, was ERK-dependent. Interestingly, in contrast to decreased VEGF protein expression, NE accelerated VEGF transcription by CSE, suggesting post-translational modification. When cells were incubated with purified NE, it was detected in the cytoplasm, suggesting the intracellular translocation of NE. Furthermore, NE fragmented rhVEGF in vitro, but not rhIL-8. These results indicate that VEGF down-regulation is due to direct degradation by NE which is translocated into cells. Similar to in vitro cell experiments, elastase treatment increases CSE-induced IL-8, however, it suppresses VEGF production in bronchoalveolar lavage (BAL) fluid of CSE-treated mice. Moreover, elastase treatment enhances CSE-induced emphysema in mice. Considering the actions of IL-8 and VEGF, our results suggest that NE contributes to the pathogenesis of COPD by enhancing inflammation and apoptosis.