ACTH Responses Require Actions of the Melanocortin-2 Receptor Accessory Protein on the Extracellular Surface of the Plasma Membrane [Membrane Biology]

September 30th, 2015 by Malik, S., Dolan, T. M., Maben, Z. J., Hinkle, P. M.

The melanocortin-2 (MC2) receptor is a G protein-coupled receptor that mediates responses to ACTH. The MC2 receptor acts in concert with an accessory protein (MRAP) that is absolutely required for ACTH binding and signaling. MRAP has a single transmembrane domain and forms a highly unusual anti-parallel homodimer that is stably associated with MC2 receptors at the plasma membrane. Despite the physiological importance of the interaction between the MC2 receptor and MRAP, there is little understanding of how the accessory protein works. The dual topology of MRAP has made it impossible to determine whether highly conserved and necessary regions of MRAP are required on the intracellular or extracellular face of the plasma membrane. The strategy used here was to fix the orientation of two anti-parallel MRAP molecules and then introduce inactivating mutations on one side of the membrane or the other. This was achieved by engineering proteins containing tandem copies of MRAP fused to the amino-terminus of the MC2 receptor. The data firmly establish that only the Nout copy of MRAP, oriented with critical segments on the extracellular side of the membrane, is essential. The transmembrane domain of MRAP is also required in only the Nout orientation. Finally, activity of MRAP-MRAP-MC2-receptor fusion proteins with inactivating mutations in either MRAP or the receptor is rescued by co-expression of free wild-type MRAP or free wild-type receptor. These results show that the basic MRAP/MRAP/receptor signaling unit forms higher order complexes and that these multimers signal.
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Homocitrullination is a Novel Histone H1 Epigenetic Mark Dependent on Aryl Hydrocarbon Receptor Recruitment of Carbamoyl Phosphate Synthase 1 [Gene Regulation]

September 30th, 2015 by Joshi, A. D., Mustafa, M. G., Lichti, C. F., Elferink, C. J.

The Aryl hydrocarbon Receptor (AhR), a regulator of xenobiotic toxicity, is a member of the eukaryotic Per-Arnt-Sim domain protein family of transcription factors. Recent evidence identified a novel AhR DNA recognition sequence called the non-consensus xenobiotic response element (NC-XRE). AhR binding to the NC-XRE in response to activation by the canonical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin, resulted in concomitant recruitment of Carbamoyl Phosphate Synthase 1 (CPS1) to the NC-XRE. Studies presented here demonstrate that CPS1 is a bona fide nuclear protein involved in homocitrullination (hcit), including a key lysine residue on histone H1 (H1K34hcit). H1K34hcit represents a hitherto unknown epigenetic mark implicated in enhanced gene expression of the peptidylarginine deiminase 2 gene, itself a chromatin modifying protein. Collectively, our data suggest that AhR activation promotes CPS1 recruitment to DNA enhancer sites in the genome resulting in a specific enzyme-independent post-translational modification (PTM) of the linker histone H1 protein (H1K34hcit), pivotal in altering local chromatin structure and transcriptional activation.
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DISC1 Regulates GABAA Receptor Trafficking and Inhibitory Synaptic Transmission in Cortical Neurons [Signal Transduction]

September 30th, 2015 by Wei, J., Graziane, N. M., Gu, Z., Yan, Z.

Association studies have suggested that DISC1 (Disrupted-in-Schizophrenia-1) confers a genetic risk at the level of endophenotypes that underlies many major mental disorders. Despite the progress on understanding the significance of DISC1 at neural development, the mechanisms underlying DISC1 regulation of synaptic functions remain elusive. Since alterations in the cortical GABA system have been strongly linked to the pathophysiology of schizophrenia, one potential target of DISC1 that is critically involved in the regulation of cognition and emotion is the GABAA receptor. We found that cellular knockdown of DISC1 significantly reduced GABAAR-mediated synaptic and whole-cell current, while overexpression of wild-type DISC1, but not the C-terminal-truncated DISC1 (a schizophrenia-related mutant), significantly increased GABAAR currents in pyramidal neurons of prefrontal cortex. These effects were accompanied by DISC1-induced changes in surface GABAAR expression. Moreover, the regulation of GABAARs by DISC1 knockdown or overexpression depends on the microtubule motor protein kinesin-1 (KIF5). Our results suggest that DISC1 exerts an important impact on GABAergic inhibitory transmission by regulating KIF5/microtubule-based GABAAR trafficking in the cortex. Knowledge gained from this study would shed light on how DISC1 and the GABA system are mechanistically linked and how their interactions are critical for maintaining a normal mental state.

The Silent Sway of Splicing by Synonymous Substitutions [Gene Regulation]

September 30th, 2015 by

Alternative splicing diversifies mRNA transcripts in human cells. This sequence-driven process can be influenced greatly by mutations, even those that do not change the protein coding potential of the transcript. Synonymous mutations have been shown to alter gene expression through modulation of splicing, mRNA stability, and translation. Using a synonymous position mutation library in SMN1 exon 7, we show that 23% of synonymous mutations across the exon decrease exon inclusion, suggesting that nucleotide identity across the entire exon has been evolutionarily optimized to support a particular exon inclusion level. While phylogenetic conservation scores are insufficient to identify synonymous positions important for exon inclusion, an alignment of organisms filtered based on similar exon/intron architecture is highly successful. Although many of the splicing neutral mutations are observed to occur, none of the exon inclusion reducing mutants was found in the filtered alignment. Using the modified phylogenetic comparison as an approach to evaluate the impact on pre-mRNA splicing suggests that up to 45% of synonymous SNPs are likely to alter pre-mRNA splicing. These results demonstrate that coding and pre-mRNA splicing pressures co-evolve and that a modified phylogenetic comparison based on the exon/intron architecture is a useful tool in identifying splice altering SNPs.

Epigenetic Control of Skeletal Development by the Histone Methyltransferase Ezh2 [Molecular Bases of Disease]

September 30th, 2015 by

Epigenetic control of gene expression is critical for normal fetal development. Yet, chromatin related mechanisms that activate bone-specific programs during osteogenesis have remained under-explored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose-tissue (AMSCs). Molecular analyses establish that the polycomb group protein Enhancer of Zeste Homolog 2 (EZH2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knock-down studies show that EZH2, a histone methyltransferase which catalyzes tri-methylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extra-cellular matrix (ECM) proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis and clinodactyly. Histological analysis and mRNASeq profiling suggests that these effects are attributable to growth plate abnormalities and premature cranial suture closure due to precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production.

Reciprocal Phosphorylation and Palmitoylation Control Dopamine Transporter Kinetics [Neurobiology]

September 30th, 2015 by

The dopamine transporter (DAT) is a neuronal protein that drives the presynaptic reuptake of dopamine (DA) and is the major determinant of transmitter availability in the brain. DAT function is regulated by protein kinase C (PKC) and other signaling pathways through mechanisms that are complex and poorly understood. Here we investigate the role of Ser7 phosphorylation and Cys580 palmitoylation in mediating steady-state transport kinetics and PKC-stimulated transport down-regulation. Using both mutational and pharmacological approaches we demonstrate that these post-translational modifications are reciprocally regulated, leading to transporter populations that display high phosphorylation-low palmitoylation or low phosphorylation-high palmitoylation. The balance between the modifications dictates transport capacity, as conditions that promote high phosphorylation or low palmitoylation reduce transport Vmax and enhance PKC-stimulated down-regulation, whereas conditions that promote low phosphorylation or high palmitoylation increase transport Vmax and suppress PKC-stimulated down-regulation. Transitions between these functional states occur when endocytosis is blocked or undetectable, indicating that the modifications kinetically regulate the velocity of surface transporters. These findings reveal a novel mechanism for control of DA reuptake that may represent a point of dysregulation in DA imbalance disorders

Characterization of the Pseudomonas aeruginosa Glycoside Hydrolase PslG Reveals that its Levels are Critical for Psl Polysaccharide Biosynthesis and Biofilm Formation. [Enzymology]

September 30th, 2015 by

A key component of colonization, biofilm formation, and protection of the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharide Psl. Composed of a pentameric repeating unit of mannose, glucose and rhamnose, the biosynthesis of Psl is proposed to occur via a Wzx/Wzy-dependent mechanism. Previous genetic studies have shown that the putative glycoside hydrolase PslG is essential for Psl biosynthesis. To understand the function of this protein, the apo structure of the periplasmic domain of PslG (PslG31-442) and its complex with mannose were determined to 2.0 and 1.9 Å resolution, respectively. Despite similar domain architecture and positioning of catalytic residues to other family 39 glycoside hydrolases (GH39), PslG31-442 exhibits a unique 32 Å long active site groove that is distinct from other structurally characterized family members. PslG formed a complex with two mannose monosaccharides in this groove, consistent with binding data obtained from intrinsic tryptophan fluorescence. PslG was able to catalyze the hydrolysis of surface-associated Psl and this activity was abolished in a E165Q/E276Q double catalytic variant. Surprisingly, P. aeruginosa variants with these chromosomal mutations as well as a pslG deletion mutant were still capable of forming Psl biofilms. However, overexpression of PslG in a pslG deletion background impaired biofilm formation and resulted in less surface-associated Psl, suggesting that regulation of this enzyme is important during polysaccharide biosynthesis.
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Retinal Degeneration Slow (RDS) Glycosylation Plays a Role in Cone Function and the Regulation of RDS/ROM-1 Complex Formation [Cell Biology]

September 29th, 2015 by Stuck, M. W., Conley, S. M., Naash, M. I.

The photoreceptor-specific glycoprotein retinal degeneration slow (RDS, also called PRPH2) is necessary for the formation of rod and cone outer segments. Mutations in RDS cause rod and cone dominant retinal disease, and it is well established that both cell types have different requirements for RDS. However, the molecular mechanisms for this difference remain unclear. Although RDS glycosylation is highly conserved, previous study revealed no apparent function for the glycan in rods. In light of the highly conserved nature of RDS glycosylation we hypothesized that it is important for RDS function in cones, and could underlie part of the differential requirement for RDS in the two photoreceptor subtypes. We generated a knockin mouse expressing RDS without the N-glycosylation site (N229S). Normal levels of RDS and the unglycosylated RDS binding partner rod outer segment membrane protein-1 (ROM-1) were found in N229S retinas however cone electroretinogram responses were decreased by 40% at 6 months of age. Since cones make up only 3-5% of photoreceptors in the wild-type background, N229S mice were crossed into the nrl-/- background (in which all rods are converted to cone-like cells) for biochemical analysis. In N229S/nrl-/- retinas, RDS and ROM-1 levels were decreased by ~60% each. These data suggest that glycosylation of RDS is required for RDS function or stability in cones, a difference that may be due to extracellular vs. intradiscal localization of the RDS glycan in cones vs. rods.

Structural Basis for Ligand Recognition and Functional Selectivity at Angiotensin Receptor [Signal Transduction]

September 29th, 2015 by

Angiotensin II type 1 receptor (AT1R) is the primary blood pressure regulator. AT1R blockers (ARBs) have been widely used in clinical settings as anti-hypertensive drugs, and share a similar chemical scaffold, although even minor variations can lead to distinct therapeutic efficacies towards cardiovascular etiologies. The structural basis for AT1R modulation by different peptide and non-peptide ligands has remained elusive. Here we report the crystal structure of the human AT1R in complex with an inverse agonist olmesartan (BenicarTM), a highly potent anti-hypertensive drug. Olmesartan is anchored to the receptor primarily by the residues Tyr351.39, Trp842.60, and Arg167ECL2, similar to the antagonist ZD7155, corroborating a common binding mode of different ARBs. Using docking simulations and site-directed mutagenesis we identified specific interactions between AT1R and different ARBs, including olmesartan derivatives with inverse agonist, neutral antagonist or agonist activities. We further observed that the mutation Asn1113.35Ala in the putative sodium-binding site affects binding of the endogenous peptide agonist Angiotensin II, but not the β-arrestin-biased peptide TRV120027.

Ethosuximide induces hippocampal neurogenesis and reverses cognitive deficits in amyloid-{beta} toxin induced Alzheimer’s rat model via PI3K/Akt/Wnt/{beta}-catenin pathway [Molecular Bases of Disease]

September 29th, 2015 by

Neurogenesis involves generation of new neurons through finely tuned multistep processes such as neural stem cell's (NSC) proliferation, migration, differentiation, and integration into existing neuronal circuitry in the dentate gyrus of the hippocampus and sub-ventricular zone (SVZ). Adult hippocampal neurogenesis is involved in cognitive functions and altered in various neurodegenerative disorders including Alzheimer's disease (AD). Ethosuximide (ETH), an anticonvulsant drug is used for the treatment of epileptic seizure. However, the effects of ETH on adult hippocampal neurogenesis and underlying cellular and molecular mechanism(s) are still elusive. Herein, we studied the effects of ETH on rat multipotent NSC proliferation and neuronal differentiation, and adult hippocampal neurogenesis in an amyloid beta (Aβ) toxin induced rat model of AD like phenotypes. ETH potently induced NSC proliferation and neuronal differentiation in the hippocampal derived NSC in vitro. ETH enhanced NSC proliferation and neuronal differentiation, and reduced Aβ toxin mediated toxicity and neurodegeneration, leading to behavioral recovery in rat AD model. ETH inhibited Aβ mediated suppression of neurogenic and Akt-Wnt/β-catenin pathway gene's expression in the hippocampus. ETH activated the PI3K/Akt and Wnt/β-catenin transduction pathways that are known to be involved in the regulation of neurogenesis. Inhibition of the PI3K/Akt and Wnt/β-catenin pathways effectively blocked the mitogenic and neurogenic effects of ETH. In silico molecular target prediction docking studies suggest that ETH interacts with Akt, Dkk-1 and GSK-3β. Our findings suggest that ETH stimulates NSC proliferation and differentiation in vitro and adult hippocampal neurogenesis via the PI3K/Akt and Wnt/β-catenin signaling.
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