Poly(ADP-ribosyl)ation of FOXP3 Mediated by PARP-1 Regulates the Function of Regulatory T Cells [Cell Biology]

October 1st, 2015 by Luo, X., Nie, J., Wang, S., Chen, Z., Chen, W., Li, D., Hu, H., Li, B.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an ADP-ribosylating enzyme participating in diverse cellular functions. The roles of PARP-1 in the immune system, however, have not been well understood. Here we find that PARP-1 interacts with FOXP3 and induces its poly(ADP-ribosyl)ation. By using PARP-1 inhibitors, we show that reduced poly(ADP-ribosyl)ation of FOXP3 results in not only FOXP3 stabilization and increased FOXP3 downstream genes, but also enhanced suppressive function of regulatory T cells (Treg). Our results suggest that PARP-1 negatively regulates the suppressive function of Treg cells at the post-translational level via FOXP3 poly(ADP-ribosyl)ation. This finding has implications in developing PARP-1 inhibitors as potential agents for the prevention and treatment of autoimmune diseases.

Structural Basis for Clonal Diversity of the Public T Cell Response to a Dominant Human Cytomegalovirus Epitope [Protein Structure and Folding]

October 1st, 2015 by

Cytomegalovirus (CMV) is a ubiquitous and persistent human pathogen that is kept in check by CD8+ cytotoxic T lymphocytes (CTLs). Individuals expressing the major histocompatibility complex (MHC) class I molecule HLA-A2 produce CTLs bearing T cell receptors (TCRs) that recognize the immunodominant CMV epitope NLVPMVATV (NLV). The NLV-specific T cell repertoire is characterized by a high prevalence of TCRs that are frequently observed in multiple unrelated individuals. These public TCRs feature identical, or nearly identical, complementarity-determining region 3α (CDR3α) and/or CDR3β sequences. The TCRs may express public CDR3α motifs alone, public CDR3β motifs alone, or dual public CDR3αβ motifs. In addition, the same public CDR3α motif may pair with different CDR3β motifs (and the reverse), giving rise to highly diverse NLV-specific TCR repertoires. To investigate the structural underpinnings of this clonal diversity, we determined crystal structures of two public TCRs (C7 and C25) in complex with NLV-HLA-A2. These TCRs utilize completely different CDR3α and CDR3β motifs that, in addition, can associate with multiple variable α (Vα) and Vβ regions in NLV-specific T cell repertoires. The C7-NLV-HLA-A2 and C25-NLV-HLA-A2 complexes exhibit divergent TCR footprints on peptide-MHC, such that C25 is more focused on the central portion of the NLV peptide than is C7. These structures, combined with molecular modeling, show how the public CDR3α motif of C25 may associate with different Vα regions, and how the public CDR3α motif of C7 may pair with different CDR3β motifs. This interchangeability of TCR V regions and CDR3 motifs permits multiple structural solutions to binding an identical peptide-MHC ligand, and thereby the generation of a clonally diverse public T cell response to CMV.
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Essential role of the EF-hand domain in targeting sperm phospholipase C{zeta} to membrane PIP2 [Developmental Biology]

October 1st, 2015 by

Sperm-specific PLC-zeta is widely considered to be the physiological stimulus that triggers intracellular Ca2+ oscillations and egg activation during mammalian fertilization. Although PLC-zeta is structurally similar to PLC-delta1, it lacks a PH domain and it remains unclear how PLC-zeta targets its PIP2 membrane substrate. Recently, the PLC-delta1 EF-hand domain was shown to bind to anionic phospholipids through a number of cationic residues, suggesting a potential mechanism for how PLCs might interact with their target membranes. Those critical cationic EF-hand residues in PLC-delta1 are notably conserved in PLC-zeta. We investigated the potential role of these conserved cationic residues in PLC-zeta by generating a series of mutants that sequentially neutralized three positively-charged residues (K49, K53 and R57) within the mouse PLC-zeta EF-hand domain. Microinjection of the PLC-zeta EF-hand mutants into mouse eggs enabled their Ca2+ oscillation-inducing activities to be compared with wild-type PLC-zeta. Further, the mutant proteins were purified and the in vitro PIP2 hydrolysis and binding properties were monitored. Our analysis suggests that PLC-zeta binds significantly to PIP2 but not to phosphatidic acid or phosphatidylserine, and that sequential reduction of the net positive charge within the 1st EF-hand domain of PLC-zeta significantly alters in vivo Ca2+ oscillation-inducing activity and in vitro interaction with PIP2 without affecting its Ca2+ sensitivity. Our findings are consistent with theoretical predictions provided by a mathematical model that links oocyte Ca2+ frequency and the binding ability of different PLC-zeta mutants to PIP2. Moreover, a PLC-zeta mutant with mutations in the cationic residues within the 1st EF hand domain and the XY linker region dramatically reduces the binding of PLC-zeta to PIP2, leading to complete abolishment of its Ca2+ oscillation-inducing activity.

Molecular mechanisms and kinetic effects of FXYD1 and phosphomimetic mutants on purified human Na,K-ATPase [Membrane Biology]

October 1st, 2015 by

Phospholemman (FXYD1) is a single trans-membrane protein regulator of Na,K-ATPase, expressed strongly in heart, skeletal muscle and brain, and phosphorylated by protein kinases A and C at S68 and S63, respectively. Binding of FXYD1 reduces Na,K-ATPase activity and phosphorylation at S68 or S63 relieves the inhibition. Despite the accumulated information on physiological effects, whole cell studies provide only limited information on molecular mechanisms. As a complementary approach, we utilized purified human Na,K-ATPase (α1β1 and α2β1) reconstituted with FXYD1 or mutants, S63E, S68E and S63E,S68E that mimic phosphorylation at S63 and S68. Compared to Control α1β1, FXYD1 reduces Vmax and turn-over rate and raises K0.5Na. The phosphomimetic mutants reverse these effects and reduce K0.5Na below Control K0.5Na. Effects on α2β1 are similar but smaller. Experiments in proteoliposomes reconstituted with α1β1 show analogous effects of FXYD1 on K0.5Na, which are abolished by phosphomimetic mutants and also by increasing mole fractions of DOPS in the proteoliposomes. Stopped-flow experiments using the dye RH421 show that FXYD1 slows the conformational transition E2(2K)ATP → E1(3Na)ATP but does not affect 3NaE1P → E2P3Na. This regulatory effect is explained simply by molecular modeling, which indicates that a cytoplasmic helix (residues 60-70) docks between the αN and αP domains in the E2 conformation but docking is weaker in E1 (also for phosphomimetic mutants). Taken together with previous work showing that FXYD1 also raises binding affinity for the Na selective site III, these results provide a rather comprehensive picture of the regulatory mechanism of FXYD1 that complements the physiological studies.

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.

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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