Unmasking of CD22 on germinal center B-cells occurs by alternative mechanisms in mouse and man [Glycobiology and Extracellular Matrices]

October 27th, 2015 by

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here we show in both mouse and man that loss of high-affinity ligands on GC B-cells unmask the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking since high-affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2-6Galβ1-4GlcNAc, which terminates N-glycans. The human ligand has Neu5Ac as the Sia, and the high affinity ligand on naive B cells contains 6-O-sulfate on the GlcNAc. On human GC cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mCD22 has Neu5Gc as the Sia, which is replaced on GC B cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high-affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high-affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high-affinity CD22 ligands on GC B-cells occurs in both mouse and man through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells.

Characterization of the functional roles of amino acid residues in acceptor binding subsite +1 in the active site of the glucansucrase GTF180 enzyme of Lactobacillus reuteri 180 [Microbiology]

October 27th, 2015 by

α-Glucans produced by glucansucrase enymes hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown, even with the recent elucidation of glucansucrase crystal structures. Guided by the crystal structure from glucansucrase GTF180-ΔN of Lactobacillus reuteri 180 in complex with the acceptor substrate maltose, we identified several residues (D1028 and N1029 from domain A, as well as L938, A978 and L981 from domain B) near subsite +1 that may be critical for linkage specificity determination and investigated these by random site-directed mutagenesis. First, mutants of A978 (to L, P, F or Y) and D1028 (to Y or W) with larger side chains showed reduced degrees of branching, likely due to the steric hindrance by these bulky residues. Second, L938 mutants (except L938F) and D1028 mutants showed altered linkage specificity, mostly with increased (α1→6) linkage synthesis. Third, mutation of L981 and N1029 significantly affected the transglycosylation reaction, indicating their essential roles in acceptor substrate binding. In conclusion, glucansucrase product specificity is determined by an interplay of domain A and B residues surrounding the acceptor substrate binding groove. Residues surrounding the +1 subsite thus are critical for activity and specificity of the GTF180 enzyme, and play different roles in the enzyme function. This study provides novel insights into the structure-function relationships of glucansucrase enzymes and clearly shows the potential of enzyme engineering to produce tailor-made α-glucans.
  • Posted in Journal of Biological Chemistry, Publications
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USP11 is a Negative Regulator to {gamma}H2AX Ubiquitylation by RNF8/RNF168 [Signal Transduction]

October 27th, 2015 by Yu, M., Liu, K., Mao, Z., Luo, J., Gu, W., Zhao, W.

Ubiquitin modification at double strand breaks (DSB) sites is an essential regulator of signaling and repair. γH2AX extends from DSB sites and provides a platform for subsequent recruitment and amplification of DNA repair proteins and signaling factors. Here, we found that RNF8/RNF168 ubiquitylate γH2AX. We identified that USP11 is a unique deubiquitylation enzyme (DUB) for γH2AX. USP11 deubiquitylates γH2AX both in vivo and in vitro; but not the canonical (ub)-K119-H2A and (ub)-K120-H2B in vitro. And USP11 ablation enhances the levels of γH2AX ubiquitylation. We also found that USP11 interacts with γH2AX both in vivo and in vitro. We found that 53BP1 and ubiquitin-conjugated proteins are mis-regulated to retain longer and stronger at DSB sites after knockdown of USP11. We further found that cells are hypersensitive to γ-irradiation after ablation of USP11. Together, our findings elucidates deeply and extensively the mechanism of RNF8/RNF168 and USP11 to maintain the proper status of ubiquitylation γH2AX to repair DSB.

Oligomerization and GTP-binding requirements of MxA for viral target recognition and antiviral activity against influenza A virus [Microbiology]

October 27th, 2015 by Nigg, P. E., Pavlovic, J.

The interferon (IFN)-induced human myxovirus resistance protein A (MxA) exhibits a broad antiviral activity against many viruses including influenza A virus (IAV). MxA belongs to the family of dynamin-like GTPases and assembles in vitro into dimers, tetramers and oligomeric ring-like structures. The molecular mechanism of action remains to be elucidated. Furthermore it is not clear whether MxA exerts its antiviral activity in a monomeric and/or multimeric form. Using a set of MxA mutants that form complexes with defined stoichiometry, we observed that in the presence of GTPγS, purified MxA disassembled into tetramers and dimers. Dimeric forms did not further disassemble into monomers. Infection experiments revealed that besides wild type MxA also dimeric and monomeric variants of MxA efficiently restricted IAV at a replication step after primary transcription. Moreover, only dimeric MxA was able to form stable complexes with the nucleoprotein (NP) of IAV. MxA interacted with NP independently of other viral components. Interestingly, the dimeric form of MxA was able to efficiently bind to NP from several MxA-sensitive strains but interacted much weaker with NP from the MxA-resistant PR8 strain derived from the H1N1 1918 lineage. Taken together, these data suggest that during infection a fraction of MxA disassembles into dimers that bind to NP synthesized following primary transcription in the cytoplasm, thereby preventing viral replication.
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The T210M substitution in the HLA-A*02:01 gp100 epitope strongly affects overall proteasomal cleavage site usage and antigen processing [Protein Synthesis and Degradation]

October 27th, 2015 by

MHC class I restricted epitopes, which carry a tumor-specific mutation resulting in improved MHC binding affinity, are preferred TCR targets in innovative adoptive T cell therapies. However, T cell therapy requires efficient generation of the selected epitope. How such mutations may affect proteasome mediated antigen processing has so far not been studied. Therefore, we analyzed by in vitro experiments the effect on antigen processing and recognition of a T210M exchange, which previously had been introduced into the melanoma gp100209-217 tumor epitope to improve the HLA-A*02:01-binding and its immunogenicity. A quantitative analysis of the main steps of antigen processing shows that the T210M exchange affects proteasomal cleavage site usage within the mutgp100201-230 polypeptide, leading to the generation of an unique set of cleavage products. The T210M substitution qualitatively affects the proteasome_catalyzed generation of spliced and non_spliced peptides predicted to bind HLA-A or -B complexes. The T210M substitution also induces an enhanced production of the mutgp100209-217 epitope and its N terminally extended peptides. The T210M exchange revealed no effect on ERAP1 mediated N terminal trimming of the precursor peptides. However, mutant N terminally extended peptides exhibited significantly increased HLA-A*02:01 binding affinity and elicited CD8+ T cell stimulation in vitro similar to the wtgp100209-217 epitope. Thus, our experiments demonstrate that amino acid exchanges within an epitope can result in the generation of an altered peptide pool with new antigenic peptides and in a wider CD8+ T-cell response also toward N_terminally extended versions of the minimal epitope.
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Cyclic Nucleotide-dependent Protein Kinases Target ARHGAP17 and ARHGEF6 Complexes in Platelets [Signal Transduction]

October 27th, 2015 by

Endothelial cells release prostacyclin (PGI2) and nitric oxide (NO) to inhibit platelet functions. PGI2 and NO effects are mediated by cyclic nucleotides, cAMP- and cGMP-dependent protein kinases (PKA, PKG), and largely unknown PKA and PKG substrate proteins. The small G-protein Rac1 plays a key role in platelets and was suggested to be a target of cyclic nucleotide signaling. We confirm that PKA and PKG activation reduces Rac1-GTP levels. Screening for potential mediators of this effect resulted in the identification of the Rac1-specific GTPase-activating protein ARHGAP17 and the guanine nucleotide exchange factor ARHGEF6 as new PKA and PKG substrates in platelets. We mapped the PKA/PKG phosphorylation sites to serine 702 on ARHGAP17 using Phos-tag gels and to serine 684 on ARHGEF6. We show that ARHGAP17 binds to the actin regulating CIP4 protein in platelets and that S702 phosphorylation interferes with this interaction. Reduced CIP4 binding results in enhanced inhibition of cell migration by ARHGAP17. Furthermore, we show that ARHGEF6 is constitutively linked to GIT1, a GAP of Arf family small G proteins, and that ARHGEF6 phosphorylation enables binding of the 14-3-3 adaptor protein to the ARHGEF6/GIT1 complex. PKA and PKG induced rearrangement of ARHGAP17 and ARHGEF6 associated protein complexes might contribute to Rac1 regulation and platelet inhibition.

Protein kinase D2 assembles a multiprotein complex at the Trans-Golgi-network to regulate matrix metalloproteinase secretion [Signal Transduction]

October 27th, 2015 by Eiseler, T., Wille, C., Koehler, C., Illing, A., Seufferlein, T.

Vesicle formation and fission are tightly regulated at the Trans-Golgi-network (TGN) during constitutive secretion. Two major protein families regulate these processes: members of the Adenosyl-ribosylation-factor-family of small G-proteins (ARFs) and the Protein kinase D (PKD) family of serine/threonine kinases. The functional relationship between these two key regulators of protein transport from the TGN so far is elusive. We here demonstrate the assembly of a novel functional protein complex at the TGN and its key members: Cytosolic PKD2 binds ARL1 and shuttles ARL1 to the TGN. ARL1, in turn, localizes Arfaptin2 to the TGN. At the TGN, where PKD2 interacts with active ARF1, PKD2 and ARL1 are required for the assembly of a complex comprising of ARF1 and Arfaptin2 leading to secretion of MMP2 and -7. In conclusion, our data indicate that PKD2 is a core factor in the formation of this multiprotein complex at the TGN that controls constitutive secretion of MMP cargo.
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Molecular Basis of Regulating High Voltage-Activated Calcium Channels by S-Nitrosylation [Membrane Biology]

October 27th, 2015 by Zhou, M.-H., Bavencoffe, A., Pan, H.-L.

Nitric oxide (NO) is involved in a variety of physiological processes, such as vasoregulation and neurotransmission, and has a complex role in the regulation of pain transduction and transmission. We have shown previously that NO inhibits high voltage-activated Ca2+ channels in primary sensory neurons and excitatory synaptic transmission in the spinal dorsal horn. However, the molecular mechanism involved in this inhibitory action remains unclear. In this study, we investigated the role of S-nitrosylation in the NO regulation of high voltage-activated Ca2+ channels. The NO donor S-Nitroso-N-acetyl-DL-penicillamine (SNAP) rapidly reduced N-type currents when Cav2.2 was coexpressed with the Cavβ1 or Cavβ3 subunit in HEK293 cells. In contrast, SNAP only slightly inhibited P/Q-type and L-type currents reconstituted with various Cavβ subunits. SNAP caused a depolarizing shift in voltage-dependent N-type channel activation, but it had no effect on Cav2.2 protein levels on the membrane surface. The inhibitory effect of SNAP on N-type currents was blocked by the sulfhydryl-specific modifying reagent methanethiosulfonate ethylammonium. Furthermore, the consensus motifs of S-nitrosylation were much more abundant in Cav2.2 than in Cav1.2 and Cav2.1. Site-directed mutagenesis studies showed that Cys805, Cys930, Cys1835 and Cys1045 in the II-III intracellular loop, Cys1845 and Cys2145 in the C-terminus of Cav2.2, and Cys346 in the Cavβ3 subunit were nitrosylation sites mediating NO sensitivity of N-type channels. Our findings demonstrate that the consensus motifs of S-nitrosylation in cytoplasmically accessible sites are critically involved in post-translational regulation of N-type Ca2+ channels by NO.

Mapping the Binding Site of the Inhibitor Tariquidar that Stabilizes the First Transmembrane Domain of P-glycoprotein [Protein Structure and Folding]

October 26th, 2015 by Loo, T. W., Clarke, D. M.

ABC (ATP-Binding Cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease.
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Kinetically-defined mechanisms and positions of action of two new modulators of glucocorticoid receptor regulated gene induction [Computational Biology]

October 26th, 2015 by

Most of the steps in, and many of the factors contributing to, glucocorticoid receptor (GR) regulated gene induction are currently unknown. A competition assay, based on a validated chemical kinetic model of steroid hormone action, is now used to identify two new factors (BRD4 and NELF-E) and to define their sites and mechanisms of action. BRD4 is a kinase involved in numerous initial steps of gene induction. Consistent with its complicated biochemistry, BRD4 is shown to alter both the maximal activity (Amax) and the steroid concentration required for half-maximal induction (EC50) of GR-mediated gene expression by acting at a minimum of three different kinetically-defined steps. The action at two of these steps is dependent on BRD4 concentration while the third step requires the association of BRD4 with P-TEFb. BRD4 is also found to bind to NELF-E, a component of the NELF-complex. Unexpectedly, NELF-E modifies GR induction in a manner that is independent of the NELF complex. Several of the kinetically-defined steps of BRD4 in this study are proposed to be related to its known biochemical actions. However, novel actions of BRD4 and of NELF-E in GR controlled gene induction have been uncovered. The model-based competition assay is also unique in being able to order, for the first time, the sites of action of the various reaction components, which is GR < Cdk9 < BRD4 < or at induced gene < NELF-E. This ability to order factor actions will assist efforts to reduce the side-effects of steroid treatments.
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