The Disulfide Bond, but not Zinc or Dimerization, Controls Initiation and Seeded Growth in Amyotrophic Lateral Sclerosis-linked Cu-Zn Superoxide Dismutase (SOD1) Fibrillation [Protein Structure and Folding]

October 28th, 2015 by

Aggregation of copper-zinc superoxide dismutase (SOD1) is a defining feature of familial ALS caused by inherited mutations in the sod1 gene and misfolded and aggregated forms of wild-type SOD1 are found in both sporadic and familial ALS cases. Mature SOD1 owes its exceptional stability to a number of posttranslational modifications: Formation of the intramolecular disulfide bond, binding of copper and zinc, and dimerization. Loss of stability due to the failure to acquire one or more of these modifications is proposed to lead to aggregation in vivo. Previously we showed that the presence of apo-, disulfide-reduced SOD1, the most immature form of SOD1, results in initiation of fibrillation of more mature forms that have an intact C57-C146 disulfide bond and are partially metallated. In this study, we examine the ability of each of the above posttranslational modifications to modulate fibril initiation and seeded growth. Cobalt or zinc binding, despite conferring great structural stability, neither inhibits the initiation propensity of disulfide-reduced SOD1 nor consistently protects disulfide-oxidized SOD1 from being recruited into growing fibrils across wild-type and a number of ALS mutants. In contrast, reduction of the disulfide bond, known to be necessary for fibril initiation, also allows for faster recruitment during seeded amyloid growth. These results identify separate factors that differently influence seeded growth and initiation and indicate a lack of correlation between the overall thermodynamic stability of partially mature SOD1 states and their ability to initiate fibrillation or be recruited by a growing fibril.
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Hyperosmotic Shock Engages Two Positive Feedback Loops Through Caspase-3 Dependent Proteolysis of JNK1-2 and Bid [Signal Transduction]

October 28th, 2015 by Yue, J., Ben Messaoud, N., Lopez, J. M.

Hyperosmotic shock induces early calpain activation, Smac/DIABLO release from the mitochondria and p38/JNK activation in Xenopus oocytes. These pathways regulate late cytochrome c release and caspase-3 activation. Here we show that JNK1-1 and JNK1-2 are early activated by osmostress and sustained activation of both isoforms accelerates the apoptotic program. When caspase-3 is activated JNK1-2 is proteolyzed at Asp385 increasing the release of cytochrome c and caspase-3 activity, and therefore creating a positive feedback loop. Expression of Bcl-xL markedly reduces hyperosmotic shock-induced apoptosis. In contrast, expression of Bid induces rapid caspase-3 activation, even in the absence of osmostress, which is blocked by Bcl-xL co-expression. In these conditions a significant amount of Bid in the cytosol is mono- and biubiquitinated. Caspase-3 activation by hyperosmotic shock induces proteolysis of Bid and mono-ubiquitinated Bid at Asp52 increasing the release of cytochrome c and caspase-3 activation, and thus creating a second positive feedback loop. Revealing the JNK isoforms and the loops activated by osmostress could help to design better treatments for human diseases caused by perturbations in fluid osmolarity.

Structure-function analysis of a mixed-linkage Beta-glucanase/xyloglucanase from key ruminal Bacteroidetes Prevotella bryantii B14 [Glycobiology and Extracellular Matrices]

October 27th, 2015 by

The recent classification of Glycoside Hydrolase Family 5 (GH5) members into subfamilies enhances the prediction of substrate specificity by phylogenetic analysis. However, the small number of well-characterized members is a current limitation to understanding the molecular basis of the diverse specificity observed across individual GH5 subfamilies. GH5 Subfamily 4 (GH5_4) is one of the largest, with known activities comprising (carboxymethyl)cellulases, mixed-linkage endo-glucanases, and endo-xyloglucanases. Through detailed structure-function analysis, we have revisited the characterization of a classic GH5_4 carboxymethylcellulase, PbGH5A (also known as Orf4, CMCase, and Cel5A) from the symbiotic rumen Bacteroidetes Prevotella bryantii B14. We demonstrate that CMC and phosphoric acid-swollen cellulose (PASC) are in fact strikingly poor substrates for PbGH5A, which instead exhibits clear primary specificity for the plant storage and cell wall polysaccharide, mixed-linkage β-glucan. Significant activity toward the plant cell wall polysaccharide xyloglucan was also observed. Determination of PbGH5A crystal structures in the apo form and in complex with (xylo)glucan oligosaccharides and an active-site affinity label, together with detailed kinetic analysis using a variety of well-defined oligosaccharide substrates, revealed the structural determinants of polysaccharide substrate specificity. In particular, this analysis highlighted the PbGH5A active-site motifs which engender predominant mixed-linkage endo-glucanase activity vis-a-vis predominant endo-xyloglucanases in GH5_4. However the detailed phylogenetic analysis of GH5_4 members did not delineate particular clades of enzymes sharing these sequence motifs; the phylogeny was instead dominated by bacterial taxonomy. Nonetheless, our results provide key enzyme functional and structural reference data for future bioinformatics analyses of meta(genomes) to elucidate the biology of complex gut ecosystems.
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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.
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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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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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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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