Toxoplasma gondii disrupts {beta}1 integrin signaling and focal adhesion formation during monocyte hypermotility [Immunology]

January 2nd, 2018 by Joshua H. Cook, Norikiyo Ueno, Melissa B. Lodoen

The motility of blood monocytes is orchestrated by the activity of cell surface integrins, which translate extracellular signals into cytoskeletal changes to mediate adhesion and migration. Toxoplasma gondii is an intracellular parasite that infects migratory cells and enhances their motility, but the mechanisms underlying T. gondii-induced hypermotility are incompletely understood. We have investigated the molecular basis for the hypermotility of primary human peripheral blood monocytes and THP-1 cells infected with T. gondii. Compared to uninfected monocytes, T. gondii infection of monocytes reduced cell spreading and the number of activated β1 integrin clusters in contact with fibronectin during settling, an effect not observed in monocytes treated with LPS or E. coli. Furthermore, T. gondii infection disrupted the phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 (Y397) and Y925 and of the related protein proline-rich tyrosine kinase (Pyk2) at Y402. The localization of paxillin, FAK, and vinculin to focal adhesions and the colocalization of these proteins with activated β1 integrins were also impaired in T. gondii-infected monocytes. Using time-lapse confocal microscopy of THP-1 cells expressing eGFP-FAK during settling on fibronectin, we found that T. gondii-induced monocyte hypermotility was characterized by a reduced number of eGFP-FAK-containing clusters over time compared to uninfected cells. This study demonstrates an integrin conformation-independent regulation of the β1 integrin adhesion pathway, providing further insight into the molecular mechanism of T. gondii- induced monocyte hypermotility.

The CDC50A extracellular domain is required for forming a functional complex with and chaperoning phospholipid flippases to the plasma membrane [Membrane Biology]

December 24th, 2017 by Katumori Segawa, Sachiko Kurata, Shigekazu Nagata

Flippases are enzymes that translocate phosphatidylserine (PtdSer) and phosphatidyl- ethanolamine (PtdEtn) from the outer to the inner leaflet in the lipid bilayer of the plasma membrane, leading to the asymmetric distribution of aminophospholipids in the membrane. One mammalian phospholipid flippase at the plasma membrane is ATP11C, a type IV P-type ATPase (P4-ATPase) that forms a hetero-complex with the transmembrane protein CDC50A. However, the structural features in CDC50A that support the function of ATP11C and other P4-ATPases have not been characterized. Here, using error-prone PCR-mediated mutagenesis of human CDC50A cDNA followed by functional screening and deep sequencing, we identified 14 amino acid residues that affect ATP11C’s flippase activity. These residues were all located in CDC50A’s extracellular domain and were evolutionarily well conserved. Most of the mutations decreased CDC50A’s ability to chaperone ATP11C and other P4-ATPases to their destinations. The CDC50A mutants failed to form a stable complex with ATP11C and could not induce ATP11C’s PtdSer-dependent ATPase activity. Notably, one mutant variant could form a stable complex with ATP11C and transfer ATP11C to the plasma membrane, yet the ATP11C complexed with this CDC50A variant had very weak or little PtdSer- or PtdEtn-dependent ATPase activity. These results indicated that the extracellular domain of CDC50A has important roles both in CDC50A’s ability to chaperone ATP11C to the plasma membrane and in inducing ATP11C’s ATP hydrolysis–coupled flippase activity.
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Chaperone-mediated autophagy and endosomal microautophagy: joint by a chaperone [Metabolism]

December 15th, 2017 by Kumsal A Tekirdag, Ana Maria Cuervo

A variety of mechanisms deliver cytosolic materials to the lysosomal compartment for degradation through autophagy. Here, we focus on two autophagic pathways, chaperone-mediated autophagy and endosomal microautophagy that rely on the cytosolic chaperone hsc70 for substrate targeting. Although hsc70 participates in triage of proteins for degradation by different proteolytic systems, the common characteristic shared by these two forms of autophagy, is that hsc70 binds directly to a specific five amino acids motif in the cargo protein for its autophagic targeting. We summarize the current understanding of the molecular machineries behind each of these types of autophagy.

Nonhomologous DNA End Joining for Repair of DNA Double-Strand Breaks [Protein Structure and Folding]

December 14th, 2017 by Nicholas R Pannunzio, Go Watanabe, Michael R. Lieber

Nonhomologous DNA end joining (NHEJ) is the predominant DSB repair pathway throughout the cell cycle and accounts for nearly all DSB repair outside of the S and G2 phases. NHEJ relies on Ku to thread onto DNA termini and thereby improve the affinity of the NHEJ enzymatic components consisting of polymerases (Pol μ and Pol λ), a nuclease (the Artemis·DNA-PKcs complex), and a ligase (XLF·XRCC4·Lig4 complex). Each of the enzymatic components is distinctive for its versatility in acting on diverse incompatible DNA end configurations coupled with a flexibility in loading order, resulting in many possible junctional outcomes from one DSB. DNA ends can either be directly ligated or, if the ends are incompatible, processed until a ligatable configuration is achieved that is often stabilized by up to 4 bp of terminal microhomology. Processing of DNA ends results in nucleotide loss or addition, explaining why DSBs repaired by NHEJ are rarely restored to their original DNA sequence. Thus, NHEJ is a single pathway with multiple enzymes at its disposal to repair DSBs, resulting in a diversity of repair outcomes.

The ubiquitin E3 ligase CHIP promotes proteasomal degradation of the serine/threonine protein kinase PINK1 during staurosporine-induced cell death [Cell Biology]

December 14th, 2017 by Lang Yoo, Kwang Chul Chung

Mutations in the gene for the serine/threonine protein kinase PTEN-induced putative kinase 1 (PINK1) are the second most frequent cause of autosomal recessive Parkinson disease (PD). Via its kinase activity, PINK1 regulates neuronal cell survival and mitochondrial quality control. Numerous reports have revealed that PINK1 has diverse and physiologically significant functions, and, therefore, its activity should be tightly regulated. However, the molecular mechanisms regulating PINK1 stability and the modulator(s) involved have not been elucidated. In this study, we demonstrate that the ubiquitin E3 ligase CHIP promotes PINK1 ubiquitination and decreases its steady-state levels. Moreover, PINK1 levels were strongly reduced in HEK293 and SH-SY5Y cells exposed to the apoptosis-inducer staurosporine. Of note, we found that this reduction resulted from CHIP-mediated PINK1 ubiquitination. Accordingly, siRNA-mediated CHIP knockdown reduced susceptibility to staurosporine-induced cell death. Taken together, these findings suggest that CHIP plays a role in negative regulation of PINK1 stability and may suppress PINK1 cytoprotective effect during staurosporine-induced mammalian cell death. We propose that this PINK1 regulatory pathway might contribute to PD pathogenesis.
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Subtle changes at the variable domain interface of the T-cell receptor can strongly increase affinity [Immunology]

December 14th, 2017 by Preeti Sharma, David M Kranz

Most affinity-maturation campaigns for antibodies and T cell receptors (TCRs) operate on the residues at the binding site, located within the loops known as complementarity determining regions (CDRs). Accordingly, mutations in contact residues, or so-called "second shell" residues, that increase affinity are typically identified by group at the α:β interface, at a significant distance from the TCR/pepMHC binding site, remarkably affected ligand binding. The variant retained a high degree of specificity for MART- 1/HLA-A2, indicating that our approach provides a general strategy for engineering improvements in either soluble or cell-based TCRs for therapeutic purposes.directed evolution involving combinatorial libraries. To determine the impact of residues located at a distance from the binding site, here we used single codon libraries of both CDR and non- CDR residues to generate a deep mutational scan of a human TCR against the cancer antigen MART-1/HLA-A2. Non-CDR residues included those at the interface of the TCR variable domains (α and β) and surface-exposed framework residues. Mutational analyses showed that both α:β interface and CDR residues were important in maintaining binding to MART- 1/HLA-A2, likely due to either structural requirements for proper α:β association or direct contact with the ligand. More surprisingly, many α:β interface substitutions yielded improved binding to MART-1/HLA-A2. To further explore this finding, we constructed interface libraries and selected them for improved stability or affinity. Among the variants identified, one conservative substitution (β) was most prevalent. Further analysis of β showed that it enhanced thermostability and increased affinity by 60-fold. Thus, introducing a single hydroxyl group at the α:β interface, at a significant distance from the TCR/pepMHC binding site, remarkably affected ligand binding. The variant retained a high degree of specificity for MART- 1/HLA-A2, indicating that our approach provides a general strategy for engineering improvements in either soluble or cell-based TCRs for therapeutic purposes.

The inducible microRNA-203 in fish represses the inflammatory responses to Gram-negative bacteria by targeting IL-1 receptor-associated kinase 4 [Gene Regulation]

December 14th, 2017 by Tianjun Xu, Qing Chu, Junxia Cui, Xueyan Zhao

Innate immune responses are the first defense against pathogenic invaders. Activation and termination of these immune responses are regulated by several mechanisms. MicroRNAs (miRNAs), a group of small non-coding RNAs, have been implicated in the regulation of a spectrum of both physiological and pathological conditions, including immune responses. Although immune regulatory miRNAs networks in higher vertebrates have been well described, regulation of these responses in fish species is poorly understood in fish species. In the present study, we investigated the role of the miRNA miR-203 involved in inflammatory responses in miiuy croaker (Miichthys miiuy). We found that the Gram-negative bacterium, Vibrio anguillarum and LPS significantly upregulated host miR-203 expression. The increased miR-203 expression suppressed the production of inflammatory cytokines and thereby prevented mounting of a full immune response. Mechanistically, we identified and validated IL-1 receptor-associated kinase 4 (IRAK4) as a target of miR-203. We observed that miR-203 could post-transcriptionally controls IRAK4 expression and thereby inhibits the activation of nuclear factor kappa B (NF-κB) signaling. In summary, our findings reveal that miR-203 in fish is a critical suppressor of innate immune responses to bacterial infection by suppressing a feedback to IRAK4-NF-κB-mediated signaling in fish.
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Structural and Functional Effects of Cytochrome b5 Interactions with Human Cytochrome P450 Enzymes [Enzymology]

October 27th, 2017 by Aaron G. Bart, Emily E. Scott

The small heme-containing protein cytochrome b5 can facilitate, inhibit, or have no effect on cytochrome P450 catalysis, often in a P450-dependent and substrate-dependent manner that is not well understood. Herein solution NMR was used to identify b5 residues interacting with different human drug-metabolizing P450 enzymes. NMR results revealed that P450 enzymes bound to either b5 α4-5 (CYP2A6 and CYP2E1) or this region and α2-3 (CYP2D6 and CYP3A4) and suggested variation in the affinity for b5. Mutations of key b5 residues suggest that not only are different b5 surfaces responsible for binding different P450 enzymes, but that these different complexes are relevant to the observed effects on P450 catalysis.

Structural analyses of the bacterial primosomal protein DnaB reveal that it is a tetramer and forms a complex with a primosomal re-initiation protein [Protein Structure and Folding]

August 14th, 2017 by Yi-Ching Li, Vankadari Naveen, Min-Guan Lin, Chwan-Deng Hsiao

The DnaB primosomal protein from Gram-positive bacteria plays a key role in DNA replication and restart as a loader protein for the recruitment of replisome cascade proteins. Previous investigations have established that DnaB is composed of an N-terminal domain, a middle domain, and a C-terminal domain. However, structural evidence for how DnaB functions at the atomic level is lacking. Here, we report the crystal structure of DnaB, encompassing the N-terminal and middle domains (residues 1-300), from Geobacillus stearothermophilus (GstDnaB1-300) at 2.8 Å resolution. Our structure revealed that GstDnaB1-300 forms a tetramer with two basket-like architecture, a finding s consistent with those from solution studies using analytical ultracentrifugation. Furthermore, our results from both GST pull-down assays and analytical ultracentrifugation show that GstDnaB1-300 is sufficient to form a complex with PriA, the primosomal re-initiation protein. Moreover, with the aid of small angle X-ray scattering (SAXS) experiments, we also determined the structural envelope of full-length DnaB (GstDnaBFL) in solution. These SAXS studies indicated that GstDnaBFL has an elongated conformation and that the protruding density envelopes originating from GstDnaB1-300 could completely accommodate the GstDnaB C-terminal domain (residues 301-461) . Taken together with biochemical assays, our results suggest that GstDnaB uses different domains to distinguish the PriA-interaction and ssDNA-binding. This finding can further extend our understanding of primosomal assembly in replication restart.
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Efficient reduction of CO2 by the molybdenum-containing formate dehydrogenase from Cupriavidus necator (Ralstonia eutropha). [Molecular Biophysics]

August 7th, 2017 by Xuejun Yu, Dimitri Niks, Ashok Mulchandani, Russ Hille

The ability of the FdsABG formate dehydrogenase from Cupriavidus necator (formerly known as Ralstonia eutropha) to catalyze the reverse of the physiological reaction, the reduction of CO2 to formate utilizing NADH as electron donor, has been investigated. Contrary to previous studies of this enzyme, we demonstrate that it is in fact effective in catalyzing the reverse reaction, with a kcat of 11 ± 0.4 s-1. We also quantify the stoichiometric accumulation of formic acid as the product of the reaction and demonstrate that the observed kinetic parameters for catalysis in the forward and reverse reaction are thermodynamically consistent, complying with the expected Haldane relationships. Finally, we demonstrate the reaction conditions necessary for gauging the ability of a given formate dehydrogenase or other CO2-utilizing enzyme to catalyze the reverse direction so as to avoid false negative results. In conjunction with our earlier studies on the reaction mechanism of this enzyme (Niks et al. (2016) J. Biol. Chem. 291, 1162- 1174), and on the basis of the present work we conclude that all molybdenum- and tungsten-containing formate dehydrogenases and related enzymes likely operate via a simple hydride transfer mechanism and are effective in catalysing the reversible interconversion of CO2 and formate under the appropriate experimental conditions.
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