Journal of Biological Chemistry

doi: 10.1016/s0021-9258(20)48682-xpmid: N/A

doi: 10.1016/s0021-9258(20)48681-8pmid: N/A

Wang, Zhaowei;Qiu, Yang;Liu, Yongxiang;Qi, Nan;Si, Jie;Xia, Xiaoling;Wu, Di;Hu, Yuanyang;Zhou, Xi

doi: 10.1074/jbc.m113.492728pmid: 24019510

<p>Nodaviruses are a family of positive-stranded RNA viruses with a bipartite genome of RNAs. In nodaviruses, genomic RNA1 encodes protein A, which is recognized as an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for its RNA replication. Although nodaviral RNA replication has been studied in considerable detail, and nodaviruses are well recognized models for investigating viral RNA replication, the mechanism(s) governing the initiation of nodaviral RNA synthesis have not been determined. In this study, we characterized the RdRP activity of Wuhan nodavirus (WhNV) protein A in detail and determined that this nodaviral protein A initiates RNA synthesis via a <i>de novo</i> mechanism, and this RNA synthesis initiation could be independent of other viral or cellular factors. Moreover, we uncovered that WhNV protein A contains a terminal nucleotidyltransferase (TNTase) activity, which is the first time such an activity has been identified in nodaviruses. We subsequently found that the TNTase activity could function <i>in vitro</i> to repair the 3′ initiation site, which may be digested by cellular exonucleases, to ensure the efficiency and accuracy of viral RNA synthesis initiation. Furthermore, we determined the <i>cis</i>-acting elements for RdRP or TNTase activity at the 3′-end of positive or negative strand RNA1. Taken together, our data establish the <i>de novo</i> synthesis initiation mechanism and the TNTase activity of WhNV protein A, and this work represents an important advance toward understanding the mechanism(s) of nodaviral RNA replication.</p><p>Background: RNA synthesis initiation and 3′-terminal RNA integrity are pivotal for the replication of (+)-RNA viruses.</p><p>Results: A nodaviral replicase can initiate RNA synthesis in a primer-independent manner and contains a terminal nucleotidyltransferase activity.</p><p>Conclusion: These activities collaborate to initiate viral RNA synthesis.</p><p>Significance: This study revealed the initiation mechanism and terminal repair function of the replicase in <i>Nodaviridae</i>.</p>

Martinez-Sanchez, Aida;Murphy, Chris L.

doi: 10.1074/jbc.m113.496729pmid: 24014021

<p>microRNAs are a large and essential class of gene regulators that play key roles in development, homeostasis, and disease. They are necessary for normal skeletal development, and their expression is altered in arthritis. However, the specific role of individual microRNAs is only beginning to be unraveled. Using microRNA expression profiling in healthy human articular cartilage cells (chondrocytes), we identified miR-1247 expression as highly correlated with that of the differentiated cell phenotype. Transcribed from the DLK1-DIO3 locus, the function of miR-1247 is completely unknown. In mice its expression level was relatively high in cartilage tissue, and correlated with cartilage-associated microRNA miR-675 across a range of 15 different mouse tissues. To further probe miR-1247 function, overexpression and inhibition studies were performed in isolated human chondrocytes. Modulation of miR-1247 was found to exert profound phenotypic effects altering expression levels of cartilage master regulator transcription factor SOX9. SOX9 is essential for cartilage development and subsequent function throughout life, and mutations in this gene result in severe dwarfism. Putative miR-1247 binding sites were further investigated using luciferase reporter assays, which indicated binding of miR-1247 to a highly conserved region in the coding sequence of SOX9 but not in its 3′-UTR. Interestingly, depletion of SOX9 in human chondrocytes resulted in increased levels of the mature, processed microRNA, suggesting a negative feedback loop between miR-1247 and its target SOX9.</p><p>Background: The function of miR-1247 was heretofore unknown.</p><p>Results: We show that miR-1247 directly targets SOX9, a transcription factor essential for cartilage formation and function.</p><p>Conclusion: miR-1247 may be an important regulator of cartilage function.</p><p>Significance: miR-1247 is a potential new target for joint repair.</p>

Gadepalli, Ravisekhar;Kotla, Sivareddy;Heckle, Mark R.;Verma, Shailendra K.;Singh, Nikhlesh K.;Rao, Gadiparthi N.

doi: 10.1074/jbc.m113.463414pmid: 24025335

<p>To understand the role of thrombin in inflammation, we tested its effects on migration of THP-1 cells, a human monocytic cell line. Thrombin induced THP-1 cell migration in a dose-dependent manner. Thrombin induced tyrosine phosphorylation of Pyk2, Gab1, and p115 RhoGEF, leading to Rac1- and RhoA-dependent Pak2 activation. Downstream to Pyk2, Gab1 formed a complex with p115 RhoGEF involving their pleckstrin homology domains. Furthermore, inhibition or depletion of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, or Pak2 levels substantially attenuated thrombin-induced THP-1 cell F-actin cytoskeletal remodeling and migration. Inhibition or depletion of PAR1 also blocked thrombin-induced activation of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2, resulting in diminished THP-1 cell F-actin cytoskeletal remodeling and migration. Similarly, depletion of Gα₁₂ negated thrombin-induced Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2 activation, leading to attenuation of THP-1 cell F-actin cytoskeletal remodeling and migration. These novel observations reveal that thrombin induces monocyte/macrophage migration via PAR1-Gα12-dependent Pyk2-mediated Gab1 and p115 RhoGEF interactions, leading to Rac1- and RhoA-targeted Pak2 activation. Thus, these findings provide mechanistic evidence for the role of thrombin and its receptor PAR1 in inflammation.</p><p>Background: The major goal of this study is to test the hypothesis that thrombin plays a role in inflammation.</p><p>Results: Thrombin stimulates monocyte F-actin cytoskeletal remodeling and migration by PAR1, Gα12, Pyk2, Gab1, Rac1, and RhoA-dependent Pak2 activation.</p><p>Conclusion: Pak2 mediates thrombin-PAR1-induced monocyte/macrophage migration.</p><p>Significance: PAR1 could be a potential target for the development of anti-inflammatory drugs.</p>

Yuan, Gang;Ma, Ben;Yuan, Wen;Zhang, Zhuqiang;Chen, Ping;Ding, Xiaojun;Feng, Li;Shen, Xiaohua;Chen, She;Li, Guohong;Zhu, Bing

doi: 10.1074/jbc.m113.475996pmid: 24019522

<p>Histone H3 lysine 27 (H3K27) methylation and H2A monoubiquitination (ubH2A) are two closely related histone modifications that regulate Polycomb silencing. Previous studies reported that H3K27 trimethylation (H3K27me3) rarely coexists with H3K36 di- or tri-methylation (H3K36me2/3) on the same histone H3 tails, which is partially controlled by the direct inhibition of the enzymatic activity of H3K27-specific methyltransferase PRC2. By contrast, H3K27 methylation does not affect the catalytic activity of H3K36-specific methyltransferases, suggesting other Polycomb mechanism(s) may negatively regulate the H3K36-specific methyltransferase(s). In this study, we established a simple protocol to purify milligram quantities of ubH2A from mammalian cells, which were used to reconstitute nucleosome substrates with fully ubiquitinated H2A. A number of histone methyltransferases were then tested on these nucleosome substrates. Notably, all of the H3K36-specific methyltransferases, including ASH1L, HYPB, NSD1, and NSD2 were inhibited by ubH2A, whereas the other histone methyltransferases, including PRC2, G9a, and Pr-Set7 were not affected by ubH2A. Together with previous reports, these findings collectively explain the mutual repulsion of H3K36me2/3 and Polycomb modifications.</p><p>Background: H3K36 methylation antagonizes Polycomb function, but it is not clear whether the reverse is true.</p><p>Results: H3K36-specific histone methyltransferases display poor enzymatic activities on nucleosome substrates containing H2A ubiquitination, an important Polycomb modification.</p><p>Conclusion: H3K36-specific histone methyltransferases can respond to chromatin environment.</p><p>Significance: It provides additional understanding about interplays among chromatin modifications and their roles in transcription regulation.</p>

Kinder, Michelle;Greenplate, Allison R.;Grugan, Katharine D.;Soring, Keri L.;Heeringa, Katharine A.;McCarthy, Stephen G.;Bannish, Gregory;Perpetua, Meredith;Lynch, Frank;Jordan, Robert E.;Strohl, William R.;Brezski, Randall J.

doi: 10.1074/jbc.m113.486142pmid: 23986451

<p>Molecularly engineered antibodies with fit-for-purpose properties will differentiate next generation antibody therapeutics from traditional IgG1 scaffolds. One requirement for engineering the most appropriate properties for a particular therapeutic area is an understanding of the intricacies of the target microenvironment in which the antibody is expected to function. Our group and others have demonstrated that proteases secreted by invasive tumors and pathological microorganisms are capable of cleaving human IgG1, the most commonly adopted isotype among monoclonal antibody therapeutics. Specific cleavage in the lower hinge of IgG1 results in a loss of Fc-mediated cell-killing functions without a concomitant loss of antigen binding capability or circulating antibody half-life. Proteolytic cleavage in the hinge region by tumor-associated or microbial proteases is postulated as a means of evading host immune responses, and antibodies engineered with potent cell-killing functions that are also resistant to hinge proteolysis are of interest. Mutation of the lower hinge region of an IgG1 resulted in protease resistance but also resulted in a profound loss of Fc-mediated cell-killing functions. In the present study, we demonstrate that specific mutations of the C_H2 domain in conjunction with lower hinge mutations can restore and sometimes enhance cell-killing functions while still retaining protease resistance. By identifying mutations that can restore either complement- or Fcγ receptor-mediated functions on a protease-resistant scaffold, we were able to generate a novel protease-resistant platform with selective cell-killing functionality.</p><p>Background: Proteases can cleave human IgG1 antibodies, resulting in loss of cell-killing functions.</p><p>Results: Mutation of the lower hinge of IgG1 confers protease resistance but disrupts Fc effector functions.</p><p>Conclusion: Compensating mutations in the C_H2 domain can selectively restore Fc effector functions on a protease-resistant backbone.</p><p>Significance: Protease-resistant antibodies may be desirable for microenvironments with high protease content and/or when selected cell-killing functions are needed.</p>

Hu, Shaoliang;Wang, Mingrong;Cai, Guoping;He, Mingyue

doi: 10.1074/jbc.m113.467977pmid: 24003234

<p>Universal genetic codes are degenerated with 61 codons specifying 20 amino acids, thus creating synonymous codons for a single amino acid. Synonymous codons have been shown to affect protein properties in a given organism. To address this issue and explore how <i>Escherichia coli</i> selects its "codon-preferred" DNA template(s) for synthesis of proteins with required properties, we have designed synonymous codon libraries based on an antibody (scFv) sequence and carried out bacterial expression and screening for variants with altered properties. As a result, 342 codon variants have been identified, differing significantly in protein solubility and functionality while retaining the identical original amino acid sequence. The soluble expression level varied from completely insoluble aggregates to a soluble yield of ∼2.5 mg/liter, whereas the antigen-binding activity changed from no binding at all to a binding affinity of > 10⁻⁸ m. Not only does our work demonstrate the involvement of genetic codes in regulating protein synthesis and folding but it also provides a novel screening strategy for producing improved proteins without the need to substitute amino acids.</p><p>Background: Synonymous codon usage affects protein properties in a given organism.</p><p>Results: A total of 342 antibody codon variants were identified, differing significantly in solubility and functionality while retaining the identical original amino acid sequence.</p><p>Conclusion: Genetic codes control protein synthesis and folding. "Codon-preferred" DNA template(s) can be generated by functional screening.</p><p>Significance: Protein properties can be considerably altered by synonymous codons without substituting amino acids.</p>

Huo, Lu;Davis, Ian;Chen, Lirong;Liu, Aimin

doi: 10.1074/jbc.m113.496869pmid: 24019523

<p>Although the crystal structure of α-amino-β-carboxymuconate-ϵ-semialdehyde decarboxylase from <i>Pseudomonas fluorescens</i> was solved as a dimer, this enzyme is a mixture of monomer, dimer, and higher order structures in solution. In this work, we found that the dimeric state, not the monomeric state, is the functionally active form. Two conserved arginine residues are present in the active site: Arg-51 and an intruding Arg-239* from the neighboring subunit. In this study, they were each mutated to alanine and lysine, and all four mutants were catalytically inactive. The mutants were also incapable of accommodating pyridine-2,6-dicarboxylic acid, a competitive inhibitor of the native enzyme, suggesting that the two Arg residues are involved in substrate binding. It was also observed that the decarboxylase activity was partially recovered in a heterodimer hybridization experiment when inactive R51(A/K) and R239(A/K) mutants were mixed together. Of the 20 crystal structures obtained from mixing inactive R51A and R239A homodimers that diffracted to a resolution lower than 3.00 Å, two structures are clearly R51A/R239A heterodimers and belong to the C2 space group. They were refined to 1.80 and 2.00 Å resolutions, respectively. Four of the remaining crystals are apparently single mutants and belong to the P4₂2₁2 space group. In the heterodimer structures, one active site is shown to contain dual mutation of Ala-51 and Ala-239*, whereas the other contains the native Arg-51 and Arg-239* residues, identical to the wild-type structure. Thus, these observations provide the foundation for a molecular mechanism by which the oligomerization state of α-amino-β-carboxymuconate-ϵ-semialdehyde decarboxylase could regulate the enzyme activity.</p><p>Background: α-Amino-β-carboxymuconate-ϵ-semialdehyde decarboxylase is a key enzyme that controls quinolinic acid levels.</p><p>Results: Two arginines, including one from a neighboring subunit, are required for substrate binding.</p><p>Conclusion: Dimerization and two arginine residues are required for activity.</p><p>Significance: This study provides the first structurally proven example of a functionally active heterodimer hybrid resulting from a simple mixing of two inactive homodimer mutants.</p>

Lonjedo, Marta;Poch, Enric;Mocholí, Enric;Hernández-Sánchez, Marta;Ivorra, Carmen;Franke, Thomas F.;Guasch, Rosa M.;Pérez-Roger, Ignacio

doi: 10.1074/jbc.m113.511105pmid: 24045951

<p>RhoE/Rnd3 is an atypical member of the Rho family of small GTPases. In addition to regulating actin cytoskeleton dynamics, RhoE is involved in the regulation of cell proliferation, survival, and metastasis. We examined RhoE expression levels during cell cycle and investigated mechanisms controlling them. We show that RhoE accumulates during G₁, in contact-inhibited cells, and when the Akt pathway is inhibited. Conversely, RhoE levels rapidly decrease at the G₁/S transition and remain low for most of the cell cycle. We also show that the half-life of RhoE is shorter than that of other Rho proteins and that its expression levels are regulated by proteasomal degradation. The expression patterns of RhoE overlap with that of the cell cycle inhibitor p27. Consistently with an involvement of RhoE in cell cycle regulation, RhoE and p27 levels decrease after overexpression of the F-box protein Skp2. We have identified a region between amino acids 231 and 240 of RhoE as the Skp2-interacting domain and Lys²³⁵ as the substrate for ubiquitylation. Based on our results, we propose a mechanism according to which proteasomal degradation of RhoE by Skp2 regulates its protein levels to control cellular proliferation.</p><p>Background: RhoE is an atypical Rho protein that lacks GTPase activity.</p><p>Results: RhoE is degraded at the G₁/S cell cycle transition in a proteasome-dependent manner.</p><p>Conclusion: Cell cycle progression requires the proteasomal degradation of RhoE.</p><p>Significance: This new mechanism of controlling RhoE protein levels can regulate cellular proliferation and may be related to cancer.</p>