N6-methyladenosine seqencing highlights the involvement of mRNA methylation in oocyte meiotic maturation and embryo development by regulating translation in Xenopus laevis [Developmental Biology]

September 9th, 2016 by Qi, S.-T., Ma, J.-Y., Wang, Z.-B., Guo, L., Hou, Y., Sun, Q.-Y.

During the oogenesis of Xenopus laevis, oocytes accumulate maternal materials for early embryo development. As the transcription activity of oocyte is silenced at the fully-grown stage and the global genome is reactivated only by the mid-blastula embryo stage, the translation of maternal mRNAs accumulated during oocyte growth should be accurately regulated. Previous evidence has illustrated that the poly(A) tail length and RNA binding elements mediate RNA translation regulation in oocyte. Recently, RNA methylation is found to exist in various systems. In the present study, we sequenced the N6-methyladenosine (m6A) modified mRNAs in fully-grown germinal vesicle (GV) stage and metaphase II (MII) stage oocytes. As a result, we identified 4207 mRNAs with m6A peaks in the GV stage or MII stage oocytes. When we integrated the mRNA methylation data with transcriptome and proteome data, we found that the highly methylated mRNAs showed significantly lower protein levels than those of the hypomethylated mRNAs, although the RNA levels showed no significant difference. We also found that the hypomethylated mRNAs were mainly enriched in the cell cycle and translation pathways, whereas the highly methylated mRNAs were mainly associated with the protein phosphorylation. Our results suggest that the oocyte mRNA methylation can regulate the cellular translation and cell division during oocyte meiotic maturation and early embryo development.
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Identification of the plant ribokinase and discovery of a role for Arabidopsis ribokinase in nucleoside metabolism [Plant Biology]

September 6th, 2016 by Riggs, J. W., Rockwell, N. C., Cavales, P. C., Callis, J.

Ribose can be used for energy or as a component of several important biomolecules but in order for it to be used in either capacity it must first be phosphorylated by ribokinase (RBSK). RBSK proteins are part of the phosphofructokinase-B (pfkB) family of carbohydrate kinases. Sequence comparisons of pfkB proteins from the model plant Arabidopsis thaliana with the human and E. coli RBSK identified a single candidate RBSK, At1g17160 (AtRBSK). AtRBSK is more similar to predicted RBSKs from other plant species and to known mammalian and prokaryotic RBSK than to all other PfkB proteins in Arabidopsis. AtRBSK contains a predicted chloroplast transit peptide, and we confirmed plastid localization using AtRBSK fused to YFP. Structure prediction software verified that the AtRBSK sequence mapped onto a known RBSK structure. Kinetic parameters of purified recombinant AtRBSK were determined to be Kmribose = 153 μM +/- 17 μM, KmATP = 45.9 μM +/- 5.6 μM, kcat = 2.0 s-1. Substrate inhibition was observed for AtRBSK (KiATP = 2.44 mM +/- 0.36 mM), as has been demonstrated for other RBSK proteins. Ribose accumulated in Arabidopsis plants lacking AtRBSK. Such plants grew normally unless media was supplemented with ribose, which led to chlorosis and growth inhibition. Ribose accumulated in plants lacking AtRBSK. Both chlorosis and ribose accumulation were abolished upon the introduction of a transgene expressing AtRBSK-MYC, demonstrating that the loss of protein is responsible for the ribose hypersensitivity. Ribose accumulation in plants lacking AtRBSK was reduced in plants also deficient in the nucleoside ribohydrolase NSH1, linking AtRBSK activity to nucleoside metabolism.

Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-Like Proteins Support a Highly Dynamic Domain Architecture [Protein Structure and Folding]

September 5th, 2016 by Miller, B. R., Drake, E. J., Shi, C., Aldrich, C. C., Gulick, A. M.

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein (PCP), an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might derive from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full length NRPS EntF bound to the MLPs from E. coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatic support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain.
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Leptin Enhances TH2 and ILC2 Responses in Allergic Airway Disease [Molecular Bases of Disease]

August 26th, 2016 by Zheng, H., Zhang, X., Castillo, E. F., Luo, Y., Liu, M., Yang, X. O.

Allergic asthma and obesity are the leading health problems in the world. Many studies have shown that obesity is a risk factor of development of asthma. However, the underlying mechanism has not been well established. In this study, we demonstrate that leptin, an adipokine elevated in obese individuals, promoted proliferation and survival of pro-allergic type 2 helper T cells and group 2 innate lymphoid cells and production of type 2 cytokines, which together contribute to allergic responses. Leptin activates mTORC1, MAPK and STAT3 pathways in TH2 cells. The effects of leptin on TH2 cell proliferation, survival and cytokine production are dependent on the mTORC1 and MAPK pathways as revealed by specific inhibitors. In vivo, leptin-deficiency led to attenuated experimental allergic airway inflammation. Our results thus support that obesity associated elevation of leptin may increase the susceptibility of asthma via modulation of pro-allergic lymphocyte responses.

Proof of Principle: Coronin 1A – An Intrinsic Modulator of T Lymphocyte Function [Signal Transduction]

August 26th, 2016 by Siegmund, K., Klepsch, V., Hermann-Kleiter, N., Baier, G.

Coronins are evolutionarily conserved proteins that were originally identified as modulators of actin-dependent processes. Studies analyzing complete Coronin 1a knockout mice have shown that this molecule is an important regulator of T cell homeostasis and it has been linked to immune deficiencies as well as autoimmune disorders. Nevertheless, since Coronin 1A is strongly expressed in all leukocyte subsets, it is not conclusive whether or not this phenotype is attributed to a T cell-intrinsic function of Coronin 1A. To address this research question, we have generated a T cell-specific Coronin 1a knockout mouse (Coro1afl/fl x Cd4[Cre]). Deletion of Coro1a specifically in T cells led to a strong reduction in T cell number and a shift towards the effector/memory phenotype in peripheral lymphoid organs when compared to Cd4[Cre] mice expressing wild-type Coro1a. In contrast to peripheral lymphoid tissue, thymocyte number and subsets were not affected by the deletion of Coro1a. Furthermore, T cell-specific Coro1a knockout mice were largely resistant to the induction of autoimmunity when tested in the MOG-induced EAE mouse model of multiple sclerosis. Thus, the phenotype of T cell-specific Coro1a deletion resembles the phenotype observed with conventional (whole body) Coro1a knockout mice. In summary, our findings provide formal proof of the predominant T cell-intrinsic role of Coronin 1A.

Severe Molecular Defects Exhibited by the R179H Mutation in Human Vascular Smooth Muscle {alpha}-Actin [Protein Structure and Folding]

August 22nd, 2016 by Lu, H., Fagnant, P. M., Krementsova, E. B., Trybus, K. M.

Mutations in vascular smooth muscle α-actin (SM α-actin), encoded by ACTA2, are the most common cause of familial thoracic aortic aneurysms that lead to dissection (TAAD). The R179H mutation has a poor patient prognosis, and is unique in causing multisystemic smooth muscle dysfunction (1). Here we characterize this mutation in expressed human SM α-actin. R179H actin shows severe polymerization defects, with a 40-fold higher critical concentration for assembly than WT SM α-actin, driven by a high disassembly rate. The mutant filaments are more readily severed by cofilin. Both defects are attenuated by copolymerization with WT. The R179H monomer binds more tightly to profilin, and formin binding suppresses nucleation and slows polymerization rates. Linear filaments will thus not be readily formed, and cells expressing R179H actin will likely have increased levels of monomeric G-actin. The co-transcription factor myocardin-related transcription factor-A (MRTF-A), which affects cellular phenotype, binds R179H actin with less cooperativity than WT actin. Smooth muscle myosin moves R179H filaments more slowly than WT, even when copolymerized with equimolar amounts of WT. The marked decrease in the ability to form filaments may contribute to the poor patient prognosis, and explain why R179H disrupts even visceral smooth muscle cell function where the SM α-actin isoform is present in low amounts. The R179H mutation has the potential to affect actin structure and function in both the contractile domain of the cell, and the more dynamic cytoskeletal pool of actin, both of which are required for contraction.

DUSP1 maintains IRF1 and leads to increased expression of IRF1-dependent genes: A mechanism promoting glucocorticoid-insensitivity [Signal Transduction]

August 22nd, 2016 by Shah, S., King, E. M., Mostafa, M. M., Altonsy, M. O., Newton, R.

Although, the mitogen-activated protein kinase (MAPK) phosphatase, DUSP1, mediates dexamethasone-induced repression of MAPKs, 14 out of 46 interleukin-1β (IL1B)-induced mRNAs were significantly enhanced by DUSP1 over-expression in pulmonary A549 cells. These include the interferon regulatory factor, IRF1, and the chemokine, CXCL10. Of these DUSP1-enhanced mRNAs, 10, including CXCL10, were IRF1-dependent. MAPK inhibitors and DUSP1 over-expression prolonged IRF1 expression by elevating transcription, and increasing IRF1 mRNA and protein stability. Conversely, DUSP1 silencing increased IL1B-induced MAPK phosphorylation, while significantly reducing IRF1 protein expression at 4h. This confirms a regulatory network, whereby DUSP1 switches off MAPKs to maintain IRF1 expression. There was no repression of IRF1 expression by dexamethasone in primary human bronchial epithelial cells, and in A549 cells IL1B-induced IRF1 protein was only modestly and transiently repressed. While dexamethasone did not repress IL1B-induced IRF1 protein expression at 4-6h, silencing of IL1B plus dexamethasone-induced DUSP1 significantly reduced IRF1 expression. IL1B-induced expression of CXCL10 was largely insensitive to dexamethasone, whereas other DUSP1-enhanced, IRF1-dependent mRNAs showed various degrees of repression. With IL1B plus dexamethasone, CXCL10 expression was also IRF1-dependent and expression was reduced by DUSP1 silencing. Thus, IL1B plus dexamethasone-induced DUSP1 maintains expression of IRF1 and the IRF1-dependent gene, CXCL10. This is supported by chromatin immunoprecipitation showing IRF1 recruitment to be essentially unaffected by dexamethasone at the CXCL10 promoter or at the promoters of more highly repressed IRF1-dependent genes. Since, IRF1-dependent genes, such as CXCL10, are central to host defence, these data may help explain the reduced effectiveness of glucocorticoids during asthma exacerbations.
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A Glutaredoxin-BolA Complex Serves as an Iron-Sulfur Cluster Chaperone for the Cytosolic Cluster Assembly Machinery [Metabolism]

August 12th, 2016 by Frey, A. G., Palenchar, D. J., Wildemann, J. D., Philpott, C. C.

Cells contain hundreds of proteins that require iron cofactors for activity. Iron cofactors are synthesized in the cell, but the pathways involved in distributing heme, iron-sulfur clusters, and ferrous/ferric ions to apo-proteins remain incompletely defined. In particular, cytosolic monothiol glutaredoxins and BolA-like proteins have been identified as [2Fe-2S]-coordinating complexes in vitro and iron-regulatory proteins in fungi, but it is not clear how these proteins function in mammalian systems or how this complex might affect Fe-S proteins or the cytosolic Fe-S assembly machinery. To explore these questions, we use quantitative immunoprecipitation and live-cell, proximity-dependent biotinylation, to monitor interactions between Glrx3, BolA2, and components of the cytosolic iron-sulfur cluster assembly system. We characterize cytosolic Glrx3-BolA2 as a [2Fe-2S] chaperone complex in human cells. Unlike complexes formed by fungal orthologs, human Glrx3-BolA2 interaction required the coordination of Fe-S clusters, while Glrx3 homodimer formation did not. Cellular Glrx3-BolA2 complexes increased 6-8-fold in response to increasing iron, forming a rapidly-expandable pool of Fe-S clusters. Fe-S coordination by Glrx3-BolA2 did not depend on Ciapin1 or Ciao1, proteins that bind Glrx3 and are involved in cytosolic Fe-S cluster assembly and distribution. Instead, Glrx3 and BolA2 bound and facilitated Fe-S incorporation into Ciapin1, a [2Fe-2S] protein functioning early in the cytosolic Fe-S assembly pathway. Thus, Glrx3-BolA is a [2Fe-2S] chaperone complex capable of transferring [2Fe-2S] clusters to apo-proteins in human cells.

Allostery Wiring Map for Kinesin Energy Transduction and its Evolution [Enzymology]

August 8th, 2016 by Richard, J., Kim, E. D., Nguyen, H., Kim, C. D., Kim, S.

How signals between the kinesin active- and cytoskeletal-binding sites are transmitted is an open question and an allosteric question. By extracting correlated evolutionary changes within 700+ sequences, we built a model of residues that are energetically coupled and that define molecular routes for signal transmission. Typically, these coupled residues are located at multiple distal sites and, thus, are predicted to form a complex, nonlinear network that wires together different functional sites in the protein. Of note, our model connected the site for ATP hydrolysis with sites that ultimately utilize its free energy, such as the microtubule-binding site, drug-binding loop-5, and necklinker. To confirm the calculated energetic connectivity between non-adjacent residues, double-mutant cycle analysis was conducted with 22 kinesin mutants. There was a direct correlation between thermodynamic coupling in experiment and evolutionarily-derived energetic coupling. We conclude that energy transduction is coordinated by multiple distal sites in the protein, rather than only being relayed through adjacent residues. Moreover, this allosteric map forecasts how energetic orchestration gives rise to different nanomotor behaviors within the superfamily.

Different Divalent Cations Alter the Kinetics and Fidelity of DNA Polymerases [DNA and Chromosomes]

August 8th, 2016 by Vashishtha, A. K., Wang, J., Konigsberg, W. H.

Divalent metal ions are essential components of DNA polymerases both for catalysis of the nucleotidyl transfer reaction and for base excision. They occupy two sites, A and B for DNA synthesis. Recently, a third metal ion was shown to be essential for phosphoryl transfer reaction. The metal ion in the A site is coordinated by the carboxylate of two highly conserved acidic residues, water molecules, and the 3' hydroxyl group of the primer so that the A metal is in an octahedral complex. Its catalytic function is to lower the pKa of the hydroxyl group making it a highly effective nucleophile that can attack the alpha phosphorous atom of the incoming dNTP. The metal ion in the B site is coordinated by the same two carboxylates that are affixed to the A metal ion as well as the non-bridging oxygen atoms of the incoming dNTP. The carboxyl oxygen of an adjacent peptide bond serves as the sixth ligand that completes the octahedral coordination geometry of the B metal ion. Similarly two metal ions are required for proof-reading, one helps to lower the pKa of the attacking water molecule and the other helps to stabilize the transition state for nucleotide excision. The role of different divalent cations are discussed in relation to these two activities as well as their influence on base selectivity and misincorporation by DNA polymerases. Some, but not all of the effects of these different metal ions can be rationalized based on their intrinsic properties which are tabulated in this review.