Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo

Citation: Lageix, S., Zhang, J. W., Rothenburg, S., & Hinnebusch, A. G. (2015). Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo. Plos Genetics, 11(2), 28. doi:10.1371/journal.pgen.1004991The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2 alpha. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd(-) phenotype), while other substitutions block kinase activation (Gcn(-) phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn(-) substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd(-) substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction

Stress-strain behavior and geometrical properties of packings of elongated particles

We present a numerical analysis of the effect of particle elongation on the quasistatic behavior of sheared granular media by means of the Contact Dynamics method. The particle shapes are rounded-cap rectangles characterized by their elongation. The macroscopic and microstructural properties of several packings subjected to biaxial compression are analyzed as a function of particle elongation. We find that the shear strength is an increasing linear function of elongation. Performing an additive decomposition of the stress tensor based on a harmonic approximation of the angular dependence of branch vectors, contact normals and forces, we show that the increasing mobilization of friction force and the associated anisotropy are key effects of particle elongation. These effects are correlated with partial nematic ordering of the particles which tend to be oriented perpendicular to the major principal stress direction and form side-to-side contacts. However, the force transmission is found to be mainly guided by cap-to-side contacts, which represent the largest fraction of contacts for the most elongated particles. Another interesting finding is that, in contrast to shear strength, the solid fraction first increases with particle elongation, but declines as the particles become more elongated. It is also remarkable that the coordination number does not follow this trend so that the packings of more elongated particles are looser but more strongly connected.Comment: Submited to Physical Review

Force transmission in a packing of pentagonal particles

We perform a detailed analysis of the contact force network in a dense confined packing of pentagonal particles simulated by means of the contact dynamics method. The effect of particle shape is evidenced by comparing the data from pentagon packing and from a packing with identical characteristics except for the circular shape of the particles. A counterintuitive finding of this work is that, under steady shearing, the pentagon packing develops a lower structural anisotropy than the disk packing. We show that this weakness is compensated by a higher force anisotropy, leading to enhanced shear strength of the pentagon packing. We revisit "strong" and "weak" force networks in the pentagon packing, but our simulation data provide also evidence for a large class of "very weak" forces carried mainly by vertex-to-edge contacts. The strong force chains are mostly composed of edge-to-edge contacts with a marked zig-zag aspect and a decreasing exponential probability distribution as in a disk packing

Solid behavior of anisotropic rigid frictionless bead assemblies

We investigate the structure and mechanical behavior of assemblies of frictionless, nearly rigid equal-sized beads, in the quasistatic limit, by numerical simulation. Three different loading paths are explored: triaxial compression, triaxial extension and simple shear. Generalizing recent results [1], we show that the material, despite rather strong finite sample size effects, is able to sustain a finite deviator stress in the macroscopic limit, along all three paths, without dilatancy. The shape of the yield surface is adequately described by a Lade-Duncan (rather than Mohr-Coulomb) criterion. While scalar state variables keep the same values as in isotropic systems, fabric and force anisotropies are each characterized by one parameter and are in one-to-one correspondence with principal stress ratio along all three loading paths.The anisotropy of the pair correlation function extends to a distance between bead surfaces on the order of 10% of the diameter. The tensor of elastic moduli is shown to possess a nearly singular, uniaxial structure related to stress anisotropy. Possible stress-strain relations in monotonic loading paths are also discussed

Sensitivity of the stress response function to packing preparation

A granular assembly composed of a collection of identical grains may pack under different microscopic configurations with microscopic features that are sensitive to the preparation history. A given configuration may also change in response to external actions such as compression, shearing etc. We show using a mechanical response function method developed experimentally and numerically, that the macroscopic stress profiles are strongly dependent on these preparation procedures. These results were obtained for both two and three dimensions. The method reveals that, under a given preparation history, the macroscopic symmetries of the granular material is affected and in most cases significant departures from isotropy should be observed. This suggests a new path toward a non-intrusive test of granular material constitutive properties.Comment: 15 pages, 11 figures, some numerical data corrected, to appear in J. Phys. Cond. Mat. special issue on Granular Materials (M. Nicodemi Editor

Double-stranded RNA-activated protein kinase PKR of fishes and amphibians: Varying the number of double-stranded RNA binding domains and lineage-specific duplications

BackgroundDouble-stranded (ds) RNA, generated during viral infection, binds and activates the mammalian anti-viral protein kinase PKR, which phosphorylates the translation initiation factor eIF2alpha leading to the general inhibition of protein synthesis. Although PKR-like activity has been described in fish cells, the responsible enzymes eluded molecular characterization until the recent discovery of goldfish and zebrafish PKZ, which contain Z-DNA-binding domains instead of dsRNA-binding domains (dsRBDs). Fish and amphibian PKR genes have not been described so far.ResultsHere we report the cloning and identification of 13 PKR genes from 8 teleost fish and amphibian species, including zebrafish, demonstrating the coexistence of PKR and PKZ in this latter species. Analyses of their genomic organization revealed up to three tandemly arrayed PKR genes, which are arranged in head-to-tail orientation. At least five duplications occurred independently in fish and amphibian lineages. Phylogenetic analyses reveal that the kinase domains of fish PKR genes are more closely related to those of fish PKZ than to the PKR kinase domains of other vertebrate species. The duplication leading to fish PKR and PKZ genes occurred early during teleost fish evolution after the divergence of the tetrapod lineage. While two dsRBDs are found in mammalian and amphibian PKR, one, two or three dsRBDs are present in fish PKR. In zebrafish, both PKR and PKZ were strongly upregulated after immunostimulation with some tissue-specific expression differences. Using genetic and biochemical assays we demonstrate that both zebrafish PKR and PKZ can phosphorylate eIF2alpha in yeast.ConclusionConsidering the important role for PKR in host defense against viruses, the independent duplication and fixation of PKR genes in different lineages probably provided selective advantages by leading to the recognition of an extended spectrum of viral nucleic acid structures, including both dsRNA and Z-DNA/RNA, and perhaps by altering sensitivity to viral PKR inhibitors. Further implications of our findings for the evolution of the PKR family and for studying PKR/PKZ interactions with viral gene products and their roles in viral infections are discussed

Effect of heterogeneity on the elastic properties of auxetic materials

Copyright © 2003 American Institute of Physics. This article may be downloaded for personal use only. Any other use requires prior permission of the author and the American Institute of Physics. The following article appeared in Journal of Applied Physics 94 (2003) and may be found at http://link.aip.org/link/?jap/94/6143Auxetic materials are gaining practical interest for their unusual and sometimes extreme mechanical response. The process of modeling these materials so far has highlighted a number of microstructural properties that are key to these materials. However these models often rely on the assumption of homogeneity and order within the materials. Practically, a homogeneous auxetic material such as foam is unlikely to be manufactured. This work seeks to analyze the effect of fluctuations within the microstructure of the material. Numerical results show the effect of fluctuations in an auxetic granular substance and analytical work indicates the relation between microscale fluctuations and the elastic moduli for a general auxetic material

Phase II study of irinotecan with bolus and high dose infusional 5-FU and folinic acid (modified de Gramont) for first or second line treatment of advanced or metastatic colorectal cancer

We investigated the activity of irinotecan given with a more convenient modified bimonthly de Gramont regimen of bolus and infusional 5-fluorouracil [IrMdG] in advanced or metastatic colorectal cancer in the first and second line setting. Irinotecan 180 mg m−2 was infused over 90 min. L-folinic acid 175 mg or d,l folinic acid 350 mg was given over 2 h followed by a bolus of 5-fluorouracil (400 mg m−2) and a 46 h continuous infusion of 5-fluorouracil (2.4–2.8 g m−2). Forty-six previously untreated patients (Group A) and 36 who had received 5-fluorouracil for metastatic disease (Group B) were recruited. Seventy-eight patients were evaluable for response. A partial response was seen in 13 out of 43 (30% [95%CI 28.1–31.9%]) in Group A and 8/35 (23% [95% CI 17.9–28.1%]) in Group B. 40% (95%CI 38.1–41.9%) of Group A and 26% (95% CI 20.9–31.1%) of Group B patients achieved disease stabilisation. The median progression free survival from the start of this treatment was 7 months (95% CI 4.4–9.6 months) in Group A and 5 months (95% CI 2.8–7.2 months) in Group B. Median overall survival was 14 months (95% CI 9.0–18.9) in Group A and 11 months (95% CI 5.9–16.1) in Group B. Grade 3–4 toxicity in both treatment groups were similar; leucopenia 17% and diarrhoea 7–8%. Grade 3–4 mucositis was not seen and severe alopecia affected only three patients. IrMdG is an active and well-tolerated regimen for both the first and second line treatment of advanced colorectal cancer