Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) on serine 51 integrates general translation repression with activation of stress-inducible genes such as ATF4, CHOP, and BiP in the unfolded protein response. We sought to identify new genes active in this phospho-eIF2alpha-dependent signaling pathway by screening a library of recombinant retroviruses for clones that inhibit the expression of a CHOP::GFP reporter. A retrovirus encoding the COOH terminus of growth arrest and DNA damage gene (GADD)34, also known as MYD116 (Fornace, A.J., D.W. Neibert, M.C. Hollander, J.D. Luethy, M. Papathanasiou, J. Fragoli, and N.J. Holbrook. 1989. Mol. Cell. Biol. 9:4196-4203; Lord K.A., B. Hoffman-Lieberman, and D.A. Lieberman. 1990. Nucleic Acid Res. 18:2823), was isolated and found to attenuate CHOP (also known as GADD153) activation by both protein malfolding in the endoplasmic reticulum, and amino acid deprivation. Despite normal activity of the cognate stress-inducible eIF2alpha kinases PERK (also known as PEK) and GCN2, phospho-eIF2alpha levels were markedly diminished in GADD34-overexpressing cells. GADD34 formed a complex with the catalytic subunit of protein phosphatase 1 (PP1c) that specifically promoted the dephosphorylation of eIF2alpha in vitro. Mutations that interfered with the interaction with PP1c prevented the dephosphorylation of eIF2alpha and blocked attenuation of CHOP by GADD34. Expression of GADD34 is stress dependent, and was absent in PERK(-)/- and GCN2(-)/- cells. These findings implicate GADD34-mediated dephosphorylation of eIF2alpha in a negative feedback loop that inhibits stress-induced gene expression, and that might promote recovery from translational inhibition in the unfolded protein response.     

Four new Candida species from geographically diverse locations

Four new species of Candida are described based on their unique nucleotide sequences in the D1/D2 domain of large subunit (26S) ribosomal DNA. Candida peoriaensis (type strain NRRL YB-1497, CBS 8800) and C. ponderosae (type strain NRRL YB-2307, CBS 8801) are members of the Pichia anomala clade and were isolated in the U.S. from, respectively, the stump of an elm tree (Ulmus sp.) and from insect frass of a Ponderosa pine (Pinus ponderosa). Candida ghanaensis (type strain NRRL YB-1486, CBS 8798) is a phylogenetically divergent species from soil in Ghana and appears related to the Dipodascus/Geotrichum clade. Candida litsaeae (type strain NRRL YB-3246, CBS 8799) was isolated from the frass of an insect-infested Litsaea polyantha tree from India, and is a divergent species that is most closely related to Candida ontarioensis.     

Bacterial diversity in soil samples from two uranium waste piles as determined by rep-APD, RISA and 16S rDNA retrieval

In a study of the mycobiota associated with bark beetles, a dimorphic fungus producing longitudinally septate basidia of the Tremella-type and yeast cells budding off from stalks, was collected. Detailed morphological, physiological and molecular studies revealed that this fungus represents the teleomorph of Sterigmatosporidium polymorphum. Consequently, a new genus, Cuniculitrema gen. nov., and a new species, C. polymorpha sp. nov., are proposed. Comparative morphological and molecular studies indicated that the new taxon belongs to a group that also comprises species of the stalk-forming anamorphic genera Fellomyces and Kockovaella. The new family Cuniculitremaceae is proposed for this group.     

Molecular taxonomy and biodiversity of rock fungal communities in an urban environment (Vienna, Austria)

The diversity of fungal communities on three different historical monuments in the city of Vienna (Austria) was analyzed and compared to the fungal diversity of microfungi on rock in the original quarry located in a rural area (Zogelsdorf, Austria). The fungal strains isolated were characterized by morphology and the complete rock fungal community was identified based on molecular data, that is, by sequencing parts of the small ribosomal subunit (18S) and internal transcribed spacer region 1 (ITS1). The genera Coniothyrium, Epicoccum and Phoma were found to be dominant {on} monument and rock surfaces. Additionally, black yeasts such as Exophiala species and microcolonial fungi like Sarcinomyces and Coniosporium which hitherto were regarded as typical rock inhabitants in semi-arid environments are frequently found on all rock surfaces in Vienna. The biodiversity of the fungi in the urban environment was much higher than on the same rock type in a rural environment, this difference can be attributed to the elevated organic pollution in the city.     

The evolution of sexual size dimorphism in the house finch. IV. Population divergence in ontogeny

Differences among taxa in sexual size dimorphism of adults can be produced by changes in distinct developmental processes and thus may reflect different evolutionary histories. Here we examine whether divergence in sexual dimorphism of adults between recently established Montana and Alabama populations of the house finch (Carpodacus mexicanus) can be attributed to population differences in growth of males and females. In both populations, males and females were similar at hatching, but as a result of sex-specific growth attained sexual size dimorphism by the time of independence. Timing and extent of growth varied between the sexes: Females maintained maximum rates of growth for a longer time than males, whereas males had higher initial growth rates and achieved maximum growth earlier and at smaller sizes than females. Ontogeny of sexual dimorphism differed between populations, but in each population, sexual dimorphism in growth parameters and sexual dimorphism at the time of nest leaving were similar to sexual dimorphism of adults. Variation in growth of females contributed more to population divergence than did growth of males. In each population, we found close correspondence between patterns of sexual dimorphism in growth and population divergence in morphology of adults: Traits that were the most sexually dimorphic in growth in each population contributed the most to population divergence in both sexes. We suggest that sex-specific expression of phenotypic and genetic variation throughout the ontogeny of house finches can result in different responses to selection between males and females of the same age, and thus produce fast population divergence in the sexual size dimorphism.     

Rapid evolution in the Nebria gregaria group (Coleoptera: Carabidae) and the paleogeography of the Queen Charlotte Islands

Morphological differentiation in the ground beetles of the Nebria gregaria group, found on the Queen Charlotte Islands, has been used as support for the glacial refugium proposed for the northwest coast of North America. Two members of this species group, N. charlottae and N. louiseae, are restricted to cobble beaches in this archipelago. A third, N. haida, is found only in alpine regions of the archipelago and the adjacent mainland. The remaining two species of the gregaria group, N. lituyae and N. gregaria, show highly restricted distributions in the mountains of the Alaska panhandle and on the beaches of the Aleutian Islands, respectively. To determine the relationships of the five species, we conducted phylogenetic analyses on nucleotide sequence data obtained from five regions of the mitochondrial DNA. In total, 1835 bp were analyzed. The results suggest that one species, N. lituyae, does not belong in the gregaria group, and that only seven mutations separated the two most divergent of the four remaining species. We also conducted random amplified polymorphic DNA fingerprinting analyses on genomic DNA extracted from the five species. Analyses of genetic diversity revealed a lack of molecular differentiation among the Queen Charlotte species, suggesting that these populations may be postglacial in origin and that together N. gregaria, N. charlottae, N. louiseae, and N. haida might represent local variations of a single species. These results are consistent with conclusions derived for the morphological and genetical differentiation among Gasterosteus populations in the archipelago.     

Protein structure determination using a database of interatomic distance probabilities

The accelerated pace of genomic sequencing has increased the demand for structural models of gene products. Improved quantitative methods are needed to study the many systems (e.g., macromolecular assemblies) for which data are scarce. Here, we describe a new molecular dynamics method for protein structure determination and molecular modeling. An energy function, or database potential, is derived from distributions of interatomic distances obtained from a database of known structures. X-ray crystal structures are refined by molecular dynamics with the new energy function replacing the Van der Waals potential. Compared to standard methods, this method improved the atomic positions, interatomic distances, and side-chain dihedral angles of structures randomized to mimic the early stages of refinement. The greatest enhancement in side-chain placement was observed for groups that are characteristically buried. More accurate calculated model phases will follow from improved interatomic distances. Details usually seen only in high-resolution refinements were improved, as is shown by an R-factor analysis. The improvements were greatest when refinements were carried out using X-ray data truncated at 3.5 A. The database potential should therefore be a valuable tool for determining X-ray structures, especially when only low-resolution data are available.     

Acetylcholinesterase of Schistosoma mansoni--functional correlates. Contributed in honor of Professor Hans Neurath's 90th birthday

Acetylcholinesterase (AChE) is an enzyme broadly distributed in many species, including parasites. It occurs in multiple molecular forms that differ in their quaternary structure and mode of anchoring to the cell surface. This review summarizes biochemical and immunological investigations carried out in our laboratories on AChE of the helmint, Schistosoma mansoni. AChE appears in S. mansoni in two principal molecular forms, both globular, with sedimentation coefficients of approximately 6.5 and 8 S. On the basis of their substrate specificity and sensitivity to inhibitors, both are "true" acetylcholinesterases. Approximately half of the AChE activity of S. mansoni is located on the outer surface of the parasite, attached to the tegumental membrane via a covalently attached glycosylphosphatidylinositol anchor. The remainder is located within the parasite, mainly associated with muscle tissue. Whereas the internal enzyme is most likely involved in termination of neurotransmission at cholinergic synapses, the role of the surface enzyme remains to be established; there are, however, indications that it is involved in signal transduction. The two forms of AChE differ in their heparin-binding properties, only the internal 8 S form of the AChE being retained on a heparin column. The two forms differ also in their immunological specificity, since they are selectively recognized by different monoclonal antibodies. Polyclonal antibodies raised against S. mansoni AChE purified by affinity chromatography are specific for the parasite AChE, reacting with both molecular forms, but do not recognize AChE from other species. They interact with the surface-localized enzyme on the intact organism, and produce almost total complement-dependent killing of the parasite. S. mansoni AChE is thus demonstrated to be a functional protein, involved in multifaceted activities, which can serve as a suitable candidate for diagnostic purposes, vaccine development, and drug design.     

Protein-protein recognition: an experimental and computational study of the R89K mutation in Raf and its effect on Ras binding

Binding of the protein Raf to the active form of Ras promotes activation of the MAP kinase signaling pathway, triggering cell growth and differentiation. Raf/Arg89 in the center of the binding interface plays an important role determining Ras-Raf binding affinity. We have investigated experimentally and computationally the Raf-R89K mutation, which abolishes signaling in vivo. The binding to [gamma-35S]GTP-Ras of a fusion protein between the Raf-binding domain (RBD) of Raf and GST was reduced at least 175-fold by the mutation, corresponding to a standard binding free energy decrease of at least 3.0 kcal/mol. To compute this free energy and obtain insights into the microscopic interactions favoring binding, we performed alchemical simulations of the RBD, both complexed to Ras and free in solution, in which residue 89 is gradually mutated from Arg into Lys. The simulations give a standard binding free energy decrease of 2.9+/-1.9 kcal/mol, in agreement with experiment. The use of numerous runs with three different force fields allows insights into the sources of uncertainty in the free energy and its components. The binding decreases partly because of a 7 kcal/mol higher cost to desolvate Lys upon binding, compared to Arg, due to better solvent interactions with the more concentrated Lys charge in the unbound state. This effect is expected to be general, contributing to the lower propensity of Lys to participate in protein-protein interfaces. Large contributions to the free energy change also arise from electrostatic interactions with groups up to 8 A away, namely residues 37-41 in the conserved effector domain of Ras (including 4 kcal/mol from Ser39 which loses a bifurcated hydrogen bond to Arg89), the conserved Lys84 and Lys87 of Raf, and 2-3 specific water molecules. This analysis will provide insights into the large experimental database of Ras-Raf mutations.