Predicting repeat protein folding kinetics from an experimentally determined folding energy landscape

The Notch ankyrin domain is a repeat protein whose folding has been characterized through equilibrium and kinetic measurements. In previous work, equilibrium folding free energies of truncated constructs were used to generate an experimentally determined folding energy landscape (Mello and Barrick, Proc Natl Acad Sci USA 2004;101:14102–14107). Here, this folding energy landscape is used to parameterize a kinetic model in which local transition probabilities between partly folded states are based on energy values from the landscape. The landscape‐based model correctly predicts highly diverse experimentally determined folding kinetics of the Notch ankyrin domain and sequence variants. These predictions include monophasic folding and biphasic unfolding, curvature in the unfolding limb of the chevron plot, population of a transient unfolding intermediate, relative folding rates of 19 variants spanning three orders of magnitude, and a change in the folding pathway that results from C‐terminal stabilization. These findings indicate that the folding pathway(s) of the Notch ankyrin domain are thermodynamically selected: the primary determinants of kinetic behavior can be simply deduced from the local stability of individual repeats.     

pH-Dependent Conformational Changes in Bacterial Hsp90 Reveal a Grp94-Like Conformation at pH 6 That Is Highly Active in Suppression of Citrate Synthase Aggregation

The molecular chaperone Hsp90 depends upon large conformational rearrangements for its function. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, structural and kinetic studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis shift the equilibrium between a preexisting set of conformational states in an organism-dependent manner. While many conformations of Hsp90 have been described, little is known about how they relate to chaperone function. In this study, we show that the conformational equilibrium of the bacterial Hsp90, HtpG, can be shifted with pH. Using small-angle X-ray scattering, we identify a two-state pH-dependent conformational equilibrium for apo HtpG. Our structural modeling reveals that this equilibrium is observed between the previously observed extended state and a second state that is strikingly similar to the recently solved Grp94 crystal structure. In the presence of nonhydrolyzable 5′-adenylyl-β,γ-imidodiphosphate, a third state, which is identical with the solved AMPPNP-bound structure from yeast Hsp90, is populated. Electron microscopy confirmed the observed conformational equilibria. We also identify key histidine residues that control this pH-dependent equilibrium; using mutagenesis, we successfully modulate the conformational equilibrium at neutral pH. Using these mutations, we show that the Grp94-like state provides stronger aggregation protection compared to the extended apo conformation in the context of a citrate synthase aggregation assay. These studies provide a more detailed view of HtpG's conformational dynamics and provide the first linkage between a specific conformation and chaperone function.     

Genetic Detection and Characterization of Lujo Virus,a New Hemorrhagic Fever–Associated Arenavirus from Southern Africa

Lujo virus (LUJV), a new member of the family Arenaviridae and the first hemorrhagic fever–associated arenavirus from the Old World discovered in three decades, was isolated in South Africa during an outbreak of human disease characterized by nosocomial transmission and an unprecedented high case fatality rate of 80% (4/5 cases). Unbiased pyrosequencing of RNA extracts from serum and tissues of outbreak victims enabled identification and detailed phylogenetic characterization within 72 hours of sample receipt. Full genome analyses of LUJV showed it to be unique and branching off the ancestral node of the Old World arenaviruses. The virus G1 glycoprotein sequence was highly diverse and almost equidistant from that of other Old World and New World arenaviruses, consistent with a potential distinctive receptor tropism. LUJV is a novel, genetically distinct, highly pathogenic arenavirus.     

PDA: Pooled DNA analyzer

Background  

Association mapping using abundant single nucleotide polymorphisms is a powerful tool for identifying disease susceptibility genes for complex traits and exploring possible genetic diversity. Genotyping large numbers of SNPs individually is performed routinely but is cost prohibitive for large-scale genetic studies. DNA pooling is a reliable and cost-saving alternative genotyping method. However, no software has been developed for complete pooled-DNA analyses, including data standardization, allele frequency estimation, and single/multipoint DNA pooling association tests. This motivated the development of the software, 'PDA' (Pooled DNA Analyzer), to analyze pooled DNA data.     

Cancer dormancy. VII. A regulatory role for CD8+ T cells and IFN-gamma in establishing and maintaining the tumor-dormant state

Dormant tumor cells resistant to ablative cancer therapy represent a significant clinical obstacle due to later relapse. Experimentally, the murine B cell lymphoma (BCL1) is used as a model of tumor dormancy in mice vaccinated with the BCL1 Ig. Here, we used this model to explore the cellular mechanisms underlying dormancy. Our previous studies have demonstrated that T cell-mediated immunity is an important component in the regulation of tumor dormancy because Id-immune T cells adoptively transferred into passively immunized SCID mice challenged with BCL1 cells significantly increased the incidence and duration of the dormant state. We have extended these observations and demonstrate that CD8+, but not CD4+, T cells are required for the maintenance of dormancy in BCL1 Ig-immunized BALB/c mice. In parallel studies, the transfer of Id-immune CD8+ cells, but not Id-immune CD4+ cells, conferred significant protection to SCID mice passively immunized with nonprotective levels of polyclonal anti-Id and then challenged with BCL1 cells. Furthermore, the ability of CD8+ T cells to induce a state of dormancy in passively immunized SCID mice was completely abrogated by treatment with neutralizing alpha-IFN-gamma mAbs in vivo. In vitro studies demonstrated that IFN-gamma alone or in combination with reagents to cross-link the surface Ig induced both cell cycle arrest and apoptosis in a BCL1 cell line. Collectively, these data demonstrate a role for CD8+ T cells via endogenous production of IFN-gamma in collaboration with humoral immunity to both induce and maintain a state of tumor dormancy.     

Cutting edge: granzymes A and B are not essential for perforin-mediated tumor rejection

Controversy still exists regarding the biological function of granzyme serine proteases released with perforin from the cytotoxic granules of NK cells and CTLs. In particular, it is not clear whether the major granzymes, A and B, play an essential role in tumor rejection mediated by the perforin pathway. We have now examined the relative importance of perforin and granzyme A and B clusters in five different tumor models that stringently distinguish their importance. We conclude that granzyme A and B clusters are not essential for CTL- and NK cell-mediated rejection of spontaneous and experimental tumors, raising the likelihood that either perforin alone or in combination with an additional granzyme or granule component(s) mediates cytotoxicity of tumor cells in vivo.     

Comment on "The first description of an archaeal hemicellulase: the xylanase from Thermococcus zilligii strain AN1": evidence that the unique N-terminal sequence proposed comes from a maltodextrin phosphorylase

Uhl and Daniel reported in this journal in 1999 (Extremophiles 3:263-267) the characterization of the first archaeal hemicellulase with a unique N-terminal sequence showing no homology with any xylanase or other protein from the databases. A genomic library of the chromosomal DNA of Thermococcus zilligii strain AN1 was screened by using a degenerate probe deduced from the N-terminal sequence. A positive clone was identified, and an amino acid sequence analysis revealed that the N-terminal sequence from this protein and the N-terminal sequence from the putative xylanase of T. zilligii were identical. However, the comparison of the amino acid sequence of the protein with sequences in the main protein databases revealed significant similarities with maltodextrin phosphorylases. In conclusion, it is likely that the N-terminal sequence proposed by Uhl and Daniel is not that of the T. zilligii xylanase, but corresponds to an archaeal T. zilligii maltodextrin phosphorylase.     

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

Histochemical characterization of monoamine oxidase in ependyma of rat hypothalamus

Sunopsis Monoamine oxidase (MAO) activity has been demonstrated histochemically in rat hypothalamic ependyma using the sulphate-tetrazolium and coupled peroxidatic techniques with tryptamine, tyramine, 5-hydroxytryptamine and benzylamine as substrates. Both methods were applied to cryostat sections with and without exposure to selective amine oxidase inhibitors, including the selective A-MAO inhibitor clorgyline, and the B-MAO inhibitor deprenyl. Our results show that both cuboidal-columnar and tanycyte ependyma contain one or more forms of MAO not generally present in the hypothalamus. It is suggested that ependymal MAO may form an amine-barrier system modulating the movement and effect within the hypothalamus of specific cerebrospinal fluid or blood monoamines.     

Palaeoecological studies at the Mesolithic site at Bedburg-Königshov near Cologne, Germany

The Late Glacial and early Holocene palaeovegetation is reconstructed by analysis of pollen and macroscopic plant remains from sediments of the lower river Erft valley, near Cologne, exposed by the Garzweiler opencast lignite mine. The study was carried out in parallel with the archaeological excavation of the Mesolithic site of Bedburg-Königshoven, located within a former meander of the Erft. During the Younger Dryas period, the study region was thinly wooded. Relatively open pine woods existed during the Preboreal, becoming more closed during the Boreal. Later in the Boreal,Corylus and taxa of the mixed oak woodland (Ulmus, Quercus, Tilia andFraxinus) appeared. The Atlantic period was characterised by mixed oak woods with dominantUlmus. At the beginning of the Subboreal,Ulmus declined andTilia became the predominant element of the woods. The Mesolithic settlement existed during the early and middle part of the Preboreal period. At that time, the woodland of the Erft valley was mainly composed ofPinits, Belula and somePopulus. The pollen results do not show any evidence of the activity of Mesolithic people. Local vegetational changes of the filling process of the Erft meander were described and interpreted. The vegetational development of the study region is compared with that of neighbouring regions.