bluuebunny/crossref_metadata_2025_split huggingface dataset:
pH‐induced folding of an apoptotic coiled coil
Authors: Dutta Kaushik , Alexandrov Andrei , Huang He , Pascal Steven M. 
Published: February 2009

AbstractPar‐4 is a 38‐kD protein pivotal to the apoptotic pathways of various cell types, most notably prostate cells and neurons, where it has been linked to prostate cancer and various neurodegenerative disorders including Alzheimer's and Huntington's diseases and HIV encephalitis. The C‐terminal region of Par‐4 is responsible for homodimerization and the ability of Par‐4 to interact with proposed effector molecules. In this study, we show that the C‐terminal 47 residues of Par‐4 are natively unfolded at physiological pH and temperature. Evidence is rapidly accumulating that natively unfolded proteins play an important role in various cellular functions and signaling pathways, and that folding can often be induced on complexation with effector molecules or alteration of environment. Here we use primarily CD studies to show that changes in the environment, particularly pH and temperature, can induce the Par‐4 C terminus to form a self‐associated coiled coil.
The hydrogen exchange core and protein folding
Authors: Li Renhao , Woodward Clare 
Published: February 2009

AbstractA database of hydrogen‐deuterium exchange results has been compiled for proteins for which there are published rates of out‐exchange in the native state, protection against exchange during folding, and out‐exchange in partially folded forms. The question of whether the slow exchange core is the folding core (Woodward C, 1993, Trends Biochem Sci 18:359–360) is reexamined in a detailed comparison of the specific amide protons (NHs) and the elements of secondary structure on which they are located. For each pulsed exchange or competition experiment, probe NHs are shown explicitly; the large number and broad distribution of probe NHs support the validity of comparing out‐exchange with pulsed‐exchange/competition experiments. There is a strong tendency for the same elements of secondary structure to carry NHs most protected in the native state, NHs first protected during folding, and NHs most protected in partially folded species. There is not a one‐to‐one correspondence of individual NHs. Proteins for which there are published data for native state out‐exchange and ϕ values are also reviewed. The elements of secondary structure containing the slowest exchanging NHs in native proteins tend to contain side chains with high ϕ values or be connected to a turn/loop with high ϕ values. A definition for a protein core is proposed, and the implications for protein folding are discussed. Apparently, during folding and in the native state, nonlocal interactions between core sequences are favored more than other possible nonlocal interactions. Other studies of partially folded bovine pancreatic trypsin inhibitor (Barbar E, Barany G, Woodward C, 1995, Biochemistry 34:11423–11434; Barber E, Hare M, Daragan V, Barany G, Woodward C, 1998, Biochemistry 37:7822–7833), suggest that developing cores have site‐specific energy barriers between microstates, one disordered, and the other(s) more ordered.
Characterization and molecular basis of the oligomeric structure of HIV‐1 Nef protein
Authors: Arold Stefan , Hoh François , Domergue Stephanie , Birck Catherine , Delsuc Marc‐André , Jullien Magali , Dumas Christian 
Published: February 2009

AbstractThe Nef protein of human immunodeficiency virus type I (HIV‐1) is an important determinant for the onset of AIDS disease. The self‐association properties of HIV‐1 Nef are analyzed by chemical cross‐linking, dynamic light scattering, equilibrium analytical ultracentrifugation, and NMR spectroscopy. The experimental data show that the HIV‐1 Nef core domain forms stable homo‐dimers and trimers in solution, but not higher oligomers. These Nef homomers are not covalently linked by disulfide bridges, and the equilibrium between these forms is dependent on the Nef concentration. We further provide the molecular basis for the Nef core dimers and trimers obtained by analysis of crystallographic models. Oligomerization of biological polypeptides is a common tool used to trigger events in cellular signaling and endocytosis, both of which are targeted by Nef. The quaternary structure of Nef may be of physiological importance and may help to connect its cellular targets or to increase affinity of the viral molecule for its ligands. The herein described models for Nef dimers and trimers will allow further mutational studies to elucidate their role in vivo. These results provide novel insight into the structural and functional relationships of this important viral protein. Moreover, the oligomer interface may represent a novel target for the design of antiviral agents.
Productive and nonproductive binding to ribonuclease A: X‐ray structure of two complexes with uridylyl(2′,5′)guanosine
Authors: Vitagliano Luigi , Merlino Antonello , Zagari Adriana , Mazzarella Lelio 
Published: February 2009

AbstractGuanine‐containing mono‐ and dinucleotides bind to the active site of ribonuclease A in a nonproductive mode (retro‐binding) (Aguilar CF, Thomas PJ, Mills A, Moss DS, Palmer RA, 1992, J Mol Biol 224:265‐267). Guanine binds to the highly specific pyrimidine site by forming hydrogen bonds with Thr45 and with the sulfate anion located in the P1 site. To investigate the influence of the anion present in the P1 site on retro‐binding, we determined the structure of two new complexes of RNase A with uridylyl(2′,5′)guanosine obtained by soaking two different forms of pre‐grown RNase A crystals. In one case, RNase A was crystallized without removing the sulfate anion strongly bound to the active site; in the other, the protein was first equilibrated with a basic solution to displace the anion from the P1 site. The X‐ray structures of the complexes with and without sulfate in P1 were refined using diffraction data up to 1.8 Å (R‐factor 0.192) and 2.0 Å (R‐factor 0.178), respectively. The binding mode of the substrate analogue to the protein differs markedly in the two complexes. When the sulfate is located in P1, we observe retro‐binding; whereas when the anion is removed from the active site, the uridine is productively bound at the B1 site. In the productive complex, the electron density is very well defined for the uridine moiety, whereas the downstream guanine is disordered. This finding indicates that the interactions of guanine in the B2 site are rather weak and that this site is essentially adenine preferring. In this crystal form, there are two molecules per asymmetric unit, and due to crystal packing, only the active site of one molecule is accessible to the ligand. Thus, in the same crystal we have a ligand‐bound and a ligand‐free RNase A molecule. The comparison of these two structures furnishes a detailed and reliable picture of the structural alterations induced by the binding of the substrate. These results provide structural information to support the hypotheses on the role of RNase A active site residues that have recently emerged from site‐directed mutagenesis studies.
A model of troponin‐I in complex with troponin‐C using hybrid experimental data: The inhibitory region is a β‐hairpin
Authors: Wall Michael E. , Gallagher Stephen C. , Trewhella Jill , Tung Chang‐Shung 
Published: February 2009

AbstractWe present a model for the skeletal muscle troponin‐C (TnC)/troponin‐I (TnI) interaction, a critical molecular switch that is responsible for calcium‐dependent regulation of the contractile mechanism. Despite concerted efforts by multiple groups for more than a decade, attempts to crystallize troponin‐C in complex with troponin‐I, or in the ternary troponin complex, have not yet delivered a high‐resolution structure. Many groups have pursued different experimental strategies, such as X‐ray crystallography, NMR, small‐angle scattering, chemical cross‐linking, and fluorescent resonance energy transfer (FRET) to gain insights into the nature of the TnC/TnI interaction. We have integrated the results of these experiments to develop a model of the TnC/TnI interaction, using an atomic model of TnC as a scaffold. The TnI sequence was fit to each of two alternate neutron scattering envelopes: one that winds about TnC in a left‐handed sense (Model L), and another that winds about TnC in a right‐handed sense (Model R). Information from crystallography and NMR experiments was used to define segments of the models. Tests show that both models are consistent with available cross‐linking and FRET data. The inhibitory region TnI (95‐114) is modeled as a flexible β‐hairpin, and in both models it is localized to the same region on the central helix of TnC. The sequence of the inhibitory region is similar to that of a β‐hairpin region of the actin‐binding protein profilin. This similarity supports our model and suggests the possibility of using an available profilin/actin crystal structure to model the TnI/actin interaction. We propose that the β‐hairpin is an important structural motif that communicates the Ca2+‐activated troponin regulatory signal to actin.
Modeling of loops in protein structures
Authors: Fiser András , Do Richard Kinh Gian , Šali Andrej 
Published: February 2009

AbstractComparative protein structure prediction is limited mostly by the errors in alignment and loop modeling. We describe here a new automated modeling technique that significantly improves the accuracy of loop predictions in protein structures. The positions of all nonhydrogen atoms of the loop are optimized in a fixed environment with respect to a pseudo energy function. The energy is a sum of many spatial restraints that include the bond length, bond angle, and improper dihedral angle terms from the CHARMM‐22 force field, statistical preferences for the main‐chain and side‐chain dihedral angles, and statistical preferences for nonbonded atomic contacts that depend on the two atom types, their distance through space, and separation in sequence. The energy function is optimized with the method of conjugate gradients combined with molecular dynamics and simulated annealing. Typically, the predicted loop conformation corresponds to the lowest energy conformation among 500 independent optimizations. Predictions were made for 40 loops of known structure at each length from 1 to 14 residues. The accuracy of loop predictions is evaluated as a function of thoroughness of conformational sampling, loop length, and structural properties of native loops. When accuracy is measured by local superposition of the model on the native loop, 100, 90, and 30% of 4–, 8–, and 12–residue loop predictions, respectively, had <2 Å RMSD error for the mainchain N, Ca, C, and O atoms; the average accuracies were 0.59 6 0.05, 1.16 6 0.10, and 2.61 6 0.16 Å, respectively. To simulate real comparative modeling problems, the method was also evaluated by predicting loops of known structure in only approximately correct environments with errors typical of comparative modeling without misalignment. When the RMSD distortion of the main‐chain stem atoms is 2.5 Å, the average loop prediction error increased by 180, 25, and 3% for 4–, 8–, and 12–residue loops, respectively. The accuracy of the lowest energy prediction for a given loop can be estimated from the structural variability among a number of low energy predictions. The relative value of the present method is gauged by (1) comparing it with one of the most successful previously described methods, and (2) describing its accuracy in recent blind predictions of protein structure. Finally, it is shown that the average accuracy of prediction is limited primarily by the accuracy of the energy function rather than by the extent of conformational sampling.
Development of a pUC19‐based recombinant plasmid to serve as a positive control in PCR for Orientia tsutsugamushi
Authors: Hanaoka Nozomu , Sakata Akiko , Takano Ai , Kawabata Hiroki , Watanabe Haruo , Kurane Ichiro , Kishimoto Toshio , Ando Shuji 
Published: February 2009

ABSTRACTA recombinant positive control plasmid was developed to be used in PCR for Orientia tsutsugamushi. This pUC19‐based plasmid contains the O. tsutsugamushi DNA sequence, part of which is replaced with Candida albicans DNA. pUC19‐Posi is a PCR control that provides several advantages over currently used positive controls for both universal and serotypical PCR.
Mitogen‐activated protein kinases are involved in tumor necrosis factor alpha production in macrophages infected with Orientia tsutsugamushi
Authors: Yun Ji‐Hyun , Koo Jung‐Eun , Koh Young‐Sang 
Published: February 2009

ABSTRACT Orientia tsutsugamushi, an obligatory intracellular bacterium, is the causative agent of scrub typhus. Here the role of MAPK in TNF‐α production in macrophages after infection with O. tsutsugamushi has been investigated. ERK1/2, JNK, and p38 MAPK became phosphorylated in Orientia‐stimulated macrophages. Selective inhibitors of MAPK cascades could all significantly reduce Orientia‐stimulated TNF‐α production. Orientia‐stimulated TNF‐α production via p38 and JNK pathways was regulated by a post‐transcriptional mechanism, whereas the ERK pathway mainly controlled the transcriptional step of TNF‐α gene expression during infection. In conclusion, our data indicate that MAPK signaling is required to induce maximal TNF‐α production in macrophages during Orientia infection.
Heavy‐ion‐induced bystander killing of human lung cancer cells: Role of gap junctional intercellular communication
Authors: Harada Kosaku , Nonaka Tetsuo , Hamada Nobuyuki , Sakurai Hideyuki , Hasegawa Masatoshi , Funayama Tomoo , Kakizaki Takehiko , Kobayashi Yasuhiko , Nakano Takashi 
Published: February 2009

The aim of the present study was to clarify the mechanisms of cell death induced by heavy‐ion irradiation focusing on the bystander effect in human lung cancer A549 cells. In microbeam irradiation, each of 1, 5, and 25 cells under confluent cell conditions was irradiated with 1, 5, or 10 particles of carbon ions (220 MeV), and then the surviving fraction of the population was measured by a clonogenic assay in order to investigate the bystander effect of heavy‐ions. In this experiment, the limited number of cells (0.0001–0.002%, 5–25 cells) under confluent cell conditions irradiated with 5 or 10 carbon ions resulted in an exaggerated 8–14% increase in cell death by clonogenic assay. However, these overshooting responses were not observed under exponentially growing cell conditions. Furthermore, these responses were inhibited in cells treated with an inhibitor of gap junctional intercellular communication (GJIC), whereas they were markedly enhanced by the addition of a stimulator of GJIC. The present results suggest that bystander cell killing by heavy‐ions was induced mainly by direct cell‐to‐cell communication, such as GJIC, which might play important roles in bystander responses. (Cancer Sci 2009; 100: 684–688)
Immunological characterization of a Blo t 12 isoallergen: identification of immunoglobulin E epitopes
Authors: Zakzuk J. , Jiménez S. , Cheong N. , Puerta L. , Lee B. W. , Chua K. Y. , Caraballo L. 
Published: February 2009

SummaryBackground
Differences in the IgE response to isoallergens could have clinical implications; therefore, its analysis will contribute to the design of better strategies for the diagnosis and treatment of allergic respiratory diseases. Several isoforms have been described from mites but there is no information about the clinical impact of Blomia tropicalis isoallergens.Objective
To evaluate the differences in the IgE response against two Blo t 12 isoallergens.Methods
The IgE‐binding properties of Blo t 12 isoallergens were analysed by ELISA, a skin prick test and ELISA cross‐inhibition. Epitope mapping was performed using synthetic overlapping peptides. Fold recognition methods were used to model the chitin‐binding domain of the two isoallergens.Results
The frequency and strength of the IgE response were greater for Blo t 12.0101 than for Blo t 12.0102. Three IgE‐binding areas were identified in Blo t 12.0101; one of them included two residues that are different in Blo t 12.0102. Modelling of the chitin‐binding domains of these allergens predicted that they have structural differences that could influence antibody recognition of one of these epitopes.Conclusion
In silico structural analysis and immunological characterization of Blo t 12 reveals that allergen polymorphism influences IgE reactivity. Blo t 12.0101 is the most IgE‐reactive isoform in Cartagena. The identified IgE epitopes could be mutated to obtain hypoallergenic molecules of potential use for immunotherapy.
Production of native and modified recombinant Der p 1 molecules in tobacco plants
Authors: Burtin D. , Chabre H. , Olagnier B. , Didierlaurent A. , Couret M.‐N. , Comeau D. , Wambre E. , Laparra H. , Van Overtvelt L. , Montandon F. , Batard T. , Jonval V. , Lorphelin A. , Merle C. , Berrouet C. , Parry L. , Gomord V. , Van Ree R. , Moingeon P. 
Published: February 2009

SummaryBackground
As a complex molecule requiring post‐translational processing, it has been difficult to produce the Der p 1 major allergen from the Dermatophagoides pteronyssinus house dust mite in a recombinant form.Objective
Here, we tested whether transgenic tobacco plants are suitable to express Der p 1, either as a wild‐type molecule or as variants lacking N‐glycosylation sites (Gly−) and/or cysteine protease activity (Enz−).Methods
Using Agrobacterium tumefaciens‐based transformation, pro Der p 1 molecules bearing mutations within either the N‐glycosylation sites (N34Q, N150Q) and/or the cysteine protease‐active site (C132V) were expressed in tobacco plants. After purification by ion exchange chromatography, allergens were characterized using immunoblotting, circular dichroism (CD), as well as basophil and T lymphocyte stimulation assays.Results
Four forms of recombinant Der p 1 (i.e. wild‐type Gly+/Enz+, as well as Gly−/Enz+, Gly+/Enz− or Gly−/Enz− variants) were successfully expressed in tobacco leaves as pro Der p 1 molecules. Spontaneous cleavage of the pro‐peptide was observed in tobacco leaf extracts for all forms of recombinant Der p 1 (r Der p 1). CD confirmed that all r Der p 1 molecules, with the exception of the Gly−/Enz− variant, exhibited secondary structures comparable to the natural protein. A cysteine protease activity was associated only with the Gly+/Enz+ form. All these molecules exhibit a profile similar to natural Der p 1 with respect to IgE immunoreactivity, basophil activation and T cell recognition.Conclusion
A tobacco plant expression system allows the production of various forms of mature Der p 1, which could be used for diagnostic or immunotherapeutic purposes.
Characterization of regulatory T cells identified as CD4+CD25highCD39+ in patients with active tuberculosis
Authors: Chiacchio T , Casetti R , Butera O , Vanini V , Carrara S , Girardi E , Di Mitri D , Battistini L , Martini F , Borsellino G , Goletti D 
Published: February 2009

SummaryForkhead box P3 (FoxP3) is a transcription factor whose expression characterizes regulatory T cells (Treg), but it is also present on activated T cells, thus hindering correct Treg identification. Using classical markers for Treg recognition, discordant results were found in terms of Treg expansion during active tuberculosis (TB) disease. Recently CD39 has been shown to be an accurate marker for Treg detection. The objectives of this study were: (i) to identify Treg expressing CD39 in patients with TB and to compare the results with those obtained by the standard phenotypic markers; (ii) to evaluate if Treg are expanded in vitro by exogenous interleukin (IL)-2 or by antigen-specific stimulation; and (iii) to characterize Treg function on the modulation of antigen-specific responses. We enrolled 13 patients with pulmonary TB and 12 healthy controls. Treg were evaluated by flow cytometry ex vivo and after antigen-specific in vitro stimulation using CD25, FoxP3, CD127 and CD39 markers. Results indicate that CD39+ cells within the CD4+CD25high cells have Treg properties (absence of interferon-γ production and transforming growth factor-β1 release upon stimulation). Ex vivo analysis did not show significant differences between TB patients and controls of Treg by classical or novel markers. In contrast, a significantly higher percentage of Treg was found in TB patients after antigen-specific stimulation both in the presence or absence of IL-2. Depletion of CD39+ Treg increased RD1-specific responses significantly. In conclusion, CD39 is an appropriate marker for Treg identification in TB. These results can be useful for future studies to monitor Mycobacterium tuberculosis-specific response during TB.
c-Src tyrosine kinase co-associates with and phosphorylates signal transducer and activator of transcription 5b which mediates the proliferation of normal human B lymphocytes
Authors: Cayer M-P , Proulx M , Ma X-Z , Sakac D , Giguère J-F , Drouin M , Néron S , Branch D R , Jung D 
Published: February 2009

Summaryc-Src is the normal human cellular protein homologue of the viral oncogene v-src. c-Src activity was reported recently to increase in CD40-activated human B lymphocytes, suggesting its involvement in proliferation. To elucidate the exact role of c-Src in this process, we investigated the effects of c-Src over-expression on normal B lymphocyte growth. B lymphocytes purified from human peripheral blood were infected with Ad5/F35 vector encoding either a constitutively active c-Src (c-Src/dominant-positive) or a dominant-negative c-Src (c-Src/DN). Little variation of B lymphocytes expansion could be observed between control enhanced yellow fluorescent protein and c-Src/dominant-positive-infected cells. In contrast, over-expression of c-Src/DN results in a 40% inhibition of B lymphocyte expansion. These results suggest that DN c-Src may compete with endogenous c-Src, resulting in partial inhibition of a transcriptional pathway involved in B lymphocyte proliferation. We demonstrate further that c-Src can phosphorylate signal transducer and activator of transcription 5b (STAT5b) on tyrosine 699 and that c-Src and STAT5b co-associate during B lymphocyte proliferation. These results confirm an important role for c-Src in the expansion of normal human B lymphocytes in vitro, in which c-Src may regulate STAT5b in the intracellular signalling pathway important for the proliferation of normal human B lymphocytes.