The ATP-binding cassette transporter-encoding gene CgSNQ2 is contributing to the CgPDR1-dependent azole resistance of Candida glabrata.

Our previous investigation on Candida glabrata azole-resistant isolates identified two isolates with unaltered expression of CgCDR1/CgCDR2, but with upregulation of another ATP-binding cassette transporter, CgSNQ2, which is a gene highly similar to ScSNQ2 from Saccharomyces cerevisiae. One of the two isolates (BPY55) was used here to elucidate this phenomenon. Disruption of CgSNQ2 in BPY55 decreased azole resistance, whereas reintroduction of the gene in a CgSNQ2 deletion mutant fully reversed this effect. Expression of CgSNQ2 in a S. cerevisiae strain lacking PDR5 mediated not only resistance to azoles but also to 4-nitroquinoline N-oxide, which is a ScSNQ2-specific substrate. A putative gain-of-function mutation, P822L, was identified in CgPDR1 from BPY55. Disruption of CgPDR1 in BPY55 conferred enhanced azole susceptibility and eliminated CgSNQ2 expression, whereas introduction of the mutated allele in a susceptible strain where CgPDR1 had been disrupted conferred azole resistance and CgSNQ2 upregulation, indicating that CgSNQ2 was controlled by CgPDR1. Finally, CgSNQ2 was shown to be involved in the in vivo response to fluconazole. Together, our data first demonstrate that CgSNQ2 contributes to the development of CgPDR1-dependent azole resistance in C. glabrata. The overlapping in function and regulation between CgSNQ2 and ScSNQ2 further highlight the relationship between S. cerevisiae and C. glabrata.

Biochemical properties of RuvBD113N: a mutation in helicase motif II of the RuvB hexamer affects DNA binding and ATPase activities.

Many DNA helicases utilise the energy derived from nucleoside triphosphate hydrolysis to fuel their actions as molecular motors in a variety of biological processes. In association with RuvA, the E. coli RuvB protein (a hexameric ring helicase), promotes the branch migration of Holliday junctions during genetic recombination and DNA repair. To analyse the relationship between ATP-dependent DNA helicase activity and branch migration, a site-directed mutation was introduced into the helicase II motif of RuvB. Over-expression of RuvBD113N in wild-type E. coli resulted in a dominant negative UVs phenotype. The biochemical properties of RuvBD113N were examined and compared with wild-type RuvB in vitro. The single amino acid substitution resulted in major alterations to the biochemical activities of RuvB, such that RuvBD113N was defective in DNA binding and ATP hydrolysis, while retaining the ability to form hexameric rings and interact with RuvA. RuvBD113N formed heterohexamers with wild-type RuvB, and could inhibit RuvB function by affecting its ability to bind DNA. However, heterohexamers exhibited an ability to promote branch migration in vitro indicating that not all subunits of the ring need to be catalytically competent.

Soluble major histocompatibility complex-peptide octamers with impaired CD8 binding selectively induce Fas-dependent apoptosis.

Fluorescence-labeled soluble major histocompatibility complex class I-peptide "tetramers" constitute a powerful tool to detect and isolate antigen-specific CD8(+) T cells by flow cytometry. Conventional "tetramers" are prepared by refolding of heavy and light chains with a specific peptide, enzymatic biotinylation at an added C-terminal biotinylation sequence, and "tetramerization" by reaction with phycoerythrin- or allophycocyanin-labeled avidin derivatives. We show here that such preparations are heterogeneous and describe a new procedure that allows the preparation of homogeneous tetra- or octameric major histocompatibility complex-peptide complexes. These compounds were tested on T1 cytotoxic T lymphocytes (CTLs), which recognize the Plasmodium berghei circumsporzoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid on Lys(259) in the context of H-2K(d). We report that mutation of the CD8 binding site of K(d) greatly impairs the binding of tetrameric but not octameric or multimeric K(d)-PbCS(ABA) complexes to CTLs. This mutation abolishes the ability of the octamer to elicit significant phosphorylation of CD3, intracellular calcium mobilization, and CTL degranulation. Remarkably, however, this octamer efficiently activates CTLs for Fas (CD95)-dependent apoptosis.

"Peptabody": a new type of high avidity binding protein.

A new type of high avidity binding molecule, termed "peptabody" was created by harnessing the effect of multivalent interaction. A short peptide ligand was fused via a semi-rigid hinge region with the coiled-coil assembly domain of the cartilage oligomeric matrix protein, resulting in a pentameric multivalent binding molecule. In the first peptabody (Pab-S) described here, a peptide (S) specific for the mouse B-cell lymphoma BCL1 surface Ig idiotype, was selected from a phage display library. A fusion gene was constructed encoding peptide S, followed by the 24 aa hinge region from camel IgG and a modified 55 aa cartilage oligomeric matrix protein pentamerization domain. The Pab-S fusion protein was expressed in Escherichia coli in a soluble form at high levels and purified in a single step by metal-affinity chromatography. Pab-S specifically bound the BCL1 surface idiotype with an avidity of about 1 nM, which corresponds to a 2 x 10(5)-fold increase compared with the affinity of the synthetic peptide S itself. Biochemical characterization showed that Pab-S is a stable homopentamer of about 85 kDa, with interchain disulfide bonds. Pab-S can be dissociated under denaturing and reducing conditions and reassociated as a pentamer with full-binding activity. This intrinsic feature provides an easy way to combine Pab molecules with two different peptide specificities, thus producing heteropentamers with bispecific and/or chelating properties.

Polarity and specific sequence requirements of peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor heterodimer binding to DNA. A functional analysis of the malic enzyme gene PPAR response element.

The malic enzyme (ME) gene is a target for both thyroid hormone receptors and peroxisome proliferator-activated receptors (PPAR). Within the ME promoter, two direct repeat (DR)-1-like elements, MEp and MEd, have been identified as putative PPAR response elements (PPRE). We demonstrate that only MEp and not MEd is able to bind PPAR/retinoid X receptor (RXR) heterodimers and mediate peroxisome proliferator signaling. Taking advantage of the close sequence resemblance of MEp and MEd, we have identified crucial determinants of a PPRE. Using reciprocal mutation analyses of these two elements, we show the preference for adenine as the spacing nucleotide between the two half-sites of the PPRE and demonstrate the importance of the two first bases flanking the core DR1 in 5'. This latter feature of the PPRE lead us to consider the polarity of the PPAR/RXR heterodimer bound to its cognate element. We demonstrate that, in contrast to the polarity of RXR/TR and RXR/RAR bound to DR4 and DR5 elements respectively, PPAR binds to the 5' extended half-site of the response element, while RXR occupies the 3' half-site. Consistent with this polarity is our finding that formation and binding of the PPAR/RXR heterodimer requires an intact hinge T region in RXR while its integrity is not required for binding of the RXR/TR heterodimer to a DR4.

Experimental analysis and computer prediction of CTF/NFI transcription factor DNA binding sites.

Accurate prediction of transcription factor binding sites is needed to unravel the function and regulation of genes discovered in genome sequencing projects. To evaluate current computer prediction tools, we have begun a systematic study of the sequence-specific DNA-binding of a transcription factor belonging to the CTF/NFI family. Using a systematic collection of rationally designed oligonucleotides combined with an in vitro DNA binding assay, we found that the sequence specificity of this protein cannot be represented by a simple consensus sequence or weight matrix. For instance, CTF/NFI uses a flexible DNA binding mode that allows for variations of the binding site length. From the experimental data, we derived a novel prediction method using a generalised profile as a binding site predictor. Experimental evaluation of the generalised profile indicated that it accurately predicts the binding affinity of the transcription factor to natural or synthetic DNA sequences. Furthermore, the in vitro measured binding affinities of a subset of oligonucleotides were found to correlate with their transcriptional activities in transfected cells. The combined computational-experimental approach exemplified in this work thus resulted in an accurate prediction method for CTF/NFI binding sites potentially functioning as regulatory regions in vivo.

Coexpression of the T-cell receptor constant alpha domain triggers tumor reactivity of single-chain TCR-transduced human T cells.

Transfer of tumor antigen-specific T-cell receptors (TCRs) into human T cells aims at redirecting their cytotoxicity toward tumors. Efficacy and safety may be affected by pairing of natural and introduced TCRalpha/beta chains potentially leading to autoimmunity. We hypothesized that a novel single-chain (sc)TCR framework relying on the coexpression of the TCRalpha constant alpha (Calpha) domain would prevent undesired pairing while preserving structural and functional similarity to a fully assembled double-chain (dc)TCR/CD3 complex. We confirmed this hypothesis for a murine p53-specific scTCR. Substantial effector function was observed only in the presence of a murine Calpha domain preceded by a TCRalpha signal peptide for shuttling to the cell membrane. The generalization to a human gp100-specific TCR required the murinization of both C domains. Structural and functional T-cell avidities of an accessory disulfide-linked scTCR gp100/Calpha were higher than those of a dcTCR. Antigen-dependent phosphorylation of the proximal effector zeta-chain-associated protein kinase 70 at tyrosine 319 was not impaired, reflecting its molecular integrity in signaling. In melanoma-engrafted nonobese diabetic/severe combined immunodeficient mice, adoptive transfer of scTCR gp100/Calpha transduced T cells conferred superior delay in tumor growth among primary and long-term secondary tumor challenges. We conclude that the novel scTCR constitutes a reliable means to immunotherapeutically target hematologic malignancies.

MMPBSA decomposition of the binding energy throughout a molecular dynamics simulation of Amyloid-Beta (Aß10-35) aggregation

Recent experiments with amyloid-beta (Aß) peptides indicate that the formation of toxic oligomers may be an important contribution to the onset of Alzheimer's disease. The toxicity of Aß oligomers depend on their structure, which is governed by assembly dynamics. However, a detailed knowledge of the structure of at the atomic level has not been achieved yet due to limitations of current experimental techniques. In this study, replica exchange molecular dynamics simulations are used to identify the expected diversity of dimer conformations of Aß10-35 monomers. The most representative dimer conformation has been used to track the dimer formation process between both monomers. The process has been characterized by means of the evolution of the decomposition of the binding free energy, which provides an energetic profile of the interaction. Dimers undergo a process of reorganization driven basically by inter-chain hydrophobic and hydrophilic interactions and also solvation/desolvation processes.

T cell division and death are segregated by mutation of TCRbeta chain constant domains.

We have studied the role of the T cell receptor (TCR) beta chain transmembrane and cytoplasmic domains (betaTM/Cyto) in T cell signaling. Upon antigen stimulation, T lymphocytes expressing a TCR with mutant and betaTM and Cyto domains accumulate in large numbers and are specifically defective in undergoing activation-induced cell death (AICD). The mutant TCR poorly recruits the protein adaptor Carma-1 and is subsequently impaired in activating NF-kappaB. This signaling defect leads to a reduced expression of Fas ligand (FasL) and to a reduction in AICD. These beta chain domains are involved in discriminating cell division and apoptosis.

Catechol-binding serines of beta(2)-adrenergic receptors control the equilibrium between active and inactive receptor states.

The binding free energy for the interaction between serines 204 and 207 of the fifth transmembrane helix of the beta(2)-adrenergic receptor (beta(2)-AR) and catecholic hydroxyl (OH) groups of adrenergic agonists was analyzed using double mutant cycles. Binding affinities for catecholic and noncatecholic agonists were measured in wild-type and mutant receptors, carrying alanine replacement of the two serines (S204A, S207A beta(2)-AR), a constitutive activating mutation, or both. The free energy coupling between the losses of binding energy attributable to OH deletion from the ligand and from the receptor indicates a strong interaction (nonadditivity) as expected for a direct binding between the two sets of groups. However, we also measured a significant interaction between the deletion of OH groups from the receptor and the constitutive activating mutation. This suggests that a fraction of the decrease in agonist affinity caused by serine mutagenesis may involve a shift in the conformational equilibrium of the receptor toward the inactive state. Direct measurements using a transient transfection assay confirm this prediction. The constitutive activity of the (S204A, S207A) beta(2)-AR mutant is 50 to 60% lower than that of the wild-type beta(2)-AR. We conclude that S204 and S207 do not only provide a docking site for the agonist, but also control the equilibrium of the receptor between active (R*) and inactive (R) forms.

T-cell hybridoma specific for a cytochrome c peptide: specific antigen binding and interleukin 2 production.

T-cell hybridomas were obtained after fusion of BW 5147 thymoma and long-term cultured T cells specific for cytochrome c peptide 66-80 derivatized with a 2,4-dinitroaminophenyl (DNAP) group. The resulting hybridomas were selected for their capacity to specifically bind to soluble radiolabeled peptide antigen. One T-cell hybrid was positive for antigen binding. This hybrid T cell exhibits surface phenotypic markers of the parent antigen-specific T cells. The binding could be inhibited either by an excess of unlabeled homologous antigen or by cytochrome c peptide 11-25 derivatized with a 2-nitrophenylsulfenyl group. Several other peptide antigens tested failed to inhibit binding of the radioactive peptide. This suggests that a specific amino acid sequence, modified by a DNAP group, is the antigenic structure recognized by the putative T-cell receptor. In addition, direct interaction of DNAP-66-80 peptide with the hybridoma cell line induced production of the T-cell growth factor interleukin 2. Furthermore, supernatants derived from syngeneic macrophages pulsed with the relevant peptide also induced the antigen-specific hybridoma to produce interleukin 2.

How do the different components of episodic memory develop? Role of executive functions and short-term feature-binding abilities.

This study investigated the development of all 3 components of episodic memory (EM), as defined by Tulving, namely, core factual content, spatial context, and temporal context. To this end, a novel, ecologically valid test was administered to 109 participants aged 4-16 years. Results showed that each EM component develops at a different rate. Ability to memorize factual content emerges early, whereas context retrieval abilities continue to improve until adolescence, due to persistent encoding difficulties (isolated by comparing results on free recall and recognition tasks). Exploration of links with other cognitive functions revealed that short-term feature-binding abilities contribute to all EM components, and executive functions to temporal and spatial context, although ability to memorize temporal context is predicted mainly by age.