Escherchia coli ribose binding protein based bioreporters revisited.

Bioreporter bacteria, i.e., strains engineered to respond to chemical exposure by production of reporter proteins, have attracted wide interest because of their potential to offer cheap and simple alternative analytics for specified compounds or conditions. Bioreporter construction has mostly exploited the natural variation of sensory proteins, but it has been proposed that computational design of new substrate binding properties could lead to completely novel detection specificities at very low affinities. Here we reconstruct a bioreporter system based on the native Escherichia coli ribose binding protein RbsB and one of its computationally designed variants, reported to be capable of binding 2,4,6-trinitrotoluene (TNT). Our results show in vivo reporter induction at 50 nM ribose, and a 125 nM affinity constant for in vitro ribose binding to RbsB. In contrast, the purified published TNT-binding variant did not bind TNT nor did TNT cause induction of the E. coli reporter system.

CCAAT/enhancer-binding protein family members recruit the coactivator CREB-binding protein and trigger its phosphorylation.

CCAAT/enhancer-binding protein (C/EBP) family members are transcription factors involved in important physiological processes, such as cellular proliferation and differentiation, regulation of energy homeostasis, inflammation, and hematopoiesis. Transcriptional activation by C/EBPalpha and C/EBPbeta involves the coactivators CREB-binding protein (CBP) and p300, which promote transcription by acetylating histones and recruiting basal transcription factors. In this study, we show that C/EBPdelta is also using CBP as a coactivator. Based on sequence homology with C/EBPalpha and -beta, we identify in C/EBPdelta two conserved amino acid segments that are necessary for the physical interaction with CBP. Using reporter gene assays, we demonstrate that mutation of these residues prevents CBP recruitment and diminishes the transactivating potential of C/EBPdelta. In addition, our results indicate that C/EBP family members not only recruit CBP but specifically induce its phosphorylation. We provide evidence that CBP phosphorylation depends on its interaction with C/EBPdelta and define point mutations within one of the two conserved amino acid segments of C/EBPdelta that abolish CBP phosphorylation as well as transcriptional activation, suggesting that this new mechanism could be important for C/EBP-mediated transcription.

Ubiquitination of the Epstein-Barr virus-encoded latent membrane protein 1 depends on the integrity of the TRAF binding site.

The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus functions as a constitutively activated receptor of the tumor necrosis factor receptor family. LMP1 is a short-lived protein that is ubiquitinated and degraded by the proteasome. We have previously shown that LMP1 recruits the adapter protein tumor necrosis factor receptor-associated factor 3 (TRAF3) to lipid rafts. To test if TRAFs are involved in LMP1's ubiquitination, we have mutated the LMP1 CTAR1 site that has been identified as a TRAF binding site. We show that the CTAR1 mutant (CTAR1(-)) is expressed after transfection at a similar level to wild-type LMP1, and behaves as wild-type LMP1 with respect to membrane localization. However, CTAR1(-) does not bind TRAF3. We demonstrate that ubiquitination of CTAR1(-) is significantly reduced when compared to wild-type LMP1. In addition, the expression of wild-type LMP1 induces the ubiquitination, an effect that is significantly reduced when the CTAR1(-) is expressed. Taken together, our results suggest that TRAF proteins are involved in the ubiquitination of LMP1, and that their binding to LMP1 may facilitate their own ubiquitination.

Penicillin-binding protein gene alterations in Streptococcus uberis isolates presenting decreased susceptibility to penicillin.

Streptococcus uberis is an environmental pathogen commonly causing bovine mastitis, an infection that is generally treated with penicillin G. No field case of true penicillin-resistant S. uberis (MIC > 16 mg/liter) has been described yet, but isolates presenting decreased susceptibility (MIC of 0.25 to 0.5 mg/liter) to this drug are regularly reported to our laboratory. In this study, we demonstrated that S. uberis can readily develop penicillin resistance in laboratory-evolved mutants. The molecular mechanism of resistance (acquisition of mutations in penicillin-binding protein 1A [PBP1A], PBP2B, and PBP2X) was generally similar to that of all other penicillin-resistant streptococci described so far. In addition, it was also specific to S. uberis in that independent resistant mutants carried a unique set of seven consensus mutations, of which only one (Q(554)E in PBP2X) was commonly found in other streptococci. In parallel, independent isolates from bovine mastitis with different geographical origins (France, Holland, and Switzerland) and presenting a decreased susceptibility to penicillin were characterized. No mosaic PBPs were detected, but they all presented mutations identical to the one found in the laboratory-evolved mutants. This indicates that penicillin resistance development in S. uberis might follow a stringent pathway that would explain, in addition to the ecological niche of this pathogen, why naturally occurring resistances are still rare. In addition, this study shows that there is a reservoir of mutated PBPs in animals, which might be exchanged with other streptococci, such as Streptococcus agalactiae, that could potentially be transmitted to humans.

c-Jun N-terminal kinase binding domain-dependent phosphorylation of mitogen-activated protein kinase kinase 4 and mitogen-activated protein kinase kinase 7 and balancing cross-talk between c-Jun N-terminal kinase and extracellular signal-regulated kinase pathways in cortical neurons

The c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase (MAPK) activated by stress-signals and involved in many different diseases. Previous results proved the powerful effect of the cell permeable peptide inhibitor d-JNKI1 (d-retro-inverso form of c-Jun N-terminal kinase-inhibitor) against neuronal death in CNS diseases, but the precise features of this neuroprotection remain unclear. We here performed cell-free and in vitro experiments for a deeper characterization of d-JNKI1 features in physiological conditions. This peptide works by preventing JNK interaction with its c-Jun N-terminal kinase-binding domain (JBD) dependent targets. We here focused on the two JNK upstream MAPKKs, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7), because they contain a JBD homology domain. We proved that d-JNKI1 prevents MKK4 and MKK7 activity in cell-free and in vitro experiments: these MAPKK could be considered not only activators but also substrates of JNK. This means that d-JNKI1 can interrupt downstream but also upstream events along the JNK cascade, highlighting a new remarkable feature of this peptide. We also showed the lack of any direct effect of the peptide on p38, MEK1, and extracellular signal-regulated kinase (ERK) in cell free, while in rat primary cortical neurons JNK inhibition activates the MEK1-ERK-Ets1/c-Fos cascade. JNK inhibition induces a compensatory effect and leads to ERK activation via MEK1, resulting in an activation of the survival pathway-(MEK1/ERK) as a consequence of the death pathway-(JNK) inhibition. This study should hold as an important step to clarify the strong neuroprotective effect of d-JNKI1.

The caveolin-binding motif of the pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is required for in vivo export of cholesteryl acetate.

Proteins belonging to the CAP superfamily are present in all kingdoms of life and have been implicated in different physiological processes. Their molecular mode of action, however, is poorly understood. Saccharomyces cerevisiae expresses three members of this superfamily, pathogen-related yeast (Pry)1, -2, and -3. We have recently shown that Pry function is required for the secretion of cholesteryl acetate and that Pry proteins bind cholesterol and cholesteryl acetate, suggesting that CAP superfamily members may generally act to bind sterols or related small hydrophobic compounds. Here, we analyzed the mode of sterol binding by Pry1. Computational modeling indicates that ligand binding could occur through displacement of a relatively poorly conserved flexible loop, which in some CAP family members displays homology to the caveolin-binding motif. Point mutations within this motif abrogated export of cholesteryl acetate but did not affect binding of cholesterol. Mutations of residues located outside the caveolin-binding motif, or mutations in highly conserved putative catalytic residues had no effect on export of cholesteryl acetate or on lipid binding. These results indicate that the caveolin-binding motif of Pry1, and possibly of other CAP family members, is crucial for selective lipid binding and that lipid binding may occur through displacement of the loop containing this motif.

Loss of the thyroid hormone-binding protein Crym renders striatal neurons more vulnerable to mutant huntingtin in Huntington's disease.

The mechanisms underlying preferential atrophy of the striatum in Huntington's disease (HD) are unknown. One hypothesis is that a set of gene products preferentially expressed in the striatum could determine the particular vulnerability of this brain region to mutant huntingtin (mHtt). Here, we studied the striatal protein µ-crystallin (Crym). Crym is the NADPH-dependent p38 cytosolic T3-binding protein (p38CTBP), a key regulator of thyroid hormone (TH) T3 (3,5,3'-triiodo-l-thyronine) transportation. It has been also recently identified as the enzyme that reduces the sulfur-containing cyclic ketimines, which are potential neurotransmitters. Here, we confirm the preferential expression of the Crym protein in the rodent and macaque striatum. Crym expression was found to be higher in the macaque caudate than in the putamen. Expression of Crym was reduced in the BACHD and Knock-in 140CAG mouse models of HD before onset of striatal atrophy. We show that overexpression of Crym in striatal medium-size spiny neurons using a lentiviral-based strategy in mice is neuroprotective against the neurotoxicity of an N-terminal fragment of mHtt in vivo. Thus, reduction of Crym expression in HD could render striatal neurons more susceptible to mHtt suggesting that Crym may be a key determinant of the vulnerability of the striatum. In addition our work points to Crym as a potential molecular link between striatal degeneration and the THs deregulation reported in HD patients.

Quantifying intracellular protein binding thermodynamics during mechanotransduction based on FRET spectroscopy.

Mechanical force modulates myriad cellular functions including migration, alignment, proliferation, and gene transcription. Mechanotransduction, the transmission of mechanical forces and its translation into biochemical signals, may be mediated by force induced protein conformation changes, subsequently modulating protein signaling. For the paxillin and focal adhesion kinase interaction, we demonstrate that force-induced changes in protein complex conformation, dissociation constant, and binding Gibbs free energy can be quantified by lifetime-resolved fluorescence energy transfer microscopy combined with intensity imaging calibrated by fluorescence correlation spectroscopy. Comparison with in vitro data shows that this interaction is allosteric in vivo. Further, spatially resolved imaging and inhibitor assays show that this protein interaction and its mechano-sensitivity are equal in the cytosol and in the focal adhesions complexes indicating that the mechano-sensitivity of this interaction must be mediated by soluble factors but not based on protein tyrosine phosphorylation.

The fibrinogen- and fibronectin-binding domains of Staphylococcus aureus fibronectin-binding protein A synergistically promote endothelial invasion and experimental endocarditis.

Staphylococcus aureus experimental endocarditis relies on sequential fibrinogen binding (for valve colonization) and fibronectin binding (for endothelial invasion) conferred by peptidoglycan-attached adhesins. Fibronectin-binding protein A (FnBPA) reconciles these two properties--as well as elastin binding--and promotes experimental endocarditis by itself. Here we attempted to delineate the minimal subdomain of FnBPA responsible for fibrinogen and fibronectin binding, cell invasion, and in vivo endocarditis. A large library of truncated constructs of FnBPA was expressed in Lactococcus lactis and tested in vitro and in animals. A 127-amino-acid subdomain spanning the hinge of the FnBPA fibrinogen-binding and fibronectin-binding regions appeared necessary and sufficient to confer the sum of these properties. Competition with synthetic peptides could not delineate specific fibrinogen- and fibronectin-binding sites, suggesting that dual binding arose from protein folding, irrespective of clearly defined binding domains. Moreover, coexpressing the 127-amino-acid subdomain with remote domains of FnBPA further increased fibrinogen binding by > or =10 times, confirming the importance of domain interactions for binding efficacy. In animals, fibrinogen binding (but not fibronectin binding) was significantly associated with endocarditis induction, whereas both fibrinogen binding and fibronectin binding were associated with disease severity. Moreover, fibrinogen binding also combined with fibronectin binding to synergize the invasion of cultured cell lines significantly, a feature correlating with endocarditis severity. Thus, while fibrinogen binding and fibronectin binding were believed to act sequentially in colonization and invasion, they appeared unexpectedly intertwined in terms of both functional anatomy and pathogenicity (in endocarditis). This unforeseen FnBPA subtlety might bear importance for the development of antiadhesin strategies.

Protein-binding microarrays: probing disease markers at the interface of proteomics and genomics.

DNA-binding proteins mediate a variety of crucial molecular functions, such as transcriptional regulation and chromosome maintenance, replication and repair, which in turn control cell division and differentiation. The roles of these proteins in disease are currently being investigated using microarray-based approaches. However, these assays can be difficult to adapt to routine diagnosis of complex diseases such as cancer. Here, we review promising alternative approaches involving protein-binding microarrays (PBMs) that probe the interaction of proteins from crude cell or tissue extracts with large collections of synthetic or natural DNA sequences. Recent studies have demonstrated the use of these novel PBM approaches to provide rapid and unbiased characterization of DNA-binding proteins as molecular markers of disease, for example cancer progression or infectious diseases.

Binding of double-strand breaks in DNA by human Rad52 protein.

Double-strand breaks (DSBs) in DNA are caused by ionizing radiation. These chromosomal breaks can kill the cell unless repaired efficiently, and inefficient or inappropriate repair can lead to mutation, gene translocation and cancer. Two proteins that participate in the repair of DSBs are Rad52 and Ku: in lower eukaryotes such as yeast, DSBs are repaired by Rad52-dependent homologous recombination, whereas vertebrates repair DSBs primarily by Ku-dependent non-homologous end-joining. The contribution of homologous recombination to vertebrate DSB repair, however, is important. Biochemical studies indicate that Ku binds to DNA ends and facilitates end-joining. Here we show that human Rad52, like Ku, binds directly to DSBs, protects them from exonuclease attack and facilitates end-to-end interactions. A model for repair is proposed in which either Ku or Rad52 binds the DSB. Ku directs DSBs into the non-homologous end-joining repair pathway, whereas Rad52 initiates repair by homologous recombination. Ku and Rad52, therefore, direct entry into alternative pathways for the repair of DNA breaks.

Artificial zinc finger fusions targeting Sp1-binding sites and the trans-activator-responsive element potently repress transcription and replication of HIV-1.

Tat activates transcription by interacting with Sp1, NF-kappaB, positive transcription elongation factor b, and trans-activator-responsive element (TAR). Tat and Sp1 play major roles in transcription by protein-protein interactions at human immunodeficiency virus, type 1 (HIV-1) long terminal repeat. Sp1 activates transcription by interacting with cyclin T1 in the absence of Tat. To disrupt the transcription activation by Tat and Sp1, we fused Sp1-inhibiting polypeptides, zinc finger polypeptide, and the TAR-binding mutant Tat (TatdMt) together. A designed or natural zinc finger and Tat mutant fusion was used to target the fusion to the key regulatory sites (GC box and TAR) on the long terminal repeat and nascent short transcripts to disrupt the molecular interaction that normally result in robust transcription. The designed zinc finger and TatdMt fusions were targeted to the TAR, and they potently repressed both transcription and replication of HIV-1. The Sp1-inhibiting POZ domain, TatdMt, and zinc fingers are key functional domains important in repression of transcription and replication. The designed artificial zinc fingers were targeted to the high affinity Sp1-binding site, and by being fused with TatdMt and POZ domain, they strongly block both Sp1-cyclin T1-dependent transcription and Tat-dependent transcription, even in the presence of excess expressed Tat.

An 18-kDa translocator protein (TSPO) polymorphism explains differences in binding affinity of the PET radioligand PBR28

[(11)C]PBR28 binds the 18-kDa Translocator Protein (TSPO) and is used in positron emission tomography (PET) to detect microglial activation. However, quantitative interpretations of signal are confounded by large interindividual variability in binding affinity, which displays a trimodal distribution compatible with a codominant genetic trait. Here, we tested directly for an underlying genetic mechanism to explain this. Binding affinity of PBR28 was measured in platelets isolated from 41 human subjects and tested for association with polymorphisms in TSPO and genes encoding other proteins in the TSPO complex. Complete agreement was observed between the TSPO Ala147Thr genotype and PBR28 binding affinity phenotype (P value=3.1 x 10(-13)). The TSPO Ala147Thr polymorphism predicts PBR28 binding affinity in human platelets. As all second-generation TSPO PET radioligands tested hitherto display a trimodal distribution in binding affinity analogous to PBR28, testing for this polymorphism may allow quantitative interpretation of TSPO PET studies with these radioligands.

Contribution of (sub)domains of Staphylococcus aureus fibronectin-binding protein to the proinflammatory and procoagulant response of human vascular endothelial cells.

The Staphylococcus aureus fibronectin (Fn) -binding protein A (FnBPA) is involved in bacterium-endothelium interactions which is one of the crucial events leading to infective endocarditis (IE). We previously showed that the sole expression of S. aureus FnBPA was sufficient to confer to non-invasive Lactococcus lactis bacteria the capacity to invade human endothelial cells (ECs) and to launch the typical endothelial proinflammatory and procoagulant responses that characterize IE. In the present study we further questioned whether these bacterium-EC interactions could be reproduced by single or combined FnBPA sub-domains (A, B, C or D) using a large library of truncated FnBPA constructs expressed in L. lactis. Significant invasion of cultured ECs was found for L. lactis expressing the FnBPA subdomains CD (aa 604-877) or A4(+16) (aa 432-559). Moreover, this correlates with the capacity of these fragments to elicit in vitro a marked increase in EC surface expression of both ICAM-1 and VCAM-1 and secretion of the CXCL8 chemokine and finally to induce a tissue factor-dependent endothelial coagulation response. We thus conclude that (sub)domains of the staphylococcal FnBPA molecule that express Fn-binding modules, alone or in combination, are sufficient to evoke an endothelial proinflammatory as well as a procoagulant response and thus account for IE severity.

Mutations in penicillin-binding protein (PBP) genes and in non-PBP genes during selection of penicillin-resistant Streptococcus gordonii.

Penicillin resistance in Streptococcus spp. involves multiple mutations in both penicillin-binding proteins (PBPs) and non-PBP genes. Here, we studied the development of penicillin resistance in the oral commensal Streptococcus gordonii. Cyclic exposure of bacteria to twofold-increasing penicillin concentrations selected for a progressive 250- to 500-fold MIC increase (from 0.008 to between 2 and 4 microg/ml). The major MIC increase (> or = 35-fold) was related to non-PBP mutations, whereas PBP mutations accounted only for a 4- to 8-fold additional increase. PBP mutations occurred in class B PBPs 2X and 2B, which carry a transpeptidase domain, but not in class A PBP 1A, 1B, or 2A, which carry an additional transglycosylase domain. Therefore, we tested whether inactivation of class A PBPs affected resistance development in spite of the absence of mutations. Deletion of PBP 1A or 2A profoundly slowed down resistance development but only moderately affected resistance in already highly resistant mutants (MIC = 2 to 4 microg/ml). Thus, class A PBPs might facilitate early development of resistance by stabilizing penicillin-altered peptidoglycan via transglycosylation, whereas they might be less indispensable in highly resistant mutants which have reestablished a penicillin-insensitive cell wall-building machinery. The contribution of PBP and non-PBP mutations alone could be individualized in DNA transformation. Both PBP and non-PBP mutations conferred some level of intrinsic resistance, but combining the mutations synergized them to ensure high-level resistance (> or = 2 microg/ml). The results underline the complexity of penicillin resistance development and suggest that inhibition of transglycosylase might be an as yet underestimated way to interfere with early resistance development.