One billion years of bZIP transcription factor evolution: conservation and change in dimerization and DNA-binding site specificity.

The genomic era has revealed that the large repertoire of observed animal phenotypes is dependent on changes in the expression patterns of a finite number of genes, which are mediated by a plethora of transcription factors (TFs) with distinct specificities. The dimerization of TFs can also increase the complexity of a genetic regulatory network manifold, by combining a small number of monomers into dimers with distinct functions. Therefore, studying the evolution of these dimerizing TFs is vital for understanding how complexity increased during animal evolution. We focus on the second largest family of dimerizing TFs, the basic-region leucine zipper (bZIP), and infer when it expanded and how bZIP DNA-binding and dimerization functions evolved during the major phases of animal evolution. Specifically, we classify the metazoan bZIPs into 19 families and confirm the ancient nature of at least 13 of these families, predating the split of the cnidaria. We observe fixation of a core dimerization network in the last common ancestor of protostomes-deuterostomes. This was followed by an expansion of the number of proteins in the network, but no major dimerization changes in interaction partners, during the emergence of vertebrates. In conclusion, the bZIPs are an excellent model with which to understand how DNA binding and protein interactions of TFs evolved during animal evolution.

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.

Aggregation of integrins and RhoA activation are required for Thy-1-induced morphological changes in astrocytes.

Thy-1, a cell adhesion molecule abundantly expressed in mammalian neurons, binds to a beta(3)-containing integrin on astrocytes and thereby stimulates the assembly of focal adhesions and stress fibers. Such events lead to morphological changes in astrocytes that resemble those occurring upon injury in the brain. Extracellular matrix proteins, typical integrin ligands, bind to integrins and promote receptor clustering as well as signal transduction events that involve small G proteins and cytoskeletal changes. Here we investigated the possibility that the cell surface protein Thy-1, when interacting with a beta(3)-containing integrin on astrocytes, could trigger signaling events similar to those generated by extracellular matrix proteins. DI-TNC(1) astrocytes were stimulated with Thy-1-Fc immobilized on beads, and increased RhoA activity was confirmed using an affinity precipitation assay. The effect of various inhibitors on the cellular response was also studied. The presence of Y-27632, an inhibitor of Rho kinase (p160ROCK), a key downstream effector of RhoA, significantly reduced focal adhesion and stress fiber formation induced by Thy-1. Similar effects were obtained when astrocytes were treated with C3 transferase, an inhibitor of RhoA. Alternatively, astrocytes were transfected with an expression vector encoding fusion proteins of enhanced green fluorescent protein with either the Rho-binding domain of Rhotekin, which blocks RhoA function, or the dominant-negative N19RhoA mutant. In both cases, Thy-1-induced focal adhesion formation was inhibited. Furthermore, we observed that RhoA activity after stimulation with soluble Thy-1-Fc molecule was augmented upon further cross-linking using protein A-Sepharose beads. The same was shown by cross-linking beta(3)-containing integrin with anti-beta(3) antibodies. Together, these results indicate that Thy-1-mediated astrocyte stimulation depended on beta(3) integrin clustering and the resulting increase in RhoA activity.

High protein intake reduces intrahepatocellular lipid deposition in humans.

BACKGROUND: High sugar and fat intakes are known to increase intrahepatocellular lipids (IHCLs) and to cause insulin resistance. High protein intake may facilitate weight loss and improve glucose homeostasis in insulin-resistant patients, but its effects on IHCLs remain unknown. OBJECTIVE: The aim was to assess the effect of high protein intake on high-fat diet-induced IHCL accumulation and insulin sensitivity in healthy young men. DESIGN: Ten volunteers were studied in a crossover design after 4 d of either a hypercaloric high-fat (HF) diet; a hypercaloric high-fat, high-protein (HFHP) diet; or a control, isocaloric (control) diet. IHCLs were measured by (1)H-magnetic resonance spectroscopy, fasting metabolism was measured by indirect calorimetry, insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp, and plasma concentrations were measured by enzyme-linked immunosorbent assay and gas chromatography-mass spectrometry; expression of key lipogenic genes was assessed in subcutaneous adipose tissue biopsy specimens. RESULTS: The HF diet increased IHCLs by 90 +/- 26% and plasma tissue-type plasminogen activator inhibitor-1 (tPAI-1) by 54 +/- 11% (P < 0.02 for both) and inhibited plasma free fatty acids by 26 +/- 11% and beta-hydroxybutyrate by 61 +/- 27% (P < 0.05 for both). The HFHP diet blunted the increase in IHCLs and normalized plasma beta-hydroxybutyrate and tPAI-1 concentrations. Insulin sensitivity was not altered, whereas the expression of sterol regulatory element-binding protein-1c and key lipogenic genes increased with the HF and HFHP diets (P < 0.02). Bile acid concentrations remained unchanged after the HF diet but increased by 50 +/- 24% after the HFHP diet (P = 0.14). CONCLUSIONS: Protein intake significantly blunts the effects of an HF diet on IHCLs and tPAI-1 through effects presumably exerted at the level of the liver. Protein-induced increases in bile acid concentrations may be involved. This trial was registered at www.clinicaltrials.gov as NCT00523562.

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.

Nuclear and cytoplasmic triiodothyronine-binding sites in primary sensory neurons and Schwann cells: radioautographic study during development.

The effects of the thyroid hormones on target cells are mediated through nuclear T3 receptors. In the peripheral nervous system, nuclear T3 receptors were previously detected with the monoclonal antibody 2B3 mAb in all the primary sensory neurons throughout neuronal life and in peripheral glia at the perinatal period only (Eur. J. Neurosci. 5, 319, 1993). To determine whether these nuclear T3 receptors correspond to functional ones able to bind T3, cryostat sections and in vitro cell cultures of dorsal root ganglion (DRG) or sciatic nerve were incubated with 0.1 nM [125I]-labeled T3, either alone to visualize the total T3-binding sites or added with a 10(3) fold excess of unlabeled T3 to estimate the part due to the non-specific T3-binding. After glutaraldehyde fixation, radioautography showed that the specific T3-binding sites were largely prevalent. The T3-binding capacity of peripheral glia in DRG and sciatic nerve was restricted to the perinatal period in vivo and to Schwann cells cultured in vitro. In all the primary sensory neurons, specific T3-binding sites were disclosed in foetal as well as adult rats. The detection of the T3-binding sites in the nucleus indicated that the nuclear T3 receptors are functional. Moreover the concomitant presence of both T3-binding sites and T3 receptors alpha isoforms in the perikaryon of DRG neurons infers that: 1) [125I]-labeled T3 can be retained on the T3-binding 'E' domain of nascent alpha 1 isoform molecules newly-synthesized on the perikaryal ribosomes; 2) the alpha isoforms translocated to the nucleus are modified by posttranslational changes and finally recognized by 2B3 mAb as nuclear T3 receptor. In conclusion, the radioautographic visualization of the T3-binding sites in peripheral neurons and glia confirms that the nuclear T3 receptors are functional and contributes to clarify the discordant intracellular localization provided by the immunocytochemical detection of nuclear T3 receptors and T3 receptor alpha isoforms.

Differential peptide binding to CD40 evokes counteractive responses.

The antigen-presenting cell-expressed CD40 is implied in the regulation of counteractive immune responses such as induction of pro-inflammatory and anti-inflammatory cytokines interleukin (IL)-12 and IL-10, respectively. The mechanism of this duality in CD40 function remains unknown. Here, we investigated whether such duality depends on ligand binding. Based on CD40 binding, we identifed two dodecameric peptides, peptide-7 and peptide-19, from the phage peptide library. Peptide-7 induces IL-10 and increases Leishmania donovani infection in macrophages, whereas peptide-19 induces IL-12 and reduces L. donovani infection. CD40-peptide interaction analyses by surface plasmon resonance and atomic force microscopy suggest that the functional differences are not associated with the studied interaction parameters. The molecular dynamic simulation of the CD40-peptides interaction suggests that these two peptides bind to two different places on CD40. Thus, we suggest for the first time that differential binding of the ligands imparts functional duality to CD40.

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.

Protein-protein interactions between adenovirus DNA polymerase and nuclear factor I mediate formation of the DNA replication preinitiation complex.

The in vitro adenovirus (Ad) DNA replication system provides an assay to study the interaction of viral and host replication proteins with the DNA template in the formation of the preinitiation complex. This initiation system requires in addition to the origin DNA sequences 1) Ad DNA polymerase (Pol), 2) Ad preterminal protein (pTP), the covalent acceptor for protein-primed DNA replication, and 3) nuclear factor I (NFI), a host cell protein identical to the CCAAT box-binding transcription factor. The interactions of these proteins were studied by coimmunoprecipitation and Ad origin DNA binding assays. The Ad Pol can bind to origin sequences only in the presence of another protein which can be either pTP or NFI. While NFI alone can bind to its origin recognition sequence, pTP does not specifically recognize DNA unless Ad Pol is present. Thus, protein-protein interactions are necessary for the targetting of either Ad Pol or pTP to the preinitiation complex. DNA footprinting demonstrated that the Ad DNA site recognized by the pTP.Pol complex was within the first 18 bases at the end of the template which constitutes the minimal origin of replication. Mutagenesis studies have defined the Ad Pol interaction site on NFI between amino acids 68-150, which overlaps the DNA binding and replication activation domain of this factor. A putative zinc finger on the Ad Pol has been mutated to a product that fails to bind the Ad origin sequences but still interacts with pTP. These results indicate that both protein-protein and protein-DNA interactions mediate specific recognition of the replication origin by Ad DNA polymerase.

Comparative genomics of the odorant-binding and chemosensory protein gene families across the arthropoda: origin and evolutionary history of the chemosensory system

Chemoreception is a biological process essential for the survival of animals, as it allows the recognition of important volatile cues for the detection of food, egg-laying substrates, mates or predators, among other purposes. Furthermore, its role in pheromone detection may contribute to evolutionary processes such as reproductive isolation and speciation. This key role in several vital biological processes makes chemoreception a particularly interesting system for studying the role of natural selection in molecular adaptation. Two major gene families are involved in the perireceptor events of the chemosensory system: the odorant-binding protein (OBP) and chemosensory protein (CSP) families. Here, we have conducted an exhaustive comparative genomic analysis of these gene families in twenty Arthropoda species. We show that the evolution of the OBP and CSP gene families is highly dynamic, with a high number of gains and losses of genes, pseudogenes and independent origins of subfamilies. Taken together, our data clearly support the birth-and-death model for the evolution of these gene families with an overall high gene-turnover rate. Moreover, we show that the genome organization of the two families is significantly more clustered than expected by chance and, more important, that this pattern appears to be actively maintained across the Drosophila phylogeny. Finally, we suggest the homologous nature of the OBP and CSP gene families, dating back their MRCA (most recent common ancestor) to 380¿420 Mya, and we propose a scenario for the origin and diversification of these families.

Comparative genomic analysis of the odorant-binding protein family in 12 Drosophila genomes: purifying selection and birth-and-death evolution

Background: Chemoreception is a widespread mechanism that is involved in critical biologic processes, including individual and social behavior. The insect peripheral olfactory system comprises three major multigene families: the olfactory receptor (Or), the gustatory receptor (Gr), and the odorant-binding protein (OBP) families. Members of the latter family establish the first contact with the odorants, and thus constitute the first step in the chemosensory transduction pathway.Results: Comparative analysis of the OBP family in 12 Drosophila genomes allowed the identification of 595 genes that encode putative functional and nonfunctional members in extant species, with 43 gene gains and 28 gene losses (15 deletions and 13 pseudogenization events). The evolution of this family shows tandem gene duplication events, progressive divergence in DNA and amino acid sequence, and prevalence of pseudogenization events in external branches of the phylogenetic tree. We observed that the OBP arrangement in clusters is maintained across the Drosophila species and that purifying selection governs the evolution of the family; nevertheless, OBP genes differ in their functional constraints levels. Finally, we detect that the OBP repertoire evolves more rapidly in the specialist lineages of the Drosophila melanogaster group (D. sechellia and D. erecta) than in their closest generalists.Conclusion: Overall, the evolution of the OBP multigene family is consistent with the birth-and-death model. We also found that members of this family exhibit different functional constraints, which is indicative of some functional divergence, and that they might be involved in some of the specialization processes that occurred through the diversification of the Drosophila genus.

The TRAF3-binding site of human molluscipox virus FLIP molecule MC159 is critical for its capacity to inhibit Fas-induced apoptosis.

Members of the viral Flice/caspase-8 inhibitory protein (v-FLIP) family prevent induction of apoptosis by death receptors through inhibition of the processing and activation of procaspase-8 and -10 at the level of the receptor-associated death-inducing signaling complex (DISC). Here, we have addressed the molecular function of the v-FLIP member MC159 of the human molluscum contagiosum virus. MC159 FLIP powerfully inhibited both caspase-dependent and caspase-independent cell death induced by Fas. The C-terminal region of MC159 bound TNF receptor-associated factor (TRAF)3, was necessary for optimal TRAF2 binding, and mediated the recruitment of both TRAFs into the Fas DISC. TRAF-binding-deficient mutants of MC159 showed impaired inhibition of FasL-induced caspase-8 processing and Fas internalization, and had reduced antiapoptotic activity. Our findings provide evidence that a MC159/TRAF2/TRAF3 complex regulates a new aspect of Fas signaling, and identify MC159 FLIP as a molecule that targets multiple features of Fas-induced cell death.

Association between mannose-binding lectin (MBL) deficiency and cytomegalovirus (CMV) infection after kidney transplantation

MBLdeficiency is thought to be a risk factor for the development of viral infection, such as genital herpes and HSV-2 meningitis. However, there is limited data on the possible interaction between MBL and CMV, especially after organ transplantation. Between 2003 and 2005, we measured MBL levels in 16 kidney transplant recipients with high-risk CMV serostatus (donor positive/recipient negative, D+/R−). All patients receivedCMV prophylaxis of valganciclovir 450 mg/day for 3 months after transplantation. After stopping valganciclovir, CMV-DNA was measured in whole blood by real time PCR every 2 weeks for 3 months. CMV infections were diagnosed according to the recommendations of the AST. MBL levels were measured in stored pre-transplantation sera by an investigator blinded to the CMV complications. MBL levels below 500 ng/ml were considered as being functionally deficient. After a follow-up of at least 10 months, seven patients out of 16 developed CMV disease (three CMV syndrome, and four probable invasive disease, i.e. two colitis and two hepatitis), four patients developed asymptomatic CMV infection, and five patients never developed any sign of CMV replication. Peak CMV-DNA was higher in patients with CMV disease than in those with asymptomatic infection (4.64 versus 2.72 mean log copy CMV-DNA/106 leukocytes, p < 0.05). Overall, 9/16 patients (56%) had MBL deficiency: 5/7 (71%) of patients with CMV disease, 4/4 (100%) of patients with asymptomatic CMVinfection, and 0/5 (0%) of patients withoutCMVinfection (p < 0.005, between CMV infection/disease versus no infection or control blood donors). There were no significant differences in age, gender or immunosuppressive regimens between the groups. MBL deficiency may be a significant risk factor for the development of post-prophylaxisCMVinfection in D+/R−kidney recipients, suggesting a new role of innate immunity in the control of CMV infection after organ transplantation.

Fibrinogen and fibronectin binding cooperate for valve infection and invasion in Staphylococcus aureus experimental endocarditis.

The expression of Staphylococcus aureus adhesins in Lactococcus lactis identified clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA) as critical for valve colonization in rats with experimental endocarditis. This study further analyzed their role in disease evolution. Infected animals were followed for 3 d. ClfA-positive lactococci successfully colonized damaged valves, but were spontaneously eradicated over 48 h. In contrast, FnBPA-positive lactococci progressively increased bacterial titers in vegetations and spleens. At imaging, ClfA-positive lactococci were restricted to the vegetations, whereas FnBPA-positive lactococci also invaded the adjacent endothelium. This reflected the capacity of FnBPA to trigger cell internalization in vitro. Because FnBPA carries both fibrinogen- and fibronectin-binding domains, we tested the role of these functionalities by deleting the fibrinogen-binding domain of FnBPA and supplementing it with the fibrinogen-binding domain of ClfA in cis or in trans. Deletion of the fibrinogen-binding domain of FnBPA did not alter fibronectin binding and cell internalization in vitro. However, it totally abrogated valve infectivity in vivo. This ability was restored in cis by inserting the fibrinogen-binding domain of ClfA into truncated FnBPA, and in trans by coexpressing full-length ClfA and truncated FnBPA on two separate plasmids. Thus, fibrinogen and fibronectin binding could cooperate for S. aureus valve colonization and endothelial invasion in vivo.