Identification and properties of the crenarchaeal single-stranded DNA binding protein from Sulfolobus solfataricus

Single-stranded DNA binding proteins (SSBs) play central roles in cellular and viral processes involving the generation of single-stranded DNA. These include DNA replication, homologous recombination and DNA repair pathways. SSBs bind DNA using four ‘OB-fold’ (oligonucleotide/oligosaccharide binding fold) domains that can be organised in a variety of overall quaternary structures. Thus eubacterial SSBs are homotetrameric whilst the eucaryal RPA protein is a heterotrimer and euryarchaeal proteins vary significantly in their subunit compositions. We demonstrate that the crenarchaeal SSB protein is an abundant protein with a unique structural organisation, existing as a monomer in solution and multimerising on DNA binding. The protein binds single-stranded DNA distributively with a binding site size of ~5 nt per monomer. Sulfolobus SSB lacks the zinc finger motif found in the eucaryal and euryarchaeal proteins, possessing instead a flexible C-terminal tail, sensitive to trypsin digestion, that is not required for DNA binding. In comparison with Escherichia coli SSB, the tail may play a role in protein–protein interactions during DNA replication and repair.

Layilin, a Novel Integral Membrane Protein, Is a Hyaluronan Receptor

The actin cytoskeleton plays a significant role in changes of cell shape and motility, and interactions between the actin filaments and the cell membrane are crucial for a variety of cellular processes. Several adaptor proteins, including talin, maintain the cytoskeleton-membrane linkage by binding to integral membrane proteins and to the cytoskeleton. Layilin, a recently characterized transmembrane protein with homology to C-type lectins, is a membrane-binding site for talin in peripheral ruffles of spreading cells. To facilitate studies of layilin's function, we have generated a layilin-Fc fusion protein comprising the extracellular part of layilin joined to human immunoglobulin G heavy chain and used this chimera to identify layilin ligands. Here, we demonstrate that layilin-Fc fusion protein binds to hyaluronan immobilized to Sepharose. Microtiter plate-binding assays, coprecipitation experiments, and staining of sections predigested with different glycosaminoglycan-degrading enzymes and cell adhesion assays all revealed that layilin binds specifically to hyaluronan but not to other tested glycosaminoglycans. Layilin's ability to bind hyaluronan, a ubiquitous extracellular matrix component, reveals an interesting parallel between layilin and CD44, because both can bind to cytoskeleton-membrane linker proteins through their cytoplasmic domains and to hyaluronan through their extracellular domains. This parallelism suggests a role for layilin in cell adhesion and motility.

Human testis expresses a specific poly(A)-binding protein

In testis mRNA stability and translation initiation are extensively under the control of poly(A)-binding proteins (PABP). Here we have cloned a new human testis-specific PABP (PABP3) of 631 amino acids (70.1 kDa) with 92.5% identical residues to the ubiquitous PABP1. A northern blot of multiple human tissues hybridised with PABP3- and PABP1-specific oligonucleotide probes revealed two PABP3 mRNAs (2.1 and 2.5 kb) detected only in testis, whereas PABP1 mRNA (3.2 kb) was present in all tested tissues. In human adult testis, PABP3 mRNA expression was restricted to round spermatids, whereas PABP1 was expressed in these cells as well as in pachytene spermatocytes. PABP3-specific antibodies identified a protein of 70 kDa in human testis extracts. This protein binds poly(A) with a slightly lower affinity as compared to PABP1. The human PABP3 gene is intronless with a transcription start site 61 nt upstream from the initiation codon. A sequence of 256 bp upstream from the transcription start site drives the promoter activity of PABP3 and its tissue-specific expression. The expression of PABP3 might be a way to bypass PABP1 translational repression and to produce the amount of PABP needed for active mRNA translation in spermatids.

Functional interaction and colocalization of the herpes simplex virus 1 major regulatory protein ICP4 with EAP, a nucleolar-ribosomal protein.

The herpes simplex virus 1 infected cell protein 4 (ICP4) binds to DNA and regulates gene expression both positively and negatively. EAP (Epstein-Barr virus-encoded small nuclear RNA-associated protein) binds to small nonpolyadenylylated nuclear RNAs and is found in nucleoli and in ribosomes, where it is also known as L22. We report that EAP interacts with a domain of ICP4 that is known to bind viral DNA response elements and transcriptional factors. In a gel-shift assay, a glutathione S-transferase (GST)-EAP fusion protein disrupted the binding of ICP4 to its cognate site on DNA in a dose-dependent manner. This effect appeared to be specifically due to EAP binding to ICP4 because (i) GST alone did not alter the binding of ICP4 to DNA, (ii) GST-EAP did not bind to the probe DNA, and (iii) GST-EAP did not influence the binding of the alpha gene trans-inducing factor (alphaTIF or VP16) to its DNA cognate site. Early in infection, ICP4 was dispersed throughout the nucleoplasm, whereas EAP was localized to the nucleoli. Late in infection, EAP was translocated from nucleoli and colocalized with ICP4 in small, dense nuclear structures. The formation of dense structures and the colocalization of EAP and ICP4 did not occur if virus DNA synthesis and late gene expression were prevented by the infection of cells at the nonpermissive temperature with a mutant virus defective in DNA synthesis, or in cells infected and maintained in the presence of phosphonoacetate, which is an inhibitor of viral DNA synthesis. These results suggest that the translocation of EAP from the nucleolus to the nucleoplasm is a viral function and that EAP plays a role in the regulatory functions expressed by ICP4.

Copy-up mutants of the plasmid RK2 replication initiation protein are defective in coupling RK2 replication origins.

The broad host range plasmid RK2 replicates and regulates its copy number in a wide range of Gram-negative bacteria. The plasmid-encoded trans-acting replication protein TrfA and the origin of replication oriV are sufficient for controlled replication of the plasmid in all Gram-negative bacteria tested. The TrfA protein binds specifically to direct repeat sequences (iterons) at the origin of replication. A replication control model, designated handcuffing or coupling, has been proposed whereby the formation of coupled TrfA-oriV complexes between plasmid molecules results in hindrance of origin activity and, consequently, a shut-down of plasmid replication under conditions of higher than normal copy number. Therefore, according to this model, the coupling activity of an initiation protein is essential for copy number control and a copy-up initiation protein mutant should have reduced ability to form coupled complexes. To test this model for plasmid RK2, two previously characterized copy-up TrfA mutations, trfA-254D and trfA-267L, were combined and the resulting copy-up double mutant TFrfA protein TrfA-254D/267L was characterized. Despite initiating runaway (uncontrolled) replication in vivo, the copy-up double-mutant TrfA protein exhibited replication kinetics similar to the wild-type protein in vitro. Purified TrfA-254D, TrfA-267L, and TrfA-254D/267L proteins were then examined for binding to the iterons and for coupling activity using an in vitro ligase-catalyzed multimerization assay. It was found that both single and double TrfA mutant proteins exhibited substantially reduced (single mutants) or barely detectable (double mutant) levels of coupling activity while not being diminished in their capacity to bind to the origin of replication. These observations provide direct evidence in support of the coupling model of replication control.

Interaction between the phage HK022 Nun protein and the nut RNA of phage lambda.

The nun gene product of prophage HK022 excludes phage lambda infection by blocking the expression of genes downstream from the lambda nut sequence. The Nun protein functions both by competing with lambda N transcription-antitermination protein and by actively inducing transcription termination on the lambda chromosome. We demonstrate that Nun binds directly to a stem-loop structure within nut RNA, boxB, which is also the target for the N antiterminator. The two proteins show comparable affinities for boxB and they compete with each other. Their interactions with boxB are similar, as shown by RNase protection experiments, NMR spectroscopy, and analysis of boxB mutants. Each protein binds the 5' strand of the boxB stem and the adjacent loop. The stem does not melt upon the binding of Nun or N, as the 3' strand remains sensitive to a double-strand-specific RNase. The binding of RNA partially protects Nun from proteolysis and changes its NMR spectra. Evidently, although Nun and N bind to the same surface of boxB RNA, their respective complexes interact differently with RNA polymerase, inducing transcription termination or antitermination, respectively.

VIP21/caveolin is a cholesterol-binding protein.

VIP21/caveolin is localized to both caveolae and apical transport vesicles and presumably cycles between the cell surface and the Golgi complex. We have studied the lipid interactions of this protein by reconstituting Escherichia coli-expressed VIP21/caveolin into liposomes. Surprisingly, the protein reconstituted only with cholesterol-containing lipid mixtures. We demonstrated that the protein binds at least 1 mol of cholesterol per mole of protein and that this binding promotes formation of protein oligomers. These findings suggest that VIP21/caveolin, through its cholesterol-binding properties, serves a specific function in microdomain formation during membrane trafficking.

Elf-1 contributes to the function of the complex interleukin (IL)-2-responsive enhancer in the mouse IL-2 receptor alpha gene.

Lymphocytes regulate their responsiveness to IL-2 through the transcriptional control of the IL-2R alpha gene, which encodes a component of the high affinity IL-2 receptor. In the mouse IL-2R alpha gene this control is exerted via two regulatable elements, a promoter proximal region, and an IL-2-responsive enhancer (IL-2rE) 1.3 kb upstream. In vitro and in vivo functional analysis of the IL-2rE in the rodent thymic lymphoma-derived, CD4- CD8- cell line PC60 demonstrated that three separate elements, sites I, II, and III, were necessary for IL-2 responsiveness; these three sites demonstrate functional cooperation. Site III contains a consensus binding motif for members of the Ets family of transcription factors. Here we demonstrate that Elf-1, an Ets-like protein, binds to site III and participates in IL-2 responsiveness. In vitro site III forms a complex with a protein constitutively present in nuclear extracts from PC60 cells as well as from normal CD4- CD8- thymocytes. We have identified this molecule as Elf-1 according to a number of criteria. The complex possesses an identical electrophoretic mobility to that formed by recombinant Elf-1 protein and is super-shifted by anti-Elf-1 antibodies. Biotinylated IL-2rE probes precipitate Elf-1 from PC60 extracts provided site III is intact and both recombinant and PC60-derived proteins bind with the same relative affinities to different mutants of site III. In addition, by introducing mutations into the core of the site III Ets-like motif and comparing the corresponding effects on the in vitro binding of Elf-1 and the in vivo IL-2rE activity, we provide strong evidence that Elf-1 is directly involved in IL-2 responsiveness. The nature of the functional cooperativity observed between Elf-1 and the factors binding sites I and II remains unresolved; experiments presented here however suggest that this effect may not require direct interactions between the proteins binding these three elements.

Visualization of recombination intermediates produced by RAD52-mediated single-strand annealing.

Double-strand breaks (DSBs) occur frequently during DNA replication. They are also caused by ionizing radiation, chemical damage or as part of the series of programmed events that occur during meiosis. In yeast, DSB repair requires RAD52, a protein that plays a critical role in homologous recombination. Here we describe the actions of human RAD52 protein in a model system for single-strand annealing (SSA) using tailed (i.e. exonuclease resected) duplex DNA molecules. Purified human RAD52 protein binds resected DSBs and promotes associations between complementary DNA termini. Heteroduplex intermediates of these recombination reactions have been visualized by electron microscopy, revealing the specific binding of multiple rings of RAD52 to the resected termini and the formation of large protein complexes at heteroduplex joints formed by RAD52-mediated annealing.

The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51.

Eukaryotic cells encode two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, which are required for meiotic recombination. Rad51, like E.coli RecA, forms helical nucleoprotein filaments that promote joint molecule and heteroduplex DNA formation. Electron microscopy reveals that the human meiosis-specific recombinase Dmc1 forms ring structures that bind single-stranded (ss) and double-stranded (ds) DNA. The protein binds preferentially to ssDNA tails and gaps in duplex DNA. hDmc1-ssDNA complexes exhibit an irregular, often compacted structure, and promote strand-transfer reactions with homologous duplex DNA. hDmc1 binds duplex DNA with reduced affinity to form nucleoprotein complexes. In contrast to helical RecA/Rad51 filaments, however, Dmc1 filaments are composed of a linear array of stacked protein rings. Consistent with the requirement for two recombinases in meiotic recombination, hDmc1 interacts directly with hRad51.

Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation.

The role of Notch signaling in growth/differentiation control of mammalian epithelial cells is still poorly defined. We show that keratinocyte-specific deletion of the Notch1 gene results in marked epidermal hyperplasia and deregulated expression of multiple differentiation markers. In differentiating primary keratinocytes in vitro endogenous Notch1 is required for induction of p21WAF1/Cip1 expression, and activated Notch1 causes growth suppression by inducing p21WAF1/Cip1 expression. Activated Notch1 also induces expression of 'early' differentiation markers, while suppressing the late markers. Induction of p21WAF1/Cip1 expression and early differentiation markers occur through two different mechanisms. The RBP-Jkappa protein binds directly to the endogenous p21 promoter and p21 expression is induced specifically by activated Notch1 through RBP-Jkappa-dependent transcription. Expression of early differentiation markers is RBP-Jkappa-independent and can be induced by both activated Notch1 and Notch2, as well as the highly conserved ankyrin repeat domain of the Notch1 cytoplasmic region. Thus, Notch signaling triggers two distinct pathways leading to keratinocyte growth arrest and differentiation.

High expression of the HMG box factor sox-13 in arterial walls during embryonic development.

Members of the Sox gene family of transcription factors are defined by the presence of an 80 amino acid homology domain, the High Mobility Group (HMG) box. Here we report the cloning and initial analysis of murine Sox-13 . The 984 amino acids Sox-13 protein contains a single HMG box, a leucine zipper motif and a glutamine-rich stretch. These characteristics are shared with another member of the Sox gene family, Sox-6. High level embryonic expression of Sox-13 occurs uniquely in the arterial walls of 13.5 days post coitum (dpc) mice and later. Low level expression was observed in the inner ear of 13.5 dpc mice and in a limited number of cells in the thymus of 16.5 dpc mice, from which Sox-13 was originally cloned. At 18.5 dpc, Sox-13 is expressed in the tracheal epithelium below the vocal cord and in the hair follicles. The Sox-13 protein binds to the consensus HMG box motif, AACAAAG, but does not transactivate transcription through a concatamer of this motif. Sox-13, like other members of the Sox family likely plays an important role in development.

NAD+-dependent post-translational modification of Escherichia coli glyceraldehyde-3-phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional housekeeping protein reported to be a target of several covalent modifications in many organisms. In a previous study we showed that enterohemorragic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains secrete GAPDH and that this protein binds to human plasminogen and fibrinogen. Here we report that GAPDH of these pathogens is ADP-ribosylated either in the cytoplasm or in the extracellular medium. GAPDH catalyzes its own modification which involves Cys149 at the active site. ADP-ribosylation of extracellular GAPDH may play important role in the interaction with the host as it has been proposed in other pathogens.

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha 1 (residues Gly-9 to Arg-21), alpha 2 (residues Glu-27 to Asn-40), alpha 3 (residues Arg-44 to Thr-54), alpha 4 (residues Leu-57 to Tyr-64), and alpha 5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

Caracterização estrutural e das interações entre a Proteína G do hRSV e potenciais inibidores

Pós-graduação em Microbiologia - IBILCE