Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition.

LINE-1s are active human DNA parasites that are agents of genome dynamics in evolution and disease. These streamlined elements require host factors to complete their life cycles, whereas hosts have developed mechanisms to combat retrotransposition's mutagenic effects. As such, endogenous L1 expression levels are extremely low, creating a roadblock for detailed interactomic analyses. Here, we describe a system to express and purify highly active L1 RNP complexes from human suspension cell culture and characterize the copurified proteome, identifying 37 high-confidence candidate interactors. These data sets include known interactors PABPC1 and MOV10 and, with in-cell imaging studies, suggest existence of at least three types of compositionally and functionally distinct L1 RNPs. Among the findings, UPF1, a key nonsense-mediated decay factor, and PCNA, the polymerase-delta-associated sliding DNA clamp, were identified and validated. PCNA interacts with ORF2p via a PIP box motif; mechanistic studies suggest that this occurs during or immediately after target-primed reverse transcription.

Soluble expression, purification, and secondary structure determination of human PDX1 transcription factor.

The transcription factor PDX1 is a master regulator essential for proper development of the pancreas, duodenum and antrum. Furthermore, it is an indispensable reprogramming factor for the derivation of human ß-cells, and recently, it has been identified as a tumor suppressor protein in gastric cancer. Here, we report the soluble expression and purification of the full-length human PDX1 protein from a heterologous system. To achieve this, the 849 bp coding sequence of the PDX1 gene was first codon-optimized for expression in Escherichia coli (E. coli). This codon-optimized gene sequence was fused to a protein transduction domain, a nuclear localization sequence, and a His-tag, and this insert was cloned into the protein expression vector for expression in E. coli strain BL21(DE3). Next, screening and identification of the suitable gene construct and optimal expression conditions to obtain this recombinant fusion protein in a soluble form was performed. Further, we have purified this recombinant fusion protein to homogeneity under native conditions. Importantly, the secondary structure of the protein was retained after purification. Further, this recombinant PDX1 fusion protein was applied to human cells and showed the ability to enter the cells as well as translocate to the nucleus. This recombinant tool can be used as a safe tool and can potentially replace its genetic and viral forms in the reprogramming process to induce a ß-cell-specific transcriptional profile in an integration-free manner. Additionally, it can also be used to elucidate its role in cellular processes and for structural and biochemical studies.

Characterization of a metazoan ADA acetyltransferase complex.

The Gcn5 acetyltransferase functions in multiple acetyltransferase complexes in yeast and metazoans. Yeast Gcn5 is part of the large SAGA (Spt-Ada-Gcn5 acetyltransferase) complex and a smaller ADA acetyltransferase complex. In flies and mammals, Gcn5 (and its homolog pCAF) is part of various versions of the SAGA complex and another large acetyltransferase complex, ATAC (Ada2A containing acetyltransferase complex). However, a complex analogous to the small ADA complex in yeast has never been described in metazoans. Previous studies in Drosophila hinted at the existence of a small complex which contains Ada2b, a partner of Gcn5 in the SAGA complex. Here we have purified and characterized the composition of this complex and show that it is composed of Gcn5, Ada2b, Ada3 and Sgf29. Hence, we have named it the metazoan 'ADA complex'. We demonstrate that the fly ADA complex has histone acetylation activity on histones and nucleosome substrates. Moreover, ChIP-Sequencing experiments identified Ada2b peaks that overlap with another SAGA subunit, Spt3, as well as Ada2b peaks that do not overlap with Spt3 suggesting that the ADA complex binds chromosomal sites independent of the larger SAGA complex.

LcpRVH2 - regulating the expression of latex-clearing proteins in Gordonia polyisoprenivorans VH2.

Gordonia polyisoprenivorans VH2 harbours two latex clearing proteins, which are responsible for the cleavage of poly(cis-1,4-isoprene) into oligoisoprenes, thereby allowing growth in presence of, e.g. natural rubber. A gene coding for a putative regulator of the TetR-family (lcpRVH2) is located 131 bp upstream of lcp1VH2. We heterologously expressed lcpRVH2 in Escherichia coli, and purified and characterized the protein with respect to its ability to bind to the operator region of lcp1VH2. LcpRVH2 forms a dimer in its native state. The size of the dimer was determined to be 52.7 kDa by size exclusion chromatography, whereas the calculated size of a monomer was 24.1 kDa. Electrophoretic mobility shift assays (EMSAs) with the purified protein revealed a shift upon binding to the intergenic region between lcpRVH2 and lcp1VH2. Within this region, an inverted repeat was identified in silico, probably being the binding site of LcpRVH2. This binding sequence was confirmed by a DNase I footprinting assay. A shift also occurred in EMSAs with this 44 bp sequence only. Interestingly, no regulator was detected upstream of the second lcp (lcp2VH2). Therefore, we performed EMSA studies with LcpRVH2 and the putative operator region upstream of lcp2VH2, and discovered by DNase I footprinting another binding sequence upstream of lcp2VH2. Hence, we concluded that LcpRVH2 binds the operator region of both lcps and, most likely, regulates their expression in G. polyisoprenivorans VH2.

Structure of natural killer cell receptor KLRG1 bound to E-cadherin reveals basis for MHC-independent missing self recognition.

The cytolytic activity of natural killer (NK) cells is regulated by inhibitory receptors that detect the absence of self molecules on target cells. Structural studies of missing self recognition have focused on NK receptors that bind MHC. However, NK cells also possess inhibitory receptors specific for non-MHC ligands, notably cadherins, which are downregulated in metastatic tumors. We determined the structure of killer cell lectin-like receptor G1 (KLRG1) in complex with E-cadherin. KLRG1 mediates missing self recognition by binding to a highly conserved site on classical cadherins, enabling it to monitor expression of several cadherins (E-, N-, and R-) on target cells. This site overlaps the site responsible for cell-cell adhesion but is distinct from the integrin alpha(E)beta(7) binding site. We propose that E-cadherin may coengage KLRG1 and alpha(E)beta(7) and that KLRG1 overcomes its exceptionally weak affinity for cadherins through multipoint attachment to target cells, resulting in inhibitory signaling.

Purification and characterization of Pseudomonas aeruginosa LasR expressed in acyl-homoserine lactone free Escherichia coli cultures.

Quorum sensing systems are essential for bacterial communication. We report here the purification and characterization of the Pseudomonas aeruginosa LasR quorum sensing regulator purified from lysates of E. coli cultures grown in the absence of added acyl-homoserine lactones (AHL). We show by isothermal titration calorimetry that LasR recognizes different AHLs with an affinity of approximately 1 µM. The affinity of LasR for its cognate 3-Oxo-C12-AHL was similar to that of other AHLs, indicating that this regulator has not evolved to preferentially recognize its cognate AHL. The α-helical content as determined by CD spectroscopy was found to be in agreement with the corresponding value derived from the homology model. Analytical ultracentrifugation studies show that LasR is a mixture of monomers and dimers and that AHL binding does not alter its oligomeric state. Thermal unfolding studies indicate that LasR has a significant thermal stability and that AHL binding does not significantly alter the unfolding temperature. Two LasR-DNA complexes were observed in electrophoretic mobility shift assays using the hcnABC promoter that has two lux boxes. Taken together, data indicate that the presence of AHLs is not a requisite for correct LasR protein folding. The protein is able to bind AHL ligands in a reversible manner, revising initial concepts of this regulator. The availability of AHL-free protein will permit further studies to determine more precisely its mode of action.

Hepatitis B Virus X Protein Modulates Apoptosis in NRK-52E Cells and Activates Fas/FasL Through the MLK3-MKK7-JNK3 Signaling Pathway.

BACKGROUND/AIMS: The hepatitis B virus X protein (HBx) contributes to HBV-induced injury of renal tubular cells and induces apoptosis via Fas/FasL up-regulation. However, the mechanism of Fas/FasL activation is unknown. Recent studies indicated that HBx induction of apoptosis in hepatic cells depends on activating the MLK3-MKK7-JNKs signaling module, which then up-regulates FasL expression. In this study, we used NRK-52E cells transfected an HBx expression vector to examine the role of the MLK3-MKK7-JNKs signaling pathway on HBx-induced renal tubular cell injury. METHODS: NRK-52E cells were transfected with pc-DNA3.1(+)-HBx to establish an HBx over-expression model, and with pc-DNA3.1(+)-HBx and pSilencer3.1-shHBx to establish an HBx low expression model. One control group was not transfected and another control group was transfected with an empty plasmid. Cell proliferation was determined by the formazan dye method (Cell Counting Kit-8) and apoptosis was measured by flow cytometry and fluorescence microscopy. Western blotting was used to measure the expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins. The activity of caspase-8 was measured by spectrophotometry. RESULTS: Transfection of NRK-52E cells with pc-DNA3.1(+)-HBx inhibited cell proliferation and increased apoptosis and caspase-8 activity. The expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins were also greater in the pc-DNA3.1(+)-HBx group, but lower in RNAi group. Furthermore, the activity of MLK3-MKK7-JNKs signaling pathway, expression of Fas/FasL, and apoptosis were significantly lower in the pc-DNA3.1(+)-HBx group when treated with K252a, a known inhibitor of MLK3. CONCLUSIONS: Our results show that HBx induces apoptosis in NRK-52E cells and activates Fas/FasL via the MLK3-MKK7-JNK3-c-Jun signaling pathway.

Recombinant production of Epstein-Barr virus BZLF1 trans-activator and characterization of its DNA-binding specificity.

This paper describes the recombinant production of a biologically active Epstein-Barr virus BZLF1 trans-activator, i.e., Z-encoded broadly reactive activator (ZEBRA), that recognized specific DNA motifs. We used auto-induction for histidine-tagged BZLF1 expression in Escherichia coli and immobilized cobalt affinity membrane chromatography for protein purification under native conditions. We obtained the purified BZLF1 at a yield of 5.4mg per gram of wet weight cells at 75% purity, in which 27% of the recombinant BZLF1 remained biologically active. The recombinant BZLF1 bound to oligonucleotides containing ZEBRA response elements, either AP-1 or ZIIIB, but not a ZIIIB mutant. The recombinant BZLF1 showed a specific DNA-binding activity which could be useful for functional studies.

Three of four GlnR binding sites are essential for GlnR-mediated activation of transcription of the Amycolatopsis mediterranei nas operon.

In Amycolatopsis mediterranei U32, genes responsible for nitrate assimilation formed one operon, nasACKBDEF, whose transcription is induced by the addition of nitrate. Here, we characterized GlnR as a direct transcriptional activator for the nas operon. The GlnR-protected DNA sequences in the promoter region of the nas operon were characterized by DNase I footprinting assay, the previously deduced Streptomyces coelicolor double 22-bp GlnR binding consensus sequences comprising a1, b1, a2, and b2 sites were identified, and the sites were then mutated individually to test their roles in both the binding of GlnR in vitro and the GlnR-mediated transcriptional activation in vivo. The results clearly showed that only three GlnR binding sites (a1, b1, and b2 sites) were required by GlnR for its specific binding to the nas promoter region and efficient activation of the transcription of the nas operon in U32, while the a2 site seemed unnecessary.

Growth phase-dependent modulation of Rgg binding specificity in Streptococcus pyogenes.

Streptococcus pyogenes Rgg is a transcriptional regulator that interacts with the cofactor LacD.1 to control growth phase-dependent expression of genes, including speB, which encodes a secreted cysteine protease. LacD.1 is thought to interact with Rgg when glycolytic intermediates are abundant in a manner that prevents Rgg-mediated activation of speB expression via binding to the promoter region. When the intermediates diminish, LacD.1 dissociates from Rgg and binds to the speB promoter to activate expression. The purpose of this study was to determine if Rgg bound to chromatin during the exponential phase of growth and, if so, to identify the binding sites. Rgg bound to 62 chromosomal sites, as determined by chromatin immunoprecipitation coupled with DNA microarrays. Thirty-eight were within noncoding DNA, including sites upstream of the genes encoding the M protein (M49), serum opacity factor (SOF), fibronectin-binding protein (SfbX49), and a prophage-encoded superantigen, SpeH. Each of these sites contained a promoter that was regulated by Rgg, as determined with transcriptional fusion assays. Purified Rgg also bound to the promoter regions of emm49, sof, and sfbX49 in vitro. Results obtained with a lacD.1 mutant showed that both LacD.1 and Rgg were necessary for the repression of emm49, sof, sfbX49, and speH expression. Overall, the results indicated that the DNA binding specificity of Rgg is responsive to environmental changes in a LacD.1-dependent manner and that Rgg and LacD.1 directly control virulence gene expression in the exponential phase of growth.

RepC protein of the octopine-type Ti plasmid binds to the probable origin of replication within repC and functions only in cis.

Vegetative replication and partitioning of many plasmids and some chromosomes of alphaproteobacteria are directed by their repABC operons. RepA and RepB proteins direct the partitioning of replicons to daughter cells, while RepC proteins are replication initiators, although they do not resemble any characterized replication initiation protein. Here we show that the replication origin of an Agrobacterium tumefaciens Ti plasmid resides fully within its repC gene. Purified RepC bound to a site within repC with moderate affinity, high specificity and with twofold cooperativity. The binding site was localized to an AT-rich region that contains a large number of GANTC sites, which have been implicated in replication regulation in related organisms. A fragment of RepC containing residues 26-158 was sufficient to bind DNA, although with limited sequence specificity. This portion of RepC is predicted to have structural homology to members of the MarR family of transcription factors. Overexpression of RepC in A. tumefaciens caused large increases in copy number in cis but did not change the copy number of plasmids containing the same oriV sequence in trans, confirming other observations that RepC functions only in cis.

Purification, crystallization and preliminary X-ray analysis of the DNA-binding domain of AdpA, the central transcription factor in the A-factor regulatory cascade in the filamentous bacterium Streptomyces griseus, in complex with a duplex DNA.

Streptomyces griseus AdpA is the central transcription factor in the A-factor regulatory cascade and activates a number of genes that are required for both secondary metabolism and morphological differentiation, leading to the onset of streptomycin biosynthesis as well as aerial mycelium formation and sporulation. The DNA-binding domain of AdpA consists of two helix-turn-helix DNA-binding motifs and shows low nucleotide-sequence specificity. To reveal the molecular basis of the low nucleotide-sequence specificity, an attempt was made to obtain cocrystals of the DNA-binding domain of AdpA and several kinds of duplex DNA. The best diffracting crystal was obtained using a 14-mer duplex DNA with two-nucleotide overhangs at the 5'-ends. The crystal diffracted X-rays to 2.8 Šresolution and belonged to space group C222(1), with unit-cell parameters a = 76.86, b = 100.96, c = 101.25 Å. The Matthews coefficient (V(M) = 3.71 Å(3) Da(-1)) indicated that the crystal was most likely to contain one DNA-binding domain of AdpA and one duplex DNA in the asymmetric unit, with a solvent content of 66.8%.

Isolation and functional characterization of a transcription factor VpNAC1 from Chinese wild Vitis pseudoreticulata.

NAC (for NAM, ATAF1, 2, and CUC2) family genes encode plant-specific transcription factors that play important roles in plant development regulation and in abiotic and biotic stresses. However, the function of NAC genes in grapevines is not clear. A novel NAC transcription factor, designated as VpNAC1, was isolated from Chinese wild Vitis pseudoreticulata. It belongs to the TERN subgroup and is a nuclear-targeting protein and functions as a transcriptional activator. Moreover, VpNAC1 was induced by the fungus Erysiphe necator and the exogenous hormones, particularly salicylic acid, methyl jasmonate and ethylene. Over-expression of VpNAC1 in tobacco plants enhanced their resistance to Erysiphe cichoracearum and Phytophthora parasitica var. nicotianae Tucker. These results suggest that VpNAC1 acts as a positive regulator in biotic stresses.

Membrane topology and DNA-binding ability of the Streptococcal CpsA protein.

Many streptococcal pathogens require a polysaccharide capsule for survival in the host during systemic infection. The highly conserved CpsA protein is proposed to be a transcriptional regulator of capsule production in streptococci, although the regulatory mechanism is unknown. Hydropathy plots of CpsA predict an integral membrane protein with 3 transmembrane domains and only 27 cytoplasmic residues, whereas other members of the LytR_cpsA_psr protein family are predicted to have a single transmembrane domain. This unique topology, with the short cytoplasmic domain, membrane localization, and large extracellular domain, suggests a novel mechanism of transcriptional regulation. Therefore, to determine the actual membrane topology of CpsA, specific protein domains were fused to beta-galactosidase or alkaline phosphatase. Enzymatic assays confirmed that the predicted membrane topology for CpsA is correct. To investigate how this integral membrane protein may be functioning in regulation of capsule transcription, purified full-length and truncated forms of CpsA were used in electrophoretic mobility shift assays to characterize the ability to bind the capsule operon promoter. Assays revealed that full-length, purified CpsA protein binds specifically to DNA containing the capsule promoter region. Furthermore, the large extracellular domain is not required for DNA binding, but all cytoplasmic regions of CpsA are necessary and sufficient for specific binding to the capsule operon promoter. This is the first demonstration of a member of this protein family interacting with its target DNA. Taken together, CpsA, as well as other members of the LytR_cpsA_psr protein family, appears to utilize a unique mechanism of transcriptional regulation.

Mediator complexes and transcription.

Over the past decade, various components of the transcription machinery have been identified as potential targets for activators. Recently, metazoan versions of yeast Mediator have been isolated and found to act as key coactivators to many transcription factors. Recent work has defined the composition, function and biology of metazoan mediator complexes, which has led us to propose a new nomenclature for the variously named versions of the mediator complex.

Purification, crystallization and preliminary X-ray diffraction studies of the arsenic repressor ArsR from Corynebacterium glutamicum.

ArsR is a member of the SmtB/ArsR family of metalloregulatory proteins that regulate prokaryotic arsenic-resistance operons. Here, the crystallization and preliminary X-ray diffraction studies of a cysteine-free derivative of ArsR from Corynebacterium glutamicum (CgArsR-C15/16/55S) are reported. CgArsR-C15/16/55S was expressed, purified, crystallized and X-ray diffraction data were collected to 1.86 Å resolution. The protein crystallized in a tetragonal space group (P4), with unit-cell parameters a = b = 41.84, c = 99.47 Å.

Staphylococcus aureus isolated from patients with recurrent furunculosis carrying Panton-Valentine leukocidin genes represent agr specificity group IV.

Recurrent furunculosis (RF) caused by Staphylococcus aureus presents a difficult clinical problem and causes significant morbidity. The study aim was to characterise agr groups and detect toxin genes among S. aureus strains isolated from RF patients. Microbiological material was obtained from evacuated furuncules of 44 RF patients. Nasal swabs were obtained from both the RF patients and the controls (150 healthy volunteers with no history of RF). All strains were screened for the presence of lukS/lukF-PV, tst, sea, seb, sec, sed, eta, and etb genes. Moreover, agr specificity groups (I-IV) were identified. Antibiotic-susceptibility tests were performed by disk diffusion method and methicillin susceptibility was verified by mecA gene amplification. The investigated strains were resistant to penicillin, clindamycin, erythromycin, and tetracycline. All showed susceptibility to methicillin. Thirty-five of 44 strains tested were positive for leukocidin lukS/lukF-PV genes and 12/44 for enterotoxin seb gene. The coexistence of PVL genes and seb gene concerned 7/44 strains. The remaining toxin genes were not found. Forty-three strains belonged to agr specificity group IV including all strains with lukS/lukF-PV genes. Nasal carriage of S. aureus was observed in 27/44 (61.3%) RF patients and in 43/150 (28.6%) controls (p = 0.001). In all RF subjects, nasal strains did not differ from those isolated from furuncules in terms of lukS/lukF-PV gene status and agr specificity. To the best of our knowledge, it is the first study that shown such a predominance of agr group IV strains in RF patients.

The biochemical and genetic discovery of the SAGA complex.

One of the major advances in our understanding of gene regulation in eukaryotes was the discovery of factors that regulate transcription by controlling chromatin structure. Prominent among these discoveries was the demonstration that Gcn5 is a histone acetyltransferase, establishing a direct connection between transcriptional activation and histone acetylation. This breakthrough was soon followed by the purification of a protein complex that contains Gcn5, the SAGA complex. In this article, we review the early genetic and biochemical experiments that led to the discovery of SAGA and the elucidation of its multiple activities.

Expression, purification, and characterization of recombinant human pancreatic duodenal homeobox-1 protein in Pichia pastoris.

Pancreatic duodenal hemeobox-1 (PDX1) is essential for the development of the embryonic pancreas and plays a key role in pancreatic beta-cell differentiation, maturation, regeneration, and maintenance of normal pancreatic beta-cell insulin-producing function. Purified recombinant PDX1 (rPDX1) may be a useful tool for many research and clinical applications, however, using the Escherichia coli expression system has several drawbacks for producing quality PDX1 protein. To explore the yeast expression system for generating rPDX1 protein, the cDNA coding for the full-length human PDX1 gene was cloned into the secreting expression organism Pichia pastoris. SDS-PAGE and western blotting analysis of culture medium from methanol-induced expression yeast clones demonstrated that the rPDX1 was secreted into the culture medium, had a molecular weight by SDS-PAGE of 50kDa, and was glycosylated. The predicted size of the mature unmodified PDX1 polypeptide is 31kDa, suggesting that eukaryotic post-translational modifications are the result of the increased molecular weight. The recombinant protein was purified to greater than 95% purity using a combined ammonium sulfate precipitation with heparin-agarose chromatography. Finally, 120mug of the protein was obtained in high purity from 1L of the culture supernatant. Bioactivity of the rPDX1 was confirmed by the ability to penetrate cell membranes and activation of an insulin-luciferase reporter gene. Our results suggest that the P. pastoris expression system can be used to produce a fully functional human rPDX1 for both research and clinical application.

Isolation and characterization of a novel zinc finger gene, ZNFD, activating AP1(PMA) transcriptional activities.

ZFPs (Zinc Finger Proteins) play important roles in various cellular functions, including transcriptional activation, transcriptional repression, cell proliferation, and development. C(2)H(2) (Cys-Cys-His-His motif) ZFPs are the most abundant proteins among the founding members of the ZFP super family in eukaryotes. In this study, we isolate a novel C(2)H(2) ZNF (Zinc Finger) gene ZNFD. It contains an ORF (Open Reading Frame) with a length of 990 bp, encoding 329 amino acids. The predicted protein contains a C(2)H(2) zinc finger. RT-PCR analysis in 18 human adult tissues indicated that it was expressed in five human adult tissues. Green fluorescence protein localization analysis showed that human ZNFD was located in the nucleus of Hela cells. Overexpression of ZNFD in the COS7 cells activates the transcriptional activities of AP1(PMA) (Activator of protein 1, that responds specifically to phorbol ester). Together the data indicate that ZNFD is probably a new type of C(2)H(2) ZFP and the ZNFD protein may act as a transcriptional activator in PKC (protein kinase C) signal pathway to mediate cellular functions.