Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells.

Gene transfer in eukaryotic cells and organisms suffers from epigenetic effects that result in low or unstable transgene expression and high clonal variability. Use of epigenetic regulators such as matrix attachment regions (MARs) is a promising approach to alleviate such unwanted effects. Dissection of a known MAR allowed the identification of sequence motifs that mediate elevated transgene expression. Bioinformatics analysis implied that these motifs adopt a curved DNA structure that positions nucleosomes and binds specific transcription factors. From these observations, we computed putative MARs from the human genome. Cloning of several predicted MARs indicated that they are much more potent than the previously known element, boosting the expression of recombinant proteins from cultured cells as well as mediating high and sustained expression in mice. Thus we computationally identified potent epigenetic regulators, opening new strategies toward high and stable transgene expression for research, therapeutic production or gene-based therapies.

Quantification of Proteins and Cells: Luminometric Nonspecific Particle-Based Methods

New luminometric particle-based methods were developed to quantify protein and to count cells. The developed methods rely on the interaction of the sample with nano- or microparticles and different principles of detection. In fluorescence quenching, timeresolved luminescence resonance energy transfer (TR-LRET), and two-photon excitation fluorescence (TPX) methods, the sample prevents the adsorption of labeled protein to the particles. Depending on the system, the addition of the analyte increases or decreases the luminescence. In the dissociation method, the adsorbed protein protects the Eu(III) chelate on the surface of the particles from dissociation at a low pH. The experimental setups are user-friendly and rapid and do not require hazardous test compounds and elevated temperatures. The sensitivity of the quantification of protein (from 40 to 500 pg bovine serum albumin in a sample) was 20-500-fold better than in most sensitive commercial methods. The quenching method exhibited low protein-to-protein variability and the dissociation method insensitivity to the assay contaminants commonly found in biological samples. Less than ten eukaryotic cells were detected and quantified with all the developed methods under optimized assay conditions. Furthermore, two applications, the method for detection of the aggregation of protein and the cell viability test, were developed by utilizing the TR-LRET method. The detection of the aggregation of protein was allowed at a more than 10,000 times lower concentration, 30 μg/L, compared to the known methods of UV240 absorbance and dynamic light scattering. The TR-LRET method was combined with a nucleic acid assay with cell-impermeable dye to measure the percentage of dead cells in a single tube test with cell counts below 1000 cells/tube.

The eukaryotic Pso2/Snm1/Artemis proteins and their function as genomic and cellular caretakers

DNA double-strand breaks (DSBs) represent a major threat to the genomic stability of eukaryotic cells. DNA repair mechanisms such as non-homologous end joining (NHEJ) are responsible for the maintenance of eukaryotic genomes. Dysfunction of one or more of the many protein complexes that function in NHEJ can lead to sensitivity to DNA damaging agents, apoptosis, genomic instability, and severe combined immunodeficiency. One protein, Pso2p, was shown to participate in the repair of DSBs induced by DNA inter-strand cross-linking (ICL) agents such as cisplatin, nitrogen mustard or photo-activated bi-functional psoralens. The molecular function of Pso2p in DNA repair is unknown, but yeast and mammalian cell line mutants for PSO2 show the same cellular responses as strains with defects in NHEJ, e.g., sensitivity to ICLs and apoptosis. The Pso2p human homologue Artemis participates in V(D)J recombination. Mutations in Artemis induce a variety of immunological deficiencies, a predisposition to lymphomas, and an increase in chromosomal aberrations. In order to better understand the role of Pso2p in the repair of DSBs generated as repair intermediates of ICLs, an in silico approach was used to characterize the catalytic domain of Pso2p, which led to identification of novel Pso2p homologues in other organisms. Moreover, we found the catalytic core of Pso2p fused to different domains. In plants, a specific ATP-dependent DNA ligase I contains the catalytic core of Pso2p, constituting a new DNA ligase family, which was named LIG6. The possible functions of Pso2p/Artemis/Lig6p in NHEJ and V(D)J recombination and in other cellular metabolic reactions are discussed.

B cells expressing IL-10 mRNA modulate memory T cells after DNA-Hsp65 immunization

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection

Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira. (C) 2008 Elsevier Ltd. All rights reserved.

Cupiennin 1a exhibits a remarkably broad, non-stereospecific cytolytic activity on bacteria, protozoan parasites, insects, and human cancer cells

Cupiennin 1a, a cytolytic peptide isolated from the venom of the spider Cupiennius salei, exhibits broad membranolytic activity towards bacteria, trypanosomes, and plasmodia, as well as human blood and cancer cells. In analysing the cytolytic activity of synthesised all-d- and all-l-cupiennin 1a towards pro- and eukaryotic cells, a stereospecific mode of membrane destruction could be excluded. The importance of negatively charged sialic acids on the outer leaflet of erythrocytes for the binding and haemolytic activity of l-cupiennin 1a was demonstrated. Reducing the overall negative charges of erythrocytes by partially removing their sialic acids or by protecting them with tri- or pentalysine results in reduced haemolytic activity of the peptide.

A global view of Staphylococcus aureus whole genome expression upon internalization in human epithelial cells

BACKGROUND: Staphylococcus aureus, a leading cause of chronic or acute infections, is traditionally considered an extracellular pathogen despite repeated reports of S. aureus internalization by a variety of non-myeloid cells in vitro. This property potentially contributes to bacterial persistence, protection from antibiotics and evasion of immune defenses. Mechanisms contributing to internalization have been partly elucidated, but bacterial processes triggered intracellularly are largely unknown. RESULTS: We have developed an in vitro model using human lung epithelial cells that shows intracellular bacterial persistence for up to 2 weeks. Using an original approach we successfully collected and amplified low amounts of bacterial RNA recovered from infected eukaryotic cells. Transcriptomic analysis using an oligoarray covering the whole S. aureus genome was performed at two post-internalization times and compared to gene expression of non-internalized bacteria. No signs of cellular death were observed after prolonged internalization of Staphylococcus aureus 6850 in epithelial cells. Following internalization, extensive alterations of bacterial gene expression were observed. Whereas major metabolic pathways including cell division, nutrient transport and regulatory processes were drastically down-regulated, numerous genes involved in iron scavenging and virulence were up-regulated. This initial adaptation was followed by a transcriptional increase in several metabolic functions. However, expression of several toxin genes known to affect host cell integrity appeared strictly limited. CONCLUSION: These molecular insights correlated with phenotypic observations and demonstrated that S. aureus modulates gene expression at early times post infection to promote survival. Staphylococcus aureus appears adapted to intracellular survival in non-phagocytic cells.

Colostrogenesis: candidate genes for IgG1 transcytosis mechanisms in primary bovine mammary epithelial cells.

Bovine colostrogenesis is distinguished by the specific transfer of IgG1 from the blood to mammary secretions. The process has been shown to be initiated by hormones and occurs during the last weeks of pregnancy when steroid concentrations of estradiol (E2 ) and progesterone (P4 ) are highly elevated. Rodent intestinal uptake of immunoglobulin G is mediated by a receptor termed Fc fragment of IgG, Receptor, Transporter, alpha (FcGRT) and supported by light chain Beta-2-Microglobulin (β2M). We hypothesized that steroid hormone treatments (E2 and P4 ) of bovine mammary epithelial cells in vitro would induce up-regulation of IgG1 transcytosis candidate gene mRNA expression suggesting involvement in IgG1 transcytosis. Two different primary bovine mammary epithelial cell cultures were cultured on plastic and rat tail collagen and treated with hormonal combinations (steroids/lactogenic hormones). Evaluated mRNA components were bLactoferrin (bLf: a control), bFcGRT, β2M, and various small GTPases; the latter components are reported to direct endosomal movements in eukaryotic cells. All tested transcytosis components showed strong expression of mRNA in the cells. Expression of bFcGRT, bRab25 and bRhoB were significantly up-regulated (p < 0.05) by steroid hormones. bRab25 and bRhoB showed increased expression by steroid treatments, but also with lactogenic hormones. Analysis for the oestrogen receptor (ER) mRNA was mostly negative, but 25% of the cultures tested exhibited weak expression, while the progesterone receptor (PR) mRNA was always detected. bRab25 and bRhoB and likely bFcGRT are potential candidate genes for IgG1 transcytosis in bovine mammary cells.

Analysis of the mechanisms that maintain coordinate production of $\alpha$- and $\beta$-tubulin in mammalian cells

Alpha and beta tubulin are essential proteins in all eukaryotic cells. To study how cells maintain coordinate levels of these two interacting proteins, we have used PCR to add a 9 amino acid epitope from influenza hemagglutinin protein onto the carboxyl terminus of $\alpha$1 and $\beta$1-tubulin. The chimeric tubulin genes (HA$\alpha$1 and HA$\beta$1) were transfected into CHO cells and cell lines that stably express each gene were selected. Cells transfected with HA-tubulin do not exhibit any gross changes in growth or morphology. Immunofluorescence analysis demonstrated that HA-tubulins incorporate into both cytoplasmic and spindle microtubules. A quantitative biochemical assay was used to show that HA-tubulins incorporate into microtubules to a normal extent and do not alter the steady state distribution of endogenous tubulin between monomer and polymer pools. Two-dimensional gel analysis of pulse-labeled cells indicated that when HA$\beta$1-tubulin is expressed at high levels, it slightly represses the synthesis of the endogenous $\beta$-tubulin but produces a small increase in the synthesis of $\alpha$-tubulin. Analysis of cells labeled to steady state showed that HA$\beta$1-tubulin accumulates to a similar level as the wild-type gene product, but together these polypeptides produce only a small increase in total tubulin content consistent with the increased synthesis of $\alpha$-tubulin. It thus appears that HA$\beta$1-tubulin successfully competes with endogenous $\beta$-tubulin for heterodimer formation and that free $\beta$-tubulin subunits (endogenous and HA$\beta$1) are selectively degraded to maintain coordinate amounts of $\alpha$- and $\beta$-tubulin. In addition, the increased synthesis of $\alpha$-tubulin suggested the existence of a mechanism to ensure coordinate synthesis of $\alpha$- and $\beta$-tubulin subunits. To analyze whether reciprocal changes in endogenous tubulin synthesis occur when $\alpha$-tubulin is overexpressed, stably transfected CHO cell lines were isolated in which HA$\alpha$1-tubulin represents 50% of the total $\alpha$-tubulin, and its relative abundance can be further increased to 85-90% by treatment with sodium butyrate. In contrast with results obtained using HA$\beta$1-tubulin, transfection of HA$\alpha$1-tubulin decreased the synthesis of endogenous $\alpha$-tubulin to 60% of normal with little or no change in $\beta$-tubulin synthesis. When the transfected cells were treated with sodium butyrate to further increase HA$\beta$1-tubulin production, a larger decrease in the synthesis of endogenous $\alpha$-tubulin (to 30% of normal) was observed. The repression on the synthesis of endogenous $\alpha$-tubulin polypeptide was found to be directly proportional to the expression of HA$\alpha$1-tubulin indicating the existence of an autoregulatory loop, where $\alpha$-tubulin inhibits its own synthesis. To determine whether overproduction of HA$\alpha$1-tubulin affected the transcription, message stability or translation of endogenous $\alpha$-tubulin, the steady state levels of $\alpha$-tubulin mRNA were analyzed by ribonuclease protection assays. The results showed that the steady state level of $\alpha$-tubulin mRNA is not affected by the overexpression of HA$\alpha$1-tubulin, indicating that the repression is translational. The results are compatible with a model in which $\beta$-tubulin synthesis is largely unperturbed by overexpression of other tubulin subunits, and excess $\beta$-tubulin subunits are rapidly degraded to maintain coordinate $\alpha$- and $\beta$-tubulin levels at steady state. In contrast, free $\alpha$-tubulin represses its own synthesis at the translational level, suggesting that its level of production may be controlled by the amount of $\beta$-tubulin available for heterodimer formation. ^

CTIF is involved in mRNA export in human cells

In eukaryotic cells, translation of messenger RNA (mRNA) can be initiated either on transcripts associated with the cap-binding complex (CBC; consisting of CBP80 and CBP20) or on transcripts with the eukaryotic translation initiation factor (eIF) 4E bound to the cap. Together with eIF4G and eIF4A, eIF4E forms the eIF4F-complex, which mediates translation initiation during the bulk of cellular protein synthesis. Functionally substituting for eIF4G, the CBP80/20-dependent translation initiation factor (CTIF) has been reported to be part of the CBC-dependent translation initiation complex 1,2. CTIF consists of a N-terminal CBP80-binding domain and a conserved C-terminal MIF4G domain 1. This MIF4G domain has been shown to mediate the interaction between CTIF and different factors such as eIF3g and the stem-loop binding protein (SLBP) 2,3. Here we provide evidence that CTIF, besides its function in translation initiation, is also involved in mRNA translocation from the nucleus to the cytoplasm, possibly through a direct interaction with the nuclear export factor NFX1/TAP. Taken together our results suggest that CTIF can function as a platform that interacts with proteins involved in different steps of the mRNA metabolism.

CTIF is involved in mRNA export in human cells

In eukaryotic cells translation initiation of messenger RNA (mRNA) transcripts can be initiated either by the cap-binding complex (CBC) consisting of CBP80 and CBP20, or by the eukaryotic translation initiation factor (eIF) 4E. Together with eIF4G and eIF4A, eIF4E forms the eIF4F-complex, which mediates initiation of the bulk of cellular translation. Analogous to eIF4G, the CBP80/20-dependent translation initiation factor (CTIF) has been reported to be part of the CBC-dependent translation initiation complex. CTIF consists of a N-terminal CBP80-binding domain and a conserved C-terminal MIF4G domain. This MIF4G domain has been shown to mediate the interaction between CTIF and different factors such as eIF3g and the stem-loop binding protein (SLBP). Here we show data indicating that CTIF, besides its function in translation initiation, is involved in mRNA translocation from the nucleus to the cytoplasm, possibly through a direct interaction with the nuclear export factor NFX1/TAP. Taken together our results suggest that CTIF can function as a platform that interacts with proteins involved in different steps of mRNA metabolism.

CTIF is involved in mRNA export in human cells

In eukaryotic cells translation initiation of messenger RNA (mRNA) transcripts can be initiated either by the cap-binding complex (CBC) consisting of CBP80 and CBP20, or by the eukaryotic translation initiation factor (eIF) 4E. Together with eIF4G and eIF4A, eIF4E forms the eIF4F-complex, which mediates translation initiation during the bulk of cellular protein synthesis [1,2]. Functionally analogous to eIF4G, the CBP80/20-dependent translation initiation factor (CTIF) has been reported to be part of the CBC-dependent translation initiation complex [3,4]. CTIF consists of a N-terminal CBP80-binding domain and a conserved C-terminal MIF4G domain [3]. This MIF4G domain has been shown to mediate the interaction between CTIF and different factors such as eIF3g and the stem-loop binding protein (SLBP) [4,5]. Here we show data indicating that CTIF, besides its function in translation initiation, is involved in mRNA translocation from the nucleus to the cytoplasm, possibly through a direct interaction with the nuclear export factor NFX1/TAP. Taken together our results suggest that CTIF can function as a platform that interacts with proteins involved in different steps of the mRNA metabolism. [1] Haghighat A. and Sonenberg N. (1997) JBC 272:21677-21680 [2] Gross J.D. et al. (2003) Cell 115:739-750 [3] Kim K.M. et al. (2009) Genes Dev 23:2033-2045 [4] Choe J. et al. (2012) JBC 287:18500-18509 [5] Choe J. et al. (2013) NAR 41:1307-1318

Invasion and persistence of Mycoplasma bovis in embryonic calf turbinate cells.

Mycoplasma bovis is a wall-less bacterium causing bovine mycoplasmosis, a disease showing a broad range of clinical manifestations in cattle. It leads to enormous economic losses to the beef and dairy industries. Antibiotic treatments are not efficacious and currently no efficient vaccine is available. Moreover, mechanisms of pathogenicity of this bacterium are not clear, as few virulence attributes are known. Microscopic observations of necropsy material suggest the possibility of an intracellular stage of M. bovis. We used a combination of a gentamicin protection assay, a variety of chemical treatments to block mycoplasmas entry in eukaryotic cells, and fluorescence and transmission electron microscopy to investigate the intracellular life of M. bovis in calf turbinate cells. Our findings indicate that M. bovis invades and persists in primary embryonic calf turbinate cells. Moreover, M. bovis can multiply within these cells. The intracellular phase of M. bovis may represent a protective niche for this pathogen and contribute to its escape from the host's immune defense as well as avoidance of antimicrobial agents.

FUNCTIONAL AND BIOCHEMICAL CHARACTERIZATION OF ADHESION DEFECTIVE CHINESE HAMSTER OVARY CELLS

Cell adhesion is an intricate process involving adhesion promoting ligands such as laminin and fibronectin, surface receptors for these ligands and a complex interplay of metabolic and cytoskeletal events (Geiger, BBA 737:305, 1983). Although considerable effort has been directed towards studying adhesion molecules such as fibronectin (Fn), very little is known about the mechanisms regulating the complex process of adhesion.^ I chose to use a CHO adhesion variant clone called AD('v)F11 as a tool to study the various steps which may be involved in adhesion. AD('v)F11 cells unlike wild type (WT), do not adhere to Fn-coated substrata, but will adhere to substrata coated with other extracellular components (Harper and Juliano, J Cell Biol. 91:647, 1981). I have found that although AD('v)F11 cells can bind Fn-coated latex beads to the same extent as WT cells, AD('v)F11 cells also differed from WT cells in that they did not aggregate in the presence of Fn-beads nor internalize Fn-beads. The defect in bead induced cell aggregation and internalization seem to be specific to Fn since lectin coated beads could aggregate AD('v)F11 cells as well as WT cells, and AD('v)F11 cells can also readily internalize lectins. These observations suggest that the defect associated with AD('v)F11 cells is distal to the initial binding to Fn to its cell surface receptor. To further investigate the biochemical defect associated with AD('v)F11 cells, a panel of compounds were examined for their ability to correct the non-adhesive phenotype of AD('v)F11 cells. Among the compounds tested, only those known to increase intracellular cAMP levels were found to be effective in correcting the adhesion defect of F11CA11 cells, a subclone of AD('v)F11 cells.^ Since cAMP effects in eukaryotic cells are mediated through phosphorylation events by the cAMP-dependent protein kinase (cAdPK) system, the phosphorylation pattern and cAdPK system of the F11CA11 cells were analyzed. Comparison between the phosphorylation pattern of intact untreated F11CA11 and WT cells, revealed the presence of a 50 kd phosphoprotein(s) in WT cells but not in F11CA11 cells. Results presented in this dissertation strongly indicate that the adhesion defect in F11CA11 is associated to an altered type I cAdPK that can be corrected by raising intracellular cAMP levels. (Abstract shortened with permission of author.) ^

The eukaryotic cellular stress response: Biochemical and genetic analyses in Saccharomyces cerevisiae

All cells must have the ability to deal with a variety of environmental stresses. Failure to correctly adapt to and/or protect against adverse stress conditions can lead to cell death. In humans, stress response defects have been linked to a number of neurodegenerative diseases and cancer, underscoring the importance of developing a fundamental understanding of the eukaryotic stress response.^ In an effort to characterize cellular response to high temperature stress, I identified and described one member of a novel gene family— RTR1. I show that the RTR1 gene and its protein product genetically and biochemically interact with core subunits of the RNA polymerase II enzyme. Appropriately, loss of RTR1 results in defective transcription from multiple promoters. These data provide evidence that Rtr1, which is essential under stress conditions, acts as a key regulator of transcription.^ In addition to transcriptional regulation, cells deal with many stressors by inducing molecular chaperones. Molecular chaperones are ubiquitous in all living cells and bind unfolded or damaged proteins and catalyze refolding or degradation. Hsp90 is a unique chaperone because it targets specific clients—typically signaling proteins—for maturation. While it has been shown that Sse1, the yeast Hsp110, is a critical regulator of the Hsp90 chaperone cycle, this work describes the molecular basis for that regulation. I show that Sse1 modulates Hsp90 function through regulation of Hsp70 nucleotide exchange. Further, Hsp110-type nucleotide exchange factors (NEFs) appear to have a specific role in modulating Hsp90 function in this manner. Finally, in addition to Hsp110, the eukaryotic cytosol contains two other types of Hsp70 NEF: Snl1 (BAG-domain protein) and Fes1 (HspBP1-like protein). I investigated the cellular roles of these NEFs to better understand the reason that eukaryotic cells contain three distinct protein families that perform the same biochemical function. I show that while cytsolic Hsp70 NEFs have some degree of functional overlap, they also exhibit striking divergence. Taken together, the work presented in this dissertation provides a more detailed understanding of the eukaryotic stress response. ^