Small-fragment genomic libraries for the display of putative epitopes from clinically significant pathogens.

Taking advantage of whole genome sequences of bacterial pathogens in many thriving diseases with global impact, we developed a comprehensive screening procedure for the identification of putative vaccine candidate antigens. Importantly, this procedure relies on highly representative small-fragment genomic libraries that are expressed to display frame-selected epitope-size peptides on a bacterial cell surface and to interact directly with carefully selected disease-relevant high-titer sera. Here we describe the generation of small-fragment genomic libraries of Gram-positive and Gram-negative clinically significant pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae, Enterococcus faecalis, Helicobacter pylori, Chlamydia pneumoniae, the enterotoxigenic Escherichia coli, and Campylobacter jejuni. Large-scale sequencing revealed that the libraries, which provide an average of 20-fold coverage, were random and, as demonstrated with two S. aureus libraries, highly representative. Consistent with the comprehensive nature of this approach is the identification of epitopes that reside in both annotated and putatively novel open reading frames. The use of these libraries therefore allows for the rapid and direct identification of immunogenic epitopes with no apparent bias or difficulty that often associate with conventional expression methods.

Analysis of sequence-specific binding of RNA to Hsp70 and its various homologs indicates the involvement of N- and C-terminal interactions.

Members of the 70-kDa family of molecular chaperones assist in a number of molecular interactions that are essential under both normal and stress conditions. These functions require ATP and co-chaperone molecules and are associated with a cyclic transition of intramolecular conformational changes. As a new putative function, we have previously shown that mammalian Hsp/Hsc70 as well as a distant relative, Hsp110, selectively bind certain RNA sequences via their N-terminal ATP-binding domain. To investigate this phenomenon in more detail, here we examined RNA-binding affinity and specificity of various deletion mutants of human Hsp70. We demonstrate, that, although the N-terminal ATPase domain alone is sufficient for RNA binding, its binding affinity is considerably reduced when compared to that of the full-length protein. Additionally, we provide evidence that binding of RNA to a membrane-immobilized protein partner results in complete loss of RNA sequence specificity. Using various Hsp70 homologs, we show distinct RNA-binding properties of these proteins judged by sequence specificity, ribopolymer sensitivity, and northwestern analysis. Finally, we present data disclosing that RNA binding by DnaK, the Escherichia coli homolog, is influenced by the activity of its co-chaperones, DnaJ and GrpE. We conclude that the RNA-binding capability of this class of molecular chaperones is a conserved feature and it is strongly influenced by the structural and conformational properties. Furthermore, the notion that RNA binding of some Hsp70 family members is influenced by co-chaperones suggests an RNA-binding cycle resembling the protein-binding property of the chaperones.

Nonsense-mediated decay of mRNA for the selenoprotein phospholipid hydroperoxide glutathione peroxidase is detectable in cultured cells but masked or inhibited in rat tissues.

Previous studies of mRNA for classical glutathione peroxidase 1 (GPx1) demonstrated that hepatocytes of rats fed a selenium-deficient diet have less cytoplasmic GPx1 mRNA than hepatocytes of rats fed a selenium-adequate diet. This is because GPx1 mRNA is degraded by the surveillance pathway called nonsense-mediated mRNA decay (NMD) when the selenocysteine codon is recognized as nonsense. Here, we examine the mechanism by which the abundance of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, another selenocysteine-encoding mRNA, fails to decrease in the hepatocytes and testicular cells of rats fed a selenium-deficient diet. We demonstrate with cultured NIH3T3 fibroblasts or H35 hepatocytes transiently transfected with PHGPx gene variants under selenium-supplemented or selenium-deficient conditions that PHGPx mRNA is, in fact, a substrate for NMD when the selenocysteine codon is recognized as nonsense. We also demonstrate that the endogenous PHGPx mRNA of untransfected H35 cells is subject to NMD. The failure of previous reports to detect the NMD of PHGPx mRNA in cultured cells is likely attributable to the expression of PHGPx cDNA rather than the PHGPx gene. We conclude that 1) the sequence of the PHGPx gene is adequate to support the NMD of product mRNA, and 2) there is a mechanism in liver and testis but not cultured fibroblasts and hepatocytes that precludes or masks the NMD of PHGPx mRNA.

Differentiation-dependent cytoplasmic distribution and in vivo RNA association of proteins recognized by the 3'-UTR stability element of alpha-globin mRNA in erythroleukemic cells.

In this study, we analyzed subcytoplasmic distribution and in vivo RNA association of proteins with specific affinity to cytosine-rich stability determinant sequences of alpha-globin mRNA 3'-UTR in a MEL-707 erythroleukemic model. We took advantage of the possibility that these cells can be reversibly differentiated (as a continuous population, but not at the level of individual cells) which, therefore, allows analysis of various stages of erythroid differentiation within the same cell population. Label transfer experiments revealed four major complexes with molecular mass of 110-, 70-, 55- and 50-kDa in various cytoplasmic fractions. Using the combination of in vitro label transfer and in vivo UV-crosslinking techniques, we also demonstrated that subcytoplasmic distribution as well as in vivo RNA association of various complex-forming proteins is differentiation dependent in MEL-707 cells. These results indicate that changes in the cytoplasmic environment imposed by the differentiating stimulus might direct important biochemical signals as to how the stability determinant 3'UTR elements interact with their binding proteins. These data also suggest that stability complexes are dynamic macromolecular structures with high response capacity to various extra- and intracellular regulatory stimuli.

Identification of the NAD(+)-binding fold of glyceraldehyde-3-phosphate dehydrogenase as a novel RNA-binding domain.

There is growing evidence that metabolic enzymes may act as multifunctional proteins performing diverse roles in cellular metabolism. Among these functions are the RNA-binding activities of NAD(+)-dependent dehydrogenases. Previously, we have characterized the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an RNA-binding protein with preference to adenine-uracil-rich sequences. In this study, we used GST-GAPDH fusion proteins generated by deletion mutagenesis to search for the RNA binding domain. We established that the N-terminal 43 amino acid residues of GAPDH, which correspond to the first mononucleotide-binding domain of the NAD(+)-binding fold is sufficient to confer RNA-binding. We also provide evidence that this single domain, although it retains most of the RNA-binding activity, loses sequence specificity. Our results suggest a molecular basis for RNA-recognition by NAD(+)-dependent dehydrogenases and (di)nucleotide-binding metabolic enzymes that had been reported to have RNA-binding activity with different specificity. To support this prediction we also identified other members of the family of NAD(+)-dependent dehydrogenases with no previous history of nucleic acid binding as RNA binding proteins in vitro. Based on our findings we propose the addition of the NAD(+)-binding domain to the list of RNA binding domains/motifs.

Cytoplasmic vacuolation, adaptation and cell death: a view on new perspectives and features.

The review focuses on a widely-observed morphological phenomenon, a unique class of cytoplasmic vacuolation, found in cultured (mammalian) cells. This vacuolation is quite distinct from autophagosomal and heterophagosomal, i.e. excessive lysosomal vacuolation, and occurs in most cell types spontaneously or via a wide range of inductive stimuli. Apart from vacuolation arising artefactually (usually due to poor fixation), spontaneous vacuolation occurs in individual or small clusters of cultured cells without apparent change in their local environment, while neighbouring cells remain completely unaffected. Since spontaneous vacuolation is unpredictable, the process of vacuolation--or 'vacuolisation'--('Vacuolation' is the state of being with vacuoles; 'vacuolisation' therefore implies the process of becoming vacuolated. However, only the quicker term vacuolation will be used throughout this review to refer to the process of vacuole development.) induced experimentally, and hence relatively reproducibly by a range of substances and disturbances, offers an experimental approach which should give further insight into its physiology and pathophysiology. Unfortunately, our knowledge here remains woefully inadequate compared with the purely morphological aspects of the phenomenon. Vacuolation following disturbances could have an underlying common mechanism; however, a review of the literature suggests that this is not the case, and that it occurs via several different pathways, involving many different cell organelles and structures. All cells appear to retain the capacity to vacuolate for some physiological purpose, and it can be a permanent feature in many cell types, particularly 'lower' organisms and plants. Vacuolation in cells is generally seen as an adaptive physiological response, presumably for 'damage limitation', but very little is known about the intracellular homeostatic mechanisms which operate to restore the status quo. Where damage limitation fails, cells usually die quickly, but no clear evidence has been found that this is in any way 'programmed'. It is argued that the demise which occurs via the vacuolation route may, in fact, be a distinct form of cell death which is difficult to fit into the conventional lytic and apoptotic modes.

Mammalian Hsp70 and Hsp110 proteins bind to RNA motifs involved in mRNA stability.

In this study, in vitro RNA binding by members of the mammalian 70-kDa heat shock protein (Hsp) family was examined. We show that Hsp/Hsc70 and Hsp110 proteins preferentially bound AU-rich RNA in vitro. Inhibition of RNA binding by ATP suggested the involvement of the N-terminal ATP-binding domain. By using deletion mutants of Hsp110 protein, a diverged Hsp70 family member, RNA binding was localized to the N-terminal ATP-binding domain of the molecule. The C-terminal peptide-binding domain did not bind RNA, but its engagement by a peptide substrate abrogated RNA binding by the N terminus of the protein. Interestingly, removal of the C-terminal alpha-helical structure or the alpha-loop domain unique to Hsp110 immediately downstream of the peptide-binding domain, but not both, resulted in considerably increased RNA binding as compared with the wild type protein. Finally, a 70-kDa activity was immunoprecipitated from RNA-protein complexes formed in vitro between cytoplasmic proteins of human lymphocytes and AU-rich RNA. These findings support the idea that certain heat shock proteins may act as RNA-binding entities in vivo to guide the appropriate folding of RNA substrates for subsequent regulatory processes such as mRNA degradation and/or translation.

Microfilament-dependent modulation of cytoplasmic protein binding to TNFalpha mRNA AU-rich instability element in human lymphoid cells.

Cytoplasmic proteins with binding capability to AU-rich instability determinant sequences (ARE) of tumour necrosis factor alpha (TNFalpha) mRNA 3' untranslated region (3'UTR) were assessed in human lymphoid cells. In vitro label transfer experiments using wild type as well as mutant sequences in which the 70 nucleotide-long AUUUA pentamer-containing portion of the 3'UTR had been deleted conferred binding specificity to five major activities of 22/25-, 38/40-, 50-, 60- and 80-kDa proteins in cytoplasmic extracts of peripheral blood mononuclear cells (PBMCs). Cytochalasin-induced disarrangement of the F-actin-based microfilament system led to a Triton X-100-insoluble to soluble redistribution of these binding activities. No such changes were observed in Jurkat tumour cells. Combination of in vivo UV-crosslinking and in vitro label transfer experiments revealed considerable differences in RNA association between proteins of the same cell type as well as between proteins of identical molecular weight (Mw) derived from either PBMCs or Jurkat cells. Our findings may explain some aspects of differential regulation of interleukin 2 (IL-2) and TNFalpha mRNA stability upon microfilament disruption in human PBMCs observed in an earlier study. These results also suggest that the physical state of cytoplasmic structural environment might contribute to important regulatory processes regarding key elements of eukaryotic mRNA metabolism, such as modulation of stability. Finally, these data highlight the possibility that the often observed disorganization of the cytoskeleton in tumour cells may partly be responsible for the maintenance of the neoplastic state, a phenomenon that potentially involves ARE-AUBP interactions.

Distribution profile and in vivo RNA association of cytoplasmic AU-rich sequence binding proteins in various mammalian cells: effect of the organizational state of cellular architecture.

With the aid of sequential detergent fractionation and label transfer techniques, in this study we monitored the distribution of cytoplasmic AU-rich sequence binding proteins (AUBP) in different cell types. We show here that cells of various origin display diverse AUBP profiles when the presence and abundance of AUBPs were compared in two major cytoplasmic compartments, the non-ionic detergent-soluble and -insoluble fractions. We also demonstrate that different RNA probes derived from AU-rich 3'-UTR elements of various cytokine and proto-oncogene mRNAs detect distinct AUBPs within the same cell. When the in vivo association of these proteins with AU-rich RNA was assessed using the combination of in vivo UV-crosslinking and label transfer methods, we found that detachment of monolayer cells by trypsinization or disruption of the microfilament network by Cytochalasin treatment prior to UV light exposure, led to selective changes in both the cytoplasmic distribution and in vivo RNA association of many AUBPs. These data indicate that the organizational state of cytoarchitecture may influence processes involved in AUBP-mediated mRNA metabolism.

Interaction of AU-rich sequence binding proteins with actin: possible involvement of the actin cytoskeleton in lymphokine mRNA turnover.

In the current study, we report that cytochalasin-induced disruption of microfilaments stabilizes lymphokine mRNAs in activated human peripheral blood lymphocytes. Parallel with this, a dose- and time-dependent increase in AU-rich sequence binding protein (AUPB) activities is apparent in the nonionic detergent-resistant fractions of these cells, suggesting that cytochalasin-induced modulation of lymphokine mRNA stability might be mediated through cytoplasmic AUBPs. We provide evidence that some of the AUBPs can be immunoprecipitated with anti-actin antibodies, implicating the potential of these proteins to associate with the actin-based cytoskeleton in vivo. Moreover, disruption of the microfilament network by cytochalasins produces increased immunoprecipitable actin-AUBP complexes in the detergent-resistant cytoplasmic subfractions of lymphocytes. We show that cytochalasin-induced changes in AUBP activities are parallel with their higher binding affinity to RNA containing AU-rich instability sequence element as judged by in vitro competition and in vivo ultraviolet-crosslinking analysis. Correlation of these findings with changes in mRNA stability indicates that the actin cytoskeleton may play a physiologically important role in posttranscriptional regulation of lymphokine gene expression during early lymphocyte activation.

Vacuolar cytoplasmic phase separation in cultured mammalian cells involves the microfilament network and reduces motional properties of intracellular water.

Hep-2, human epithelial carcinoma cells, and human foreskin fibroblasts (FF9 and FF13) were exposed to either an ultrafiltrate (< 50 kD) of human sera or the weak base, procaine hydrochloride, to induce reversible cytoplasmic vacuolization. The formation of vacuoles was shown not to be due to imbibition of medium. Ultrastructural details obtained from various stages of vacuole formation were compared. In both cases of induction vacuoles were irregular and often appeared membraneless, with little in the way of electron-dense content. They started to form in the perinuclear cytoplasm and progressed towards the periphery. Osmotic stress was not involved since mitochondria remained normal throughout a vacuolization episode. Vacuoles were often seen in close contact with filamentous structures, and this association remained detectable at late stages of the phenomenon. Fluorescent visualization of F-actin confirmed that the vacuoles were frequently bordered by microfilaments. No major metabolic impairment was apparent in vacuolized cells as judged by protein synthesis measurements, but nuclear fluorescence (DNA content) and forward light scatter (nuclear volume) by flow cytometric analysis suggested late S phase and G2 retardation. 1H-nmr relaxation measurements indicated intracellular water restricted in motional characteristics in vacuolized cells. The possibility of a restricted cytoplasmic phase separation as part of a transient adaptation response is raised, and a hypothesis to explain the findings is discussed.

Combined application of in vivo UV-crosslinking and in vitro label transfer in the examination of AU-rich sequence binding protein-RNA interactions.

A combination of in vivo UV light-induced crosslinking of nucleic acids to proteins and in vitro label transfer assay was applied to investigate specific interactions between AU-rich sequences (ARE) in the 3' UTR of lymphokine mRNAs and cytoplasmic AU-rich sequence element binding proteins (AUBP) in normal human lymphoblasts and MLA 144 gibbon lymphoid tumor cells. We demonstrate that a pool of cytoplasmic AUBP can be effectively crosslinked to RNA in vivo, suggesting a close association of these proteins with ARE sequences in the cytoplasm. We also show that the UV-crosslinked AUBP pool is markedly reduced in malignantly transformed MLA 144 cells compared with normal lymphoblasts, indicating weaker interaction between lymphokine ARE and AUBP in these tumor cells. Similar differences in AUBP-RNA associations were found between the membrane-bound polysomal subfractions of the two cell types where most of the AUBP activity was localized. We suggest that the decreased AUBP-mRNA association in MLA 144 cells might reflect a process concerned with disturbances of mRNA metabolism in the neoplastic phenotype.

Selective modulation of IFN-gamma mRNA stability by IL-12/NKSF.

We investigated the role of IL-12 in regulating IL-2 and IFN-gamma production in primary culture of human T cells. Addition of neutralizing antiserum against the 40-kDa subunit of IL-12 to PHA-stimulated PBMC markedly reduced both IFN-gamma protein production and mRNA accumulation and stability. Moreover, concurrent treatment of partially purified T cells (> 90% CD3+) with PHA and rIL-12 selectively enhanced IFN-gamma mRNA stability and protein production, while IL-2 protein and mRNA levels were unaffected. These studies also show that IFN-gamma and IL-2 mRNA stability are temporally dissociated during the course of T cell activation, and we propose that this dissociation may be mediated through the production of IL-12. The effect of IL-12 on modulation of IFN-gamma mRNA turnover is not associated with detectable changes in either the levels or affinity of cytoplasmic RNA-binding proteins capable of recognizing AU-rich sequences in the 3'UTR of IFN-gamma mRNA.

Enhanced stability of interleukin-2 mRNA in MLA 144 cells. Possible role of cytoplasmic AU-rich sequence-binding proteins.

The MLA 144 gibbon T cell line is infected with a type C retrovirus and constitutively expresses interleukin-2 (IL-2) and granulocyte macrophage colony-stimulating factor (GM-CSF). IL-2 mRNA levels are 10-fold more abundant than GM-CSF in these cells. Comparable transcriptional rates for these lymphokines suggested the involvement of post-transcriptional mechanisms in selective IL-2 mRNA accumulation. IL-2 mRNA is exceptionally stable in MLA cells with a t1/2 of more than 8 h. The presence of reiterated AUUUA sequences in the 3'-untranslated region (UTR) has been shown to confer mRNA lability. The provirally altered MLA IL-2 allele encodes an mRNA in which three AUUUA motifs have been deleted. Six major cytoplasmic proteins bound in vitro transcribed RNA probes containing sequences from the 3'-UTR of normal human IL-2 (3'-IL-2), GM-CSF (delta 2R1), and the virally altered MLA IL-2 (3'-IL-2 PV) mRNA. Increased binding of these proteins to 3'-IL-2 PV was observed relative to 3'-IL-2 or delta 2R1. Northwestern blotting demonstrated similar differential ability of a 36- and 43-kDa protein to bind, as well as showed that these proteins colocalized by immunoblotting as hnRNP A1 and C, respectively. These findings suggest a direct correlation between differential binding of cytoplasmic proteins to AU-rich 3'-UTRs in vitro and lymphokine mRNA stability in vivo.

Transient cytoplasmic vacuolization in cultured normal and neoplastic cells treated with low molecular weight human serum ultrafiltrate: is out inside?

Human embryonal fibroblasts (HEF) and epithelial carcinoma cells (HEp-2) were exposed to low molecular weight (LMW) fractions (M(R) < 50-kDa) of human serum, generated by ultrafiltration. A prominent change in cytoplasmic morphology was detected by microscopic analysis of both cell types. Within 1-3 h of incubation an extensive vacuolization was induced, which phenotype was maintained for 6-12 h in the presence of the LMW filtrate in a cell type dependent manner, followed by a spontaneous reconstitution of the original cytoplasmic morphology by 24 h. Reversibility occurred by incubation in medium without filtered serum fractions. Transmission electron microscopy revealed vesicles without continuous bordering membrane or electron dense interna and demonstrated vacuoles in close contact with filamentous structures. Taken together the light- and electron microscopic characteristics of the observed vacuolization, potential interpretations of our results are discussed in the context of general cytoplasmic organization. Rearrangement of the cytoplasmic protein-water system as a consequence of an inductive effect of serum filtrates is proposed.