Helicobacter pylori Cholesteryl α-Glucosides Contribute to Its Pathogenicity and Immune Response by Natural Killer T Cells

Approximately 10–15% of individuals infected with Helicobacter pylori will develop ulcer disease (gastric or duodenal ulcer), while most people infected with H. pylori will be asymptomatic. The majority of infected individuals remain asymptomatic partly due to the inhibition of synthesis of cholesteryl α-glucosides in H. pylori cell wall by α1,4-GlcNAc-capped mucin O-glycans, which are expressed in the deeper portion of gastric mucosa. However, it has not been determined how cholesteryl α-glucosyltransferase (αCgT), which forms cholesteryl α-glucosides, functions in the pathogenesis of H. pylori infection. Here, we show that the activity of αCgT from H. pylori clinical isolates is highly correlated with the degree of gastric atrophy. We investigated the role of cholesteryl α-glucosides in various aspects of the immune response. Phagocytosis and activation of dendritic cells were observed at similar degrees in the presence of wild-type H. pylori or variants harboring mutant forms of αCgT showing a range of enzymatic activity. However, cholesteryl α-glucosides were recognized by invariant natural killer T (iNKT) cells, eliciting an immune response in vitro and in vivo. Following inoculation of H. pylori harboring highly active αCgT into iNKT cell-deficient (Jα18^−/−) or wild-type mice, bacterial recovery significantly increased in Jα18^−/− compared to wild-type mice. Moreover, cytokine production characteristic of Th1 and Th2 cells dramatically decreased in Jα18^−/− compared to wild-type mice. These findings demonstrate that cholesteryl α-glucosides play critical roles in H. pylori-mediated gastric inflammation and precancerous atrophic gastritis.     

The Alkaloid Compound Harmane Increases the Lifespan of Caenorhabditis elegans during Bacterial Infection,by Modulating the Nematode’s Innate Immune Response

The nematode Caenorhabditis elegans has in recent years been proven to be a powerful in vivo model for testing antimicrobial compounds. We report here that the alkaloid compound Harmane (2-methyl-β-carboline) increases the lifespan of nematodes infected with a human pathogen, the Shiga toxin-producing Escherichia coli O157:H7 strain EDL933 and several other bacterial pathogens. This was shown to be unrelated to the weak antibiotic effect of Harmane. Using GFP-expressing E. coli EDL933, we showed that Harmane does not lower the colonization burden in the nematodes. We also found that the expression of the putative immune effector gene F35E12.5 was up-regulated in response to Harmane treatment. This indicates that Harmane stimulates the innate immune response of the nematode; thereby increasing its lifespan during bacterial infection. Expression of F35E12.5 is predominantly regulated through the p38 MAPK pathway; however, intriguingly the lifespan extension resulting from Harmane was higher in p38 MAPK-deficient nematodes. This indicates that Harmane has a complex effect on the innate immune system of C. elegans. Harmane could therefore be a useful tool in the further research into C. elegans immunity. Since the innate immunity of C. elegans has a high degree of evolutionary conservation, drugs such as Harmane could also be possible alternatives to classic antibiotics. The C. elegans model could prove to be useful for selection and development of such drugs.     

Taking the Escherichia coli TonB Transmembrane Domain “Offline”? Nonprotonatable Asn Substitutes Fully for TonB His20

The TonB system of Gram-negative bacteria uses the proton motive force (PMF) of the cytoplasmic membrane to energize active transport of nutrients across the outer membrane. The single transmembrane domain (TMD) anchor of TonB, the energy transducer, is essential. Within that TMD, His20 is the only TMD residue that is unable to withstand alanine replacement without a loss of activity. H20 is required for a PMF-dependent conformational change, suggesting that the importance of H20 lies in its ability to be reversibly protonated and deprotonated. Here all possible residues were substituted at position 20 (H20X substitutions). The His residue was also relocated throughout the TonB TMD. Surprisingly, Asn, a structurally similar but nonprotonatable residue, supported full activity at position 20; H20S was very weakly active. All the remaining substitutions, including H20K, H20R, H20E, and H20D, the obvious candidates to mimic a protonated state or support proton translocation, were inactive. A second-site suppressor, ExbB(A39E), indiscriminately reactivated the majority of H20 substitutions and relocations, including H20V, which cannot be made protonatable. These results suggested that the TonB TMD was not on a proton conductance pathway and thus only indirectly responds to PMF, probably via ExbD.     

Staphylococcus aureus α-Hemolysin Activates the NLRP3-Inflammasome in Human and Mouse Monocytic Cells

Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal α-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus α-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood. Using highly purified recombinant α-hemolysin, we now demonstrate that α-hemolysin activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3)-inflammasome, a host inflammatory signaling complex involved in responses to pathogens and endogenous danger signals. Non-cytolytic mutant α-hemolysin molecules fail to elicit NLRP3-inflammasome signaling, demonstrating that the responses are not due to non-specific activation of this innate immune signaling system by bacterially derived proteins. In monocyte-derived cells from humans and mice, inflammasome assembly in response to α-hemolysin results in activation of the cysteine proteinase, caspase-1. We also show that inflammasome activation by α-hemolysin works in conjunction with signaling by other CA-MRSA-derived Pathogen Associated Molecular Patterns (PAMPs) to induce secretion of pro-inflammatory cytokines IL-1β and IL-18. Additionally, α-hemolysin induces cell death in these cells through an NLRP3-dependent program of cellular necrosis, resulting in the release of endogenous pro-inflammatory molecules, like the chromatin-associated protein, High-mobility group box 1 (HMGB1). These studies link the activity of a major S. aureus virulence factor to a specific host signaling pathway. The cellular events linked to inflammasome activity have clear relevance to the disease processes associated with CA-MRSA including tissue necrosis and inflammation.     

ssb Gene Duplication Restores the Viability of ΔholC and ΔholD Escherichia coli Mutants

The HolC-HolD (χψ) complex is part of the DNA polymerase III holoenzyme (Pol III HE) clamp-loader. Several lines of evidence indicate that both leading- and lagging-strand synthesis are affected in the absence of this complex. The Escherichia coli ΔholD mutant grows poorly and suppressor mutations that restore growth appear spontaneously. Here we show that duplication of the ssb gene, encoding the single-stranded DNA binding protein (SSB), restores ΔholD mutant growth at all temperatures on both minimal and rich medium. RecFOR-dependent SOS induction, previously shown to occur in the ΔholD mutant, is unaffected by ssb gene duplication, suggesting that lagging-strand synthesis remains perturbed. The C-terminal SSB disordered tail, which interacts with several E. coli repair, recombination and replication proteins, must be intact in both copies of the gene in order to restore normal growth. This suggests that SSB-mediated ΔholD suppression involves interaction with one or more partner proteins. ssb gene duplication also suppresses ΔholC single mutant and ΔholC ΔholD double mutant growth defects, indicating that it bypasses the need for the entire χψ complex. We propose that doubling the amount of SSB stabilizes HolCD-less Pol III HE DNA binding through interactions between SSB and a replisome component, possibly DnaE. Given that SSB binds DNA in vitro via different binding modes depending on experimental conditions, including SSB protein concentration and SSB interactions with partner proteins, our results support the idea that controlling the balance between SSB binding modes is critical for DNA Pol III HE stability in vivo, with important implications for DNA replication and genome stability.     

Unlipidated Outer Membrane Protein Omp16 (U-Omp16) from Brucella spp. as Nasal Adjuvant Induces a Th1 Immune Response and Modulates the Th2 Allergic Response to Cow’s Milk Proteins

The discovery of novel mucosal adjuvants will help to develop new formulations to control infectious and allergic diseases. In this work we demonstrate that U-Omp16 from Brucella spp. delivered by the nasal route (i.n.) induced an inflammatory immune response in bronchoalveolar lavage (BAL) and lung tissues. Nasal co-administration of U-Omp16 with the model antigen (Ag) ovalbumin (OVA) increased the amount of Ag in lung tissues and induced OVA-specific systemic IgG and T helper (Th) 1 immune responses. The usefulness of U-Omp16 was also assessed in a mouse model of food allergy. U-Omp16 i.n. administration during sensitization ameliorated the hypersensitivity responses of sensitized mice upon oral exposure to Cow’s Milk Protein (CMP), decreased clinical signs, reduced anti-CMP IgE serum antibodies and modulated the Th2 response in favor of Th1 immunity. Thus, U-Omp16 could be used as a broad Th1 mucosal adjuvant for different Ag formulations.     

An Important Role of α-Hemolysin in Extracellular Vesicles on the Development of Atopic Dermatitis Induced by Staphylococcus aureus

Skin barrier disruption and dermal inflammation are key phenotypes of atopic dermatitis (AD). Staphylococcus aureus secretes extracellular vesicles (EVs), which are involved in AD pathogenesis. Here, we evaluated the role of EVs-associated α-hemolysin derived from S. aureus in AD pathogenesis. α-hemolysin production from S. aureus was detected using western blot analyses. The cytotoxic activity of α-hemolysin on HaCaT keratinocytes was evaluated by measuring cell viability after treating cells with soluble and EVs-associated α-hemolysin. To determine the type of cell death, HaCaT keratinocytes were stained with annexin V and 7-AAD. The in vivo effects of α-hemolysin were evaluated by application of soluble and EV-associated α-hemolysin on the mouse skin. The present study showed that increased α-hemolysin was produced by S. aureus colonized on AD patients compared to healthy subjects. α-hemolysin production was also related to AD severity. In addition, EV-associated α-hemolysin was more cytotoxic to HaCaT keratinocytes than soluble α-hemolysin, and α-hemolysin-negative EVs did not induce keratinocyte death. EV-associated α-hemolysin induced necrosis, but soluble α-hemolysin induced apoptosis of keratinocytes. In vivo, skin barrier disruption and epidermal hyperplasia were induced by soluble and EV-associated α-hemolysin. However, AD-like dermal inflammation was only caused by EV-associated α-hemolysin. Moreover, neither skin barrier disruption nor AD-like skin inflammation was induced by α-hemolysin-negative EVs. Taken together, α-Hemolysin secreted from S. aureus, particularly the EV-associated form, induces both skin barrier disruption and AD-like skin inflammation, suggesting that EV-associated α-hemolysin is a novel diagnostic and therapeutic target for the control of AD.     

Probing the Conformation of the Fibronectin III1�C2 Domain by Fluorescence Resonance Energy Transfer

Fibronectin (FN) matrix is crucial for cell and tissue functions during embryonic development, wound healing, and oncogenesis. Assembly of FN matrix fibrils requires FN domains that mediate interactions with integrin receptors and with other FN molecules. In addition, regulation of FN matrix assembly depends on the first two FN type III modules, III₁ and III₂, which harbor FN-binding sites. We propose that interactions between these two modules sequester FN-binding sites in soluble FN and that these sites become exposed by FN conformational changes during assembly. To test the idea that III_1–2 has a compact conformation, we constructed CIIIY, a conformational sensor of III_1–2 based on fluorescent resonance energy transfer between cyan and yellow fluorescent proteins conjugated at its N and C termini. We demonstrate energy transfer in CIIIY and show that fluorescent resonance energy transfer was eliminated by proteolysis and by treatment with mild denaturants that disrupted intramolecular interactions between the two modules. We also show that mutations of key charged residues resulted in conformational changes that exposed binding sites for the N-terminal 70-kDa FN fragment. Collectively, these results support a conformation-dependent mechanism for the regulation of FN matrix assembly by III_1–2.Fibronectin (FN)³ is a 500-kDa modular dimeric protein and a major component of the extracellular matrix. It exists in the blood and other body fluids as a soluble compact molecule and undergoes cell-mediated assembly to form an insoluble three-dimensional fibrillar matrix (reviewed in Ref. 1). The process of FN matrix assembly has been implicated in embryonic development, wound healing, and cancer (2–4). FN is composed of type I–III modules, and sets of these modules comprise binding domains for cells and for other extracellular matrix components (see Fig. 1A). Three of these binding domains are essential for matrix assembly (1). Integrin receptor interactions with the cell-binding domain tether disulfide-bonded FN dimers to the cell surface, where FN-FN interactions involving the N-terminal assembly domain form dimers into fibrils. In addition to these essential domains, other FN-binding sites have been implicated in assembly. In particular, the III_1–2 FN-binding domain plays a regulatory role in matrix assembly. Within this domain reside a cryptic FN-binding site in III₁ and a site available for FN binding in the native form of III₂ (5–8). Recombinant FN lacking III₁ is assembled into a matrix at wild-type levels, but that lacking the III_1–2 domain results in short immature FN fibrils (8). Peptides derived from the III_1–2 domain or antibodies against III_1–2 block matrix assembly by cultured cells (9–11). Furthermore, FN binding to this region is enhanced when FN is mechanically stretched (12). Taken together, these results suggest that conformational changes in the III_1–2 domain may control its interactions during FN assembly.Open in a separate windowFIGURE 1.The FN III_1–2 FRET conformational sensor. A, representation of the domain structure of FN and major interaction sites. FN is composed of repeating modules that form binding domains for other FN molecules, cell receptors, and other extracellular matrix components as indicated. The first two type III modules III₁ and III₂ (black), have FN-binding sites and regulate FN matrix assembly. The N-terminal 70-kDa region contains a matrix assembly domain with FN-binding activity. The cell-binding domain (cell), the heparin-binding domain (heparin), the dimerization site (SS), and the alternatively spliced type IIIA (A), IIIB (B), and variable regions (V) are indicated. 70kD, N-terminal 70-kDa FN fragment. B, schematic of proposed model of III_1–2 domain conformation. Panel i, in solution, the FN-binding sites in III₁ and III₂ (hatched areas) are sequestered through domain orientations that are facilitated by the linker between modules (thin line). Panel ii, binding sites are exposed through conformational changes resulting from cell-mediated extension of FN (arrows). The length of the linker and the height and width of the modules are drawn to scale for a linear peptide and published data on FN type III modules, respectively. C, ribbon diagram representation of CIIIY, a FRET sensor of the model in B (panel i), oriented with N and C termini 50 Å apart. CIIIY consists of the III_1–2 domain with CFP at the N terminus and YFP at the C terminus.To more fully understand the roles of native and cryptic FN-binding sites in matrix assembly, the conformational dynamics of III_1–2 must be characterized. One approach to this problem is to tag III_1–2 with fluorescent probes, which, in conjunction with fluorescent resonance energy transfer (FRET), create a molecular conformational sensor. FRET involves the radiationless transfer of energy from an excited donor fluorophore to an acceptor fluorophore, a process that is very sensitive to the distance between the two fluorophores (13–15). Two fluorescent protein variants, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), are highly related to green fluorescent protein (GFP). Because the emission spectrum of CFP is well matched to the excitation spectrum of YFP, these two fluorophores have been widely used as a donor-acceptor pair in FRET studies (13–15).In this study, we describe a FRET conformational sensor designed to test the idea that intramolecular interactions between III₁ and III₂ sequester key FN-binding and assembly sites. We show that III_1–2 with CFP and YFP fused to the N and C termini, respectively, displays a clear FRET signal, indicating that the attached fluorescent proteins and thus the ends of III_1–2 are in close proximity. FRET data from III_1–2 mutants support the presence of a stabilizing intermodule salt bridge that regulates FN-binding activity.     

An Enhanced Immune Response of Mclk1 +/? Mutant Mice Is Associated with Partial Protection from Fibrosis,Cancer and the Development of Biomarkers of Aging

The immune response is essential for survival by destroying microorganisms and pre-cancerous cells. However, inflammation, one aspect of this response, can result in short- and long-term deleterious side-effects. Mclk1^+/− mutant mice can be long-lived despite displaying a hair-trigger inflammatory response and chronically activated macrophages as a result of high mitochondrial ROS generation. Here we ask whether this phenotype is beneficial or simply tolerated. We used models of infection by Salmonella serovars and found that Mclk1^+/− mutants mount a stronger immune response, control bacterial proliferation better, and are resistant to cell and tissue damage resulting from the response, including fibrosis and types of oxidative damage that are considered to be biomarkers of aging. Moreover, these same types of tissue damage were found to be low in untreated 23 months-old mutants. We also examined the initiation of tumour growth after transplantation of mouse LLC1 carcinoma cells into Mclk1^+/− mutants, as well as during spontaneous tumorigenesis in Mclk1^+/−Trp53^+/− double mutants. Tumour latency was increased by the Mclk1^+/− genotype in both models. Furthermore, we used the transplantation model to show that splenic CD8+ T lymphocytes from Mclk1^+/− graft recipients show enhanced cytotoxicity against LLC1 cells in vitro. Mclk1^+/− mutants thus display an association of an enhanced immune response with partial protection from age-dependent processes and from pathologies similar to those that are found with increased frequency during the aging process. This suggests that the immune phenotype of these mutants might contribute to their longevity. We discuss how these findings suggest a broader view of how the immune response might impact the aging process.     

One-Step Biosynthesis of α-Keto-γ-Methylthiobutyric Acid from L-Methionine by an Escherichia coli Whole-Cell Biocatalyst Expressing an Engineered L-Amino Acid Deaminase from Proteus vulgaris

α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl₂, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 10⁴ mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca²⁺ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.     

E-Cadherin–dependent Growth Suppression is Mediated by the Cyclin-dependent Kinase Inhibitor p27KIP1

Recent studies have demonstrated the importance of E-cadherin, a homophilic cell–cell adhesion molecule, in contact inhibition of growth of normal epithelial cells. Many tumor cells also maintain strong intercellular adhesion, and are growth-inhibited by cell– cell contact, especially when grown in three-dimensional culture. To determine if E-cadherin could mediate contact-dependent growth inhibition of nonadherent EMT/6 mouse mammary carcinoma cells that lack E-cadherin, we transfected these cells with an exogenous E-cadherin expression vector. E-cadherin expression in EMT/6 cells resulted in tighter adhesion of multicellular spheroids and a reduced proliferative fraction in three-dimensional culture. In addition to increased cell–cell adhesion, E-cadherin expression also resulted in dephosphorylation of the retinoblastoma protein, an increase in the level of the cyclin-dependent kinase inhibitor p27^kip1 and a late reduction in cyclin D1 protein. Tightly adherent spheroids also showed increased levels of p27 bound to the cyclin E-cdk2 complex, and a reduction in cyclin E-cdk2 activity. Exposure to E-cadherin–neutralizing antibodies in three-dimensional culture simultaneously prevented adhesion and stimulated proliferation of E-cadherin transfectants as well as a panel of human colon, breast, and lung carcinoma cell lines that express functional E-cadherin. To test the importance of p27 in E-cadherin–dependent growth inhibition, we engineered E-cadherin–positive cells to express inducible p27. By forcing expression of p27 levels similar to those observed in aggregated cells, the stimulatory effect of E-cadherin–neutralizing antibodies on proliferation could be inhibited. This study demonstrates that E-cadherin, classically described as an invasion suppressor, is also a major growth suppressor, and its ability to inhibit proliferation involves upregulation of the cyclin-dependent kinase inhibitor p27.     

The Block of DNA Polymerase �� Strand Displacement Activity by an Abasic Site Can Be Rescued by the Concerted Action of DNA Polymerase �� and Flap Endonuclease 1

Abasic (AP) sites are very frequent and dangerous DNA lesions. Their ability to block the advancement of a replication fork has been always viewed as a consequence of their inhibitory effect on the DNA synthetic activity of replicative DNA polymerases (DNA pols). Here we show that AP sites can also affect the strand displacement activity of the lagging strand DNA pol δ, thus preventing proper Okazaki fragment maturation. This block can be overcome through a polymerase switch, involving the combined physical and functional interaction of DNA pol β and Flap endonuclease 1. Our data identify a previously unnoticed deleterious effect of the AP site lesion on normal cell metabolism and suggest the existence of a novel repair pathway that might be important in preventing replication fork stalling.Loss of purine and pyrimidine bases is a significant source of DNA damage in prokaryotic and eukaryotic organisms. Abasic (apurinic and apyrimidinic) lesions occur spontaneously in DNA; in eukaryotes it has been estimated that about 10⁴ depurination and 10² depyrimidation events occur per genome per day. An equally important source of abasic DNA lesions results from the action of DNA glycosylases, such as uracil glycosylase, which excises uracil arising primarily from spontaneous deamination of cytosines (1). Although most AP sites are removed by the base excision repair (BER)⁵ pathway, a small fraction of lesions persists, and DNA with AP lesions presents a strong block to DNA synthesis by replicative DNA polymerases (DNA pols) (2, 3). Several studies have been performed to address the effects of AP sites on the template DNA strand on the synthetic activity of a variety of DNA pols. The major replicative enzyme of eukaryotic cells, DNA pol δ, was shown to be able to bypass an AP lesion, but only in the presence of the auxiliary factor proliferating cell nuclear antigen (PCNA) and at a very reduced catalytic efficiency if compared with an undamaged DNA template (4). On the other hand, the family X DNA pols β and λ were shown to bypass an AP site but in a very mutagenic way (5). Recent genetic evidence in Saccharomyces cerevisiae cells showed that DNA pol δ is the enzyme replicating the lagging strand (6). According to the current model for Okazaki fragment synthesis (7–9), the action of DNA pol δ is not only critical for the extension of the newly synthesized Okazaki fragment but also for the displacement of an RNA/DNA segment of about 30 nucleotides on the pre-existing downstream Okazaki fragment to create an intermediate Flap structure that is the target for the subsequent action of the Dna2 endonuclease and the Flap endonuclease 1 (Fen-1). This process has the advantage of removing the entire RNA/DNA hybrid fragment synthesized by the DNA pol α/primase, potentially containing nucleotide misincorporations caused by the lack of a proofreading exonuclease activity of DNA pol α/primase. This results in a more accurate copy synthesized by DNA pol δ. The intrinsic strand displacement activity of DNA pol δ, in conjunction with Fen-1, PCNA, and replication protein A (RP-A), has been also proposed to be essential for the S phase-specific long patch BER pathway (10, 11). Although it is clear that an AP site on the template strand is a strong block for DNA pol δ-dependent synthesis on single-stranded DNA, the functional consequences of such a lesion on the ability of DNA pol δ to carry on strand displacement synthesis have never been investigated so far. Given the high frequency of spontaneous hydrolysis and/or cytidine deamination events, any detrimental effect of an AP site on the strand displacement activity of DNA pol δ might have important consequences both for lagging strand DNA synthesis and for long patch BER. In this work, we addressed this issue by constructing a series of synthetic gapped DNA templates with a single AP site at different positions with respect to the downstream primer to be displaced by DNA pol δ (see Fig. 1A). We show that an AP site immediately upstream of a single- to double-strand DNA junction constitutes a strong block to the strand displacement activity of DNA pol δ, even in the presence of RP-A and PCNA. Such a block could be resolved only through a “polymerase switch” involving the concerted physical and functional interaction of DNA pol β and Fen-1. The closely related DNA pol λ could only partially substitute for DNA pol β. Based on our data, we propose that stalling of a replication fork by an AP site not only is a consequence of its ability to inhibit nucleotide incorporation by the replicative DNA pols but can also stem from its effects on strand displacement during Okazaki fragment maturation. In summary, our data suggest the existence of a novel repair pathway that might be important in preventing replication fork stalling and identify a previously unnoticed deleterious effect of the AP site lesion on normal cell metabolism.Open in a separate windowFIGURE 1.An abasic site immediately upstream of a double-stranded DNA region inhibits the strand displacement activity of DNA polymerase δ. The reactions were performed as described under “Experimental Procedures.” A, schematic representation of the various DNA templates used. The size of the resulting gaps is indicated in nt. The position of the AP site on the 100-mer template strand is indicated relative to the 3′ end. Base pairs in the vicinity of the lesion are indicated by dashes. The size of the gaps (35–38 nt) is consistent with the size of ssDNA covered by a single RP-A molecule, which has to be released during Okazaki fragment synthesis when the DNA pol is approaching the 5′-end of the downstream fragment. When the AP site is covered by the downstream terminator oligonucleotide (Gap-3 and Gap-1 templates) the nucleotide placed on the opposite strand is C to mimic the situation generated by spontaneous loss of a guanine or excision of an oxidized guanine, whereas when the AP site is covered by the primer (nicked AP template), the nucleotide placed on the opposite strand is A to mimic the most frequent incorporation event occurring opposite an AP site. B, human PCNA was titrated in the presence of 15 nm (lanes 2–4 and 10–12) or 30 nm (lanes 6–8 and 14–16) recombinant human four subunit DNA pol δ, on a linear control (lanes 1–8) or a 38-nt gap control (lanes 9–16) template. Lanes 1, 5, 9, and 13, control reactions in the absence of PCNA. C, human PCNA was titrated in the presence of 60 nm DNA pol δ, on a linear AP (lanes 2–4) or 38-nt gap AP (lanes 6–9) template. Lanes 1 and 5, control reactions in the absence of PCNA.     

FUS-DDIT3 Prevents the Development of Adipocytic Precursors in Liposarcoma by Repressing PPARγ and C/EBPα and Activating eIF4E

Background

FUS-DDIT3 is a chimeric protein generated by the most common chromosomal translocation t(12;16)(q13;p11) linked to liposarcomas, which are characterized by the accumulation of early adipocytic precursors. Current studies indicate that FUS-DDIT3- liposarcoma develops from uncommitted progenitors. However, the precise mechanism whereby FUS-DDIT3 contributes to the differentiation arrest remains to be elucidated.

Methodology/Principal Findings

Here we have characterized the adipocyte regulatory protein network in liposarcomas of FUS-DITT3 transgenic mice and showed that PPARγ2 and C/EBPα expression was altered. Consistent with in vivo data, FUS-DDIT3 MEFs and human liposarcoma cell lines showed a similar downregulation of both PPARγ2 and C/EBPα expression. Complementation studies with PPARγ but not C/EBPα rescued the differentiation block in committed adipocytic precursors expressing FUS-DDIT3. Our results further show that FUS-DDIT3 interferes with the control of initiation of translation by upregulation of the eukaryotic translation initiation factors eIF2 and eIF4E both in FUS-DDIT3 mice and human liposarcomas cell lines, explaining the shift towards the truncated p30 isoform of C/EBPα in liposarcomas. Suppression of the FUS-DDIT3 transgene did rescue this adipocyte differentiation block. Moreover, eIF4E was also strongly upregulated in normal adipose tissue of FUS-DDIT3 transgenic mice, suggesting that overexpression of eIF4E may be a primary event in the initiation of liposarcomas. Reporter assays showed FUS-DDIT3 is involved in the upregulation of eIF4E in liposarcomas and that both domains of the fusion protein are required for affecting eIF4E expression.

Conclusions/Significance

Taken together, this study provides evidence of the molecular mechanisms involve in the disruption of normal adipocyte differentiation program in liposarcoma harbouring the chimeric gene FUS-DDIT3.     

Drosophila TIM Binds Importin α1, and Acts as an Adapter to Transport PER to the Nucleus

Regulated nuclear entry of clock proteins is a conserved feature of eukaryotic circadian clocks and serves to separate the phase of mRNA activation from mRNA repression in the molecular feedback loop. In Drosophila, nuclear entry of the clock proteins, PERIOD (PER) and TIMELESS (TIM), is tightly controlled, and impairments of this process produce profound behavioral phenotypes. We report here that nuclear entry of PER-TIM in clock cells, and consequently behavioral rhythms, require a specific member of a classic nuclear import pathway, Importin α1 (IMPα1). In addition to IMPα1, rhythmic behavior and nuclear expression of PER-TIM require a specific nuclear pore protein, Nup153, and Ran-GTPase. IMPα1 can also drive rapid and efficient nuclear expression of TIM and PER in cultured cells, although the effect on PER is mediated by TIM. Mapping of interaction domains between IMPα1 and TIM/PER suggests that TIM is the primary cargo for the importin machinery. This is supported by attenuated interaction of IMPα1 with TIM carrying a mutation previously shown to prevent nuclear entry of TIM and PER. TIM is detected at the nuclear envelope, and computational modeling suggests that it contains HEAT-ARM repeats typically found in karyopherins, consistent with its role as a co-transporter for PER. These findings suggest that although PER is the major timekeeper of the clock, TIM is the primary target of nuclear import mechanisms. Thus, the circadian clock uses specific components of the importin pathway with a novel twist in that TIM serves a karyopherin-like role for PER.     

Coordination of Eukaryotic Translation Elongation Factor 1A (eEF1A) Function in Actin Organization and Translation Elongation by the Guanine Nucleotide Exchange Factor eEF1B��

Eukaryotic translation elongation factor 1A (eEF1A) both shuttles aminoacyl-tRNA (aa-tRNA) to the ribosome and binds and bundles actin. A single domain of eEF1A is proposed to bind actin, aa-tRNA and the guanine nucleotide exchange factor eEF1Bα. We show that eEF1Bα has the ability to disrupt eEF1A-induced actin organization. Mutational analysis of eEF1Bα F163, which binds in this domain, demonstrates effects on growth, eEF1A binding, nucleotide exchange activity, and cell morphology. These phenotypes can be partially restored by an intragenic W130A mutation. Furthermore, the combination of F163A with the lethal K205A mutation restores viability by drastically reducing eEF1Bα affinity for eEF1A. This also results in a consistent increase in actin bundling and partially corrected morphology. The consequences of the overlapping functions in this eEF1A domain and its unique differences from the bacterial homologs provide a novel function for eEF1Bα to balance the dual roles in actin bundling and protein synthesis.The final step of gene expression takes place at the ribosome as mRNA is translated into protein. In the yeast Saccharomyces cerevisiae, elongation of the polypeptide chain requires the orchestrated action of three soluble factors. The eukaryotic elongation factor 1 (eEF1)² complex delivers aminoacyl-tRNA (aa-tRNA) to the empty A-site of the elongating ribosome (1). The eEF1A subunit is a classic G-protein that acts as a “molecular switch” for the active and inactive states based on whether GTP or GDP is bound, respectively (2). Once an anticodon-codon match occurs, the ribosome acts as a GTPase-activating factor to stimulate GTP hydrolysis resulting in the release of inactive GDP-bound eEF1A from the ribosome. Because the intrinsic rate of GDP release from eEF1A is extremely slow (3, 4), a guanine nucleotide exchange factor (GEF) complex, eEF1B, is required (5, 6). The yeast S. cerevisiae eEF1B complex contains two subunits, the essential catalytic subunit eEF1Bα (5) and the non-essential subunit eEF1Bγ (7).The co-crystal structures of eEF1A:eEF1Bα C terminus:GDP: Mg²⁺ and eEF1A:eEF1Bα C terminus:GDPNP (8, 9) demonstrated a surprising structural divergence from the bacterial EF-Tu-EF-Ts (10) and mammalian mitochondrial EF-Tu_mt-EF-Ts_mt (11). While the G-proteins have a similar topology and consist of three well-defined domains, a striking difference was observed in binding sites for their GEFs. The C terminus of eEF1Bα interacts with domain I and a distinct pocket of domain II eEF1A, creating two binding interfaces. In contrast, the bacterial counterpart EF-Ts and mammalian mitochondrial EF-Ts_mt, make extensive contacts with domain I and III of EF-Tu and EF-Tu_mt, respectively. The altered binding interface of eEF1Bα to domain II of eEF1A is particularly unexpected given the functions associated with domain II of eEF1A and EF-Tu. The crystal structure of the EF-Tu:GDPNP:Phe-tRNA^Phe complex reveals aa-tRNA binding to EF-Tu requires only minor parts of both domain II and tRNA to sustain stable contacts (12). That eEF1A employs the same aa-tRNA binding site is supported by genetic and biochemical data (13-15). Interestingly, eEF1Bα contacts many domain II eEF1A residues in the region hypothesized to be involved in the binding of the aa-tRNA CCA end (8). Because, the shared binding site of eEF1Bα and aa-tRNA on domain II of eEF1A is significantly different between the eukaryotic and bacterial/mitochondrial systems, eEF1Bα may play a unique function aside from guanine nucleotide release in eukaryotes.In eukaroytes, eEF1A is also an actin-binding and -bundling protein. This noncanonical function of eEF1A was initially observed in Dictyostelium amoebae (16). It is estimated that greater than 60% of Dictyostelium eEF1A is associated with the actin cytoskeleton (17). The eEF1A-actin interaction is conserved among species from yeast to mammals, suggesting the importance of eEF1A for cytoskeleton integrity. Using a unique genetic approach, multiple eEF1A mutations were identified that altered cell growth and morphology, and are deficient in bundling actin in vitro (18, 19). Intriguingly, most mutations localized to domain II, the shared aa-tRNA and eEF1Bα binding site. Previous studies have demonstrated that actin bundling by eEF1A is significantly reduced in the presence of aa-tRNA while eEF1A bound to actin filaments is not in complex with aa-tRNA (20). Therefore, actin and aa-tRNA binding to eEF1A is mutually exclusive. In addition, overexpression of yeast eEF1A or actin-bundling deficient mutants do not affect translation elongation (18, 19, 21), suggesting eEF1A-dependent cytoskeletal organization is independent of its translation elongation function (18, 20). Thus, while aa-tRNA binding to domain II is conserved between EF-Tu and eEF1A, this actin bundling function associated with eEF1A domain II places greater importance on its relationship with the “novel” binding interface between eEF1A domain II and eEF1Bα.Based on this support for an overlapping actin bundling and eEF1Bα binding site in eEF1A domain II, we hypothesize that eEF1Bα modulates the equilibrium between actin and translation functions of eEF1A and is perhaps the result of evolutionary selective pressure to balance the eukaryotic-specific role of eEF1A in actin organization. Here, we present kinetic and biochemical evidence using a F163A mutant of eEF1Bα for the importance of the interactions between domain II of eEF1A and eEF1Bα to prevent eEF1A-dependent actin bundling as well as promoting guanine nucleotide exchange. Furthermore, altered affinities of eEF1Bα mutants for eEF1A support that this complex formation is a determining factor for eEF1A-induced actin organization. Interestingly, the F163A that reduces eEF1A affinity is an intragenic suppressor of the lethal K205A eEF1Bα mutant that displays increased affinity for eEF1A. This, along with a consistent change in the actin bundling correlated with the affinity of eEF1Bα for eEF1A, indicates that eEF1Bα is a balancer, directing eEF1A to translation elongation and away from actin, and alterations in this balance result in detrimental effects on cell growth and eEF1A function.     

Initiation of New DNA Strands by the Herpes Simplex Virus-1 Primase-Helicase Complex and Either Herpes DNA Polymerase or Human DNA Polymerase ��

A key set of reactions for the initiation of new DNA strands during herpes simplex virus-1 replication consists of the primase-catalyzed synthesis of short RNA primers followed by polymerase-catalyzed DNA synthesis (i.e. primase-coupled polymerase activity). Herpes primase (UL5-UL52-UL8) synthesizes products from 2 to ∼13 nucleotides long. However, the herpes polymerase (UL30 or UL30-UL42) only elongates those at least 8 nucleotides long. Surprisingly, coupled activity was remarkably inefficient, even considering only those primers at least 8 nucleotides long, and herpes polymerase typically elongated <2% of the primase-synthesized primers. Of those primers elongated, only 4–26% of the primers were passed directly from the primase to the polymerase (UL30-UL42) without dissociating into solution. Comparing RNA primer-templates and DNA primer-templates of identical sequence showed that herpes polymerase greatly preferred to elongate the DNA primer by 650–26,000-fold, thus accounting for the extremely low efficiency with which herpes polymerase elongated primase-synthesized primers. Curiously, one of the DNA polymerases of the host cell, polymerase α (p70-p180 or p49-p58-p70-p180 complex), extended herpes primase-synthesized RNA primers much more efficiently than the viral polymerase, raising the possibility that the viral polymerase may not be the only one involved in herpes DNA replication.Herpes simplex virus 1 (HSV-1)² encodes seven proteins essential for replicating its double-stranded DNA genome; five of these encode the heterotrimeric helicase-primase (UL5-UL52-UL8 gene products) and the heterodimeric polymerase (UL30-UL42 gene products) (1, 2). The helicase-primase unwinds the DNA at the replication fork and generates single-stranded DNA for both leading and lagging strand synthesis. Primase synthesizes short RNA primers on the lagging strand that the polymerase presumably elongates using dNTPs (i.e. primase-coupled polymerase activity). These two protein complexes are thought to replicate the viral genome on both the leading and lagging strands (1, 2).Previous studies have focused on the helicase-primase and polymerase separately. The helicase-primase contains three subunits, UL5, UL52, and UL8 in a 1:1:1 ratio (3–5). The UL5 subunit has helicase-like motifs and the UL52 subunit has primase-like motifs, yet the minimal active complex that demonstrates either helicase or primase activities contains both UL5 and UL52 (6, 7). Although the UL8 subunit has no known catalytic activity, several functions have been proposed, including enhancing helicase and primase activities, enhancing primer synthesis on ICP8 (the HSV-1 single-stranded binding protein)-coated DNA strands, and facilitating formation of the replisome (8–12). Although primase will synthesize short (2–3 nucleotides long) primers on a variety of template sequences, synthesis of longer primers up to 13 nucleotides long requires the template sequence, 3′-deoxyguanidine-pyrimidine-pyrimidine-5′ (13). Primase initiates synthesis at the first pyrimidine via the polymerization of two purine NTPs (13). Even after initiation at this sequence, however, the vast majority of products are only 2–3 nucleotides long (13, 14).The herpes polymerase consists of the UL30 subunit, which has polymerase and 3′ → 5′ exonuclease activities (1, 2), and the UL42 subunit, which serves as a processivity factor (15–17). Unlike most processivity factors that encircle the DNA, the UL42 protein binds double-stranded DNA and thus directly tethers the polymerase to the DNA (18). Using pre-existing DNA primer-templates as the substrate, the heterodimeric polymerase (UL30-UL42) incorporates dNTPs at a rate of 150 s^–1, a rate much faster than primer synthesis (for primers >7 nucleotides long, 0.0002–0.01 s^–1) (19, 20).We examined primase-coupled polymerase activity by the herpes primase and polymerase complexes. Although herpes primase synthesizes RNA primers 2–13 nucleotides long, the polymerase only effectively elongates those at least 8 nucleotides long. Surprisingly, the polymerase elongated only a small fraction of the primase-synthesized primers (<1–2%), likely because of the polymerase elongating RNA primer-templates much less efficiently than DNA primer-templates. In contrast, human DNA polymerase α (pol α) elongated the herpes primase-synthesized primers very efficiently. The biological significance of these data is discussed.     

A Human Platelet Receptor Protein Microarray Identifies the High Affinity Immunoglobulin E Receptor Subunit α (FcεR1α) as an Activating Platelet Endothelium Aggregation Receptor 1 (PEAR1) Ligand

Genome-wide association studies to identify loci responsible for platelet function and cardiovascular disease susceptibility have repeatedly identified polymorphisms linked to a gene encoding platelet endothelium aggregation receptor 1 (PEAR1), an “orphan” cell surface receptor that is activated to stabilize platelet aggregates. To investigate how PEAR1 signaling is initiated, we sought to identify its extracellular ligand by creating a protein microarray representing the secretome and receptor repertoire of the human platelet. Using an avid soluble recombinant PEAR1 protein and a systematic screening assay designed to detect extracellular interactions, we identified the high affinity immunoglobulin E (IgE) receptor subunit α (FcεR1α) as a PEAR1 ligand. FcεR1α and PEAR1 directly interacted through their membrane-proximal Ig-like and 13th epidermal growth factor domains with a relatively strong affinity (K_D ∼ 30 nm). Precomplexing FcεR1α with IgE potently inhibited the FcεR1α-PEAR1 interaction, and this was relieved by the anti-IgE therapeutic omalizumab. Oligomerized FcεR1α potentiated platelet aggregation and led to PEAR1 phosphorylation, an effect that was also inhibited by IgE. These findings demonstrate how a protein microarray resource can be used to gain important insight into the function of platelet receptors and provide a mechanistic basis for the initiation of PEAR1 signaling in platelet aggregation.Platelets play a vital role in preserving blood circulation in response to vessel injury by detecting lesions, aggregating to form a hemostatic plug, and nucleating the formation of a fibrin-rich, injury-occluding clot. Although necessary to prevent blood loss at sites of tissue trauma, clot formation must also be attenuated to prevent blockage of the vasculature serving vital organs that would cause life-threatening ischemia and infarction. Inappropriate platelet aggregation and vessel occlusion, often triggered by atherosclerotic plaque rupture, is a major pathological process that is a major contributor to cardiovascular disease, which is the leading cause of mortality worldwide (1). With the eventual aim of guiding the development of new treatments and diagnostic assays, genome-wide association studies using large patient cohorts have identified several genetic loci that are associated with cardiovascular disease susceptibility and platelet function (2, 3). Among the candidate genes identified, polymorphisms linked to PEAR1 have been repeatedly linked to natural variation in response to platelet agonists in several independent studies (3–7). PEAR1 encodes platelet endothelium activation receptor 1 (PEAR1;¹ also known as multiple epidermal growth factor-like domain protein 12 (MEGF12) or JEDI-1), a platelet cell surface receptor that was originally identified as a protein phosphorylated in response to platelet aggregation (8, 9). PEAR1 is expressed at low levels on the surface of circulating platelets but is significantly up-regulated during platelet activation when released from cytoplasmic α-granules (8). Consistent with polymorphisms linked to PEAR1 being associated with cardiovascular disease and platelet function, PEAR1-mediated signaling was shown to reinforce and stabilize the interactions between platelets within a forming aggregate (8). PEAR1 is an orphan receptor, and an important unanswered question in understanding the mechanism of PEAR1 function during platelet aggregation, therefore, is the identification of its activating ligand.Identifying interactions between membrane-embedded receptor proteins is technically challenging, and many commonly used approaches such as biochemical purifications are generally not suitable to detect them. This is largely due to the amphipathic nature of membrane-embedded proteins that makes them difficult to solubilize in detergents that retain their native conformation and the fact that their extracellular interactions are often highly transient, having half-lives of just fractions of a second (10). To address these issues, we and others have developed assays based on detecting direct protein interactions between the entire ectodomains of cell surface receptors expressed as soluble recombinant proteins in eukaryotic cells (11–14). Using this approach, binding avidity can be increased by the purposeful inclusion of oligomerizing tags to overcome the fleeting nature of these interactions. In our assay, avidity-based extracellular interaction screen (AVEXIS), arrays of monomeric biotinylated “bait” proteins are screened against multimerized, enzyme-tagged, highly avid “preys” (11, 15); a schematic of the assay is shown in supplemental Fig. S1. The likelihood that the extracellular binding functions of receptors are preserved is increased by expressing whole ectodomains in mammalian cells so that structurally critical posttranslational modifications such as disulfide bonds are faithfully added. Consequently, this method has identified interactions that have subsequently been demonstrated to be essential for cellular recognition processes in vivo (16–18).In this study, we have compiled a protein resource representing the cell surface receptor repertoire and secretome of the human platelet that will be useful to identify intercellular interactions important for platelet biology. As an example, we identify the activating ligand for PEAR1 as the high affinity immunoglobulin E (IgE) receptor subunit α (FcεR1α) and show that multimerized FcεR1α potentiated platelet aggregation and led to PEAR1 phosphorylation, an effect that was specifically inhibited by IgE.