Expression of the vault RNA protects cells from undergoing apoptosis

Non-protein-coding RNAs are a functionally versatile class of transcripts exerting their biological roles on the RNA level. Recently, we demonstrated that the vault complex-associated RNAs (vtRNAs) are significantly upregulated in Epstein-Barr virus (EBV)-infected human B cells. Very little is known about the function(s) of the vtRNAs or the vault complex. Here, we individually express latent EBV-encoded proteins in B cells and identify the latent membrane protein 1 (LMP1) as trigger for vtRNA upregulation. Ectopic expression of vtRNA1-1, but not of the other vtRNA paralogues, results in an improved viral establishment and reduced apoptosis, a function located in the central domain of vtRNA1-1. Knockdown of the major vault protein has no effect on these phenotypes revealing that vtRNA1-1 and not the vault complex contributes to general cell death resistance. This study describes a NF-κB-mediated role of the non-coding vtRNA1-1 in inhibiting both the extrinsic and intrinsic apoptotic pathways.

Development of a protective vaccine against Helicobacter pylori

Helicobacter pylori, which colonizes the stomach and causes the most common chronic infection in man, is associated with peptic ulceration, gastric carcinoma and gastric lymphoma. Studies in animals demonstrated that mucosal immunization could induce immune response against H. pylori and prevent H. pylori infection only if powerful mucosal adjuvants such as cholera toxin (CT) or heat-labile toxin of E. coli (LT) were used along with an H. pylori protein antigen. Adjuvants such as CT or LT cannot be used for humans because of their toxicity. Finding non-toxic alternative adjuvants/immunomodulators or immunization strategies that eliminates the use of adjuvants is critical for the development of efficacious human Helicobacter vaccines. We investigated whether several new adjuvants such as Muramyl Tripeptide Phosphatidylethonolamine (MTP-PE), QS21 (a Quil A derivative), Monophosphoryl lipid A (MPL) or heat shock proteins (HSP) of Mycobacterium tuberculosis could be feasible to develop a safe and effective mucosal vaccine against H. pylori using a murine model. C57/BL6 mice were immunized with liposomes incorporating each adjuvant along with urease, a major antigenic protein of H. pylori, to test their mucosal effectiveness. Since DNA vaccination eliminates both the use of adjuvants and antigens we also investigated whether immunization with plasmid DNA encoding urease could induce protective immunity to H. pylori infection in the same murine model. We found that oral vaccination with liposomal MTP-PE (6.7 m g) and urease, (100 m g) induced antigen-specific systemic and mucosal immune response and protected mice against H. pylori challenge when compared to control groups. Parenteral and mucosal immunizations with as little as 20 m g naked or formulated DNA encoding urease induced systemic and mucosal immune response against urease and partially protected mice against H. pylori infection. DNA vaccination provided long-lasting immunity and serum anti-urease IgG antibodies were elevated for up to 12 months. No toxicity was detected after immunizations with either liposomal MTP-PE and urease or plasmid DNA and both were well tolerated. We conclude that immunization liposomes containing MTP-PE and urease is a promising strategy deserving further investigation and may be considered for humans. DNA vaccination could be used to prime immune response prior to oral protein vaccination and may reduce the dose of protein and adjuvant needed to achieve protective immunity. ^

Roles of Alix in regulating cell morphology

Mammalian Alix (ALG2-interacting protein X&barbelow;) is a conserved adaptor protein that is involved in endosomal trafficking, apoptosis and growth factor receptor turnover. Accumulating evidence also indicates that Alix plays roles in promoting/maintaining spread and aligned fibroblast morphology in monolayer culture. Since cell morphology is determined by the structure and dynamics of an integrin-mediated transmembrane protein network that links extracellular matrix to intracellular cytoskeleton, we hypothesized that Alix plays direct or indirect roles in regulating certain components or steps in this transmembrane protein network. To test this hypothesis, we first examined the subcellular localization of Alix and discovered that, as a predominantly cytoplasmic protein, Alix is also present on the substratum/cell surface and in the conditioned medium of fibroblast cultures. Further, precoating of culture surfaces with recombinant Alix promotes spreading and fibronectin assembly to NIH/3T3 cells, and siRNA-mediated Alix knockdown in W138 cells has the opposite effects. These findings indicate the extracellular functions of Alix in regulating cell spreading and extracellular matrix assembly. In a separate study, we analyzed Alix immunocomplexes from normal fibroblast W138 cells by mass spectrometry and identified actin as a major partner protein of Alix. Follow-up studies demonstrated that Alix preferentially binds filamentous actin (F-actin) in vitro and is required for maintaining normal F-actin content and proper actin cytoskeleton assembly in W138 cells. These findings establish direct and essential roles of Alix in regulating actin cytoskeleton. Finally, we investigated the effects of Alix knockdown on the activation and subcellular localization of FAK and Pyk2, the focal adhesion kinases required for cell spreading/migration by promoting turnover of integrin-mediated cell adhesions. We discovered that Alix knockdown inhibits FAK and Pyk2 localizations to focal adhesions or plasma membrane, in association with characteristics of reduced turnover of focal adhesions. These findings reveal a positive role of Alix in focal adhesion turnover. Based on these results, we conclude that Alix targets both intracellularly and extracellularly components to regulate extracellular matrix remodeling, actin cytoskeleton assembly and focal adhesion turnover. A combination of these three functions of Alix explains its crucial role in regulating spread and aligned fibroblast morphology. ^

Immunomodulation of experimental autoimmune encephalomyelitis by oral administration of copolymer 1

The activity of copolymer 1 (Cop 1, Copaxone, glatiramer acetate) in suppressing experimental autoimmune encephalomyelitis (EAE) and in the treatment of multiple sclerosis patients when injected parenterally has been extensively demonstrated. In the present study we addressed the question of whether Cop 1 can induce oral tolerance to EAE similar to myelin basic protein (MBP). We now have demonstrated that oral Cop 1 inhibited EAE induction in both rats and mice. Furthermore, oral Cop 1 was more effective than oral MBP in suppressing EAE in rats. The beneficial effect of oral Cop 1 was found to be associated with specific inhibition of the proliferative and Th1 cytokine secretion responses to MBP of spleen cells from Cop 1-fed mice and rats. In all of these assays, oral Cop 1 was more effective than oral MBP. The tolerance induced by Cop 1 could be adoptively transferred with spleen cells from Cop 1-fed animals. Furthermore, Cop 1-specific T cell lines, which inhibit EAE induction in vivo, could be isolated from the above spleen cells. These T cell lines secrete the anti-inflammatory cytokines IL-10 and transforming growth factor type β, but not IL-4, in response to both Cop 1 and MBP. In conclusion, oral Cop 1 has a beneficial effect on the development of EAE that is associated with down-regulation of T cell immune responses to MBP and is mediated by Th2/3 type regulatory cells. These results suggest that oral administration of Cop 1 may modulate multiple sclerosis as well.

T cell vaccination induces T cell receptor Vβ-specific Qa-1-restricted regulatory CD8+ T cells

Vaccination of mice with activated autoantigen-reactive CD4+ T cells (T cell vaccination, TCV) has been shown to induce protection from the subsequent induction of a variety of experimental autoimmune diseases, including experimental allergic encephalomyelitis (EAE). Although the mechanisms involved in TCV-mediated protection are not completely known, there is some evidence that TCV induces CD8+ regulatory T cells that are specific for pathogenic CD4+ T cells. Previously, we demonstrated that, after superantigen administration in vivo, CD8+ T cells emerge that preferentially lyse and regulate activated autologous CD4+ T cells in a T cell receptor (TCR) Vβ-specific manner. This TCR Vβ-specific regulation is not observed in β2-microglobulin-deficient mice and is inhibited, in vitro, by antibody to Qa-1. We now show that similar Vβ8-specific Qa-1-restricted CD8+ T cells are also induced by TCV with activated CD4+ Vβ8+ T cells. These CD8+ T cells specifically lyse murine or human transfectants coexpressing Qa-1 and murine TCR Vβ8. Further, CD8+ T cell hybridoma clones generated from B10.PL mice vaccinated with a myelin basic protein-specific CD4+Vβ8+ T cell clone specifically recognize other CD4+ T cells and T cell tumors that express Vβ8 and the syngeneic Qa-1a but not the allogeneic Qa-1b molecule. Thus, Vβ-specific Qa-1-restricted CD8+ T cells are induced by activated CD4+ T cells. We suggest that these CD8+ T cells may function to specifically regulate activated CD4+ T cells during immune responses.

A T cell receptor antagonist peptide induces T cells that mediate bystander suppression and prevent autoimmune encephalomyelitis induced with multiple myelin antigens

Experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) residues 139–151 (HSLGKWLGHPDKF) can be prevented by treatment with a T cell receptor (TCR) antagonist peptide (L144/R147) generated by substituting at the two principal TCR contact residues in the encephalitogenic peptide. The TCR antagonist peptide blocks activation of encephalitogenic Th1 helper cells in vitro, but the mechanisms by which the antagonist peptide blocks EAE in vivo are not clear. Immunization with L144/R147 did not inhibit generation of PLP-(139–151)-specific T cells in vivo. Furthermore, preimmunization with L144/R147 protected mice from EAE induced with the encephalitogenic peptides PLP-(178–191) and myelin oligodendrocyte protein (MOG) residues 92–106 and with mouse myelin basic protein (MBP). These data suggest that the L144/R147 peptide does not act as an antagonist in vivo but mediates bystander suppression, probably by the generation of regulatory T cells. To confirm this we generated T cell lines and clones from animals immunized with PLP-(139–151) plus L144/R147. T cells specific for L144/R147 peptide were crossreactive with the native PLP-(139–151) peptide, produced Th2/Th0 cytokines, and suppressed EAE upon adoptive transfer. These studies demonstrate that TCR antagonist peptides may have multiple biological effects in vivo. One of the principal mechanisms by which these peptides inhibit autoimmunity is by the induction of regulatory T cells, leading to bystander suppression of EAE. These results have important implications for the treatment of autoimmune diseases where there are autopathogenic responses to multiple antigens in the target organ.

Synapsin I interacts with c-Src and stimulates its tyrosine kinase activity

Synapsin I is a synaptic vesicle-associated phosphoprotein that has been implicated in the formation of presynaptic specializations and in the regulation of neurotransmitter release. The nonreceptor tyrosine kinase c-Src is enriched on synaptic vesicles, where it accounts for most of the vesicle-associated tyrosine kinase activity. Using overlay, affinity chromatography, and coprecipitation assays, we have now shown that synapsin I is the major binding protein for the Src homology 3 (SH3) domain of c-Src in highly purified synaptic vesicle preparations. The interaction was mediated by the proline-rich domain D of synapsin I and was not significantly affected by stoichiometric phosphorylation of synapsin I at any of the known regulatory sites. The interaction of purified c-Src and synapsin I resulted in a severalfold stimulation of tyrosine kinase activity and was antagonized by the purified c-Src-SH3 domain. Depletion of synapsin I from purified synaptic vesicles resulted in a decrease of endogenous tyrosine kinase activity. Portions of the total cellular pools of synapsin I and Src were coprecipitated from detergent extracts of rat brain synaptosomal fractions using antibodies to either protein species. The interaction between synapsin I and c-Src, as well as the synapsin I-induced stimulation of tyrosine kinase activity, may be physiologically important in signal transduction and in the modulation of the function of axon terminals, both during synaptogenesis and at mature synapses.

CooB plays a chaperone-like role for the proteins involved in formation of CS1 pili of enterotoxigenic Escherichia coli

CS1 pili serve as the prototype of a class of filamentous appendages found on the surface of strains of enterotoxigenic Escherichia coli. The four genes needed to synthesize functional CS1 pili in E. coli K12 are: cooA, which encodes the major pilin protein; cooD, which encodes a minor pilin protein found at the tip of the structure; cooC, which encodes a protein found in the outer membrane of piliated bacteria; and cooB. We show here that CooB, which is required for pilus assembly but is not part of the final structure, stabilizes CooA, CooC, and CooD. We previously reported that CooB is complexed with CooA in the periplasm and show here that CooB also is found complexed with CooD in the periplasm. CooB is associated with the membrane fraction only in the presence of CooC, suggesting that these two proteins also interact. This suggests that although it has no homology to known chaperone proteins, CooB serves a chaperone-like role for assembly of CS1.

Induction of maturation of human blood dendritic cell precursors by measles virus is associated with immunosuppression

As well as inducing a protective immune response against reinfection, acute measles is associated with a marked suppression of immune functions against superinfecting agents and recall antigens, and this association is the major cause of the current high morbidity and mortality rate associated with measles virus (MV) infections. Dendritic cells (DCs) are antigen-presenting cells crucially involved in the initiation of primary and secondary immune responses, so we set out to define the interaction of MV with these cells. We found that both mature and precursor human DCs generated from peripheral blood monocytic cells express the major MV protein receptor CD46 and are highly susceptible to infection with both MV vaccine (ED) and wild-type (WTF) strains, albeit with different kinetics. Except for the down-regulation of CD46, the expression pattern of functionally important surface antigens on mature DCs was not markedly altered after MV infection. However, precursor DCs up-regulated HLA-DR, CD83, and CD86 within 24 h of WTF infection and 72 h after ED infection, indicating their functional maturation. In addition, interleukin 12 synthesis was markedly enhanced after both ED and WTF infection in DCs. On the other hand, MV-infected DCs strongly interfered with mitogen-dependent proliferation of freshly isolated peripheral blood lymphocytes in vitro. These data indicate that the differentiation of effector functions of DCs is not impaired but rather is stimulated by MV infection. Yet, mature, activated DCs expressing MV surface antigens do give a negative signal to inhibit lymphocyte proliferation and thus contribute to MV-induced immunosuppression.

Disruption of the telomerase catalytic subunit gene from Arabidopsis inactivates telomerase and leads to a slow loss of telomeric DNA

Telomerase is an essential enzyme that maintains telomeres on eukaryotic chromosomes. In mammals, telomerase is required for the lifelong proliferative capacity of normal regenerative and reproductive tissues and for sustained growth in a dedifferentiated state. Although the importance of telomeres was first elucidated in plants 60 years ago, little is known about the role of telomeres and telomerase in plant growth and development. Here we report the cloning and characterization of the Arabidopsis telomerase reverse transcriptase (TERT) gene, AtTERT. AtTERT is predicted to encode a highly basic protein of 131 kDa that harbors the reverse transcriptase and telomerase-specific motifs common to all known TERT proteins. AtTERT mRNA is 10–20 times more abundant in callus, which has high levels of telomerase activity, versus leaves, which contain no detectable telomerase. Plants homozygous for a transfer DNA insertion into the AtTERT gene lack telomerase activity, confirming the identity and function of this gene. Because telomeres in wild-type Arabidopsis are short, the discovery that telomerase-null plants are viable for at least two generations was unexpected. In the absence of telomerase, telomeres decline by approximately 500 bp per generation, a rate 10 times slower than seen in telomerase-deficient mice. This gradual loss of telomeric DNA may reflect a reduced rate of nucleotide depletion per round of DNA replication, or the requirement for fewer cell divisions per organismal generation. Nevertheless, progressive telomere shortening in the mutants, however slow, ultimately should be lethal.

The N-terminal 209-aa domain of high molecular- weight 4.1R isoforms abrogates 4.1R targeting to the nucleus

An extensive repertoire of protein 4.1R isoforms is predominantly generated by alternative pre-mRNA splicing and differential usage of two translation initiation sites. The usage of the most upstream ATG (ATG-1) generates isoforms containing N-terminal extensions of up to 209 aa compared with those translated from the downstream ATG (ATG-2). To characterize nonerythroid 4.1R proteins translated from ATG-1 and analyze their intracellular localization, we cloned 4.1R cDNAs containing this translation initiation site. Six different clones were isolated from the nucleated human MOLT-4 T-cell line by reverse transcriptase–PCR techniques. Transient expression of the six ATG-1-translated 4.1R isoforms tagged with a c-Myc epitope revealed that all of them predominantly distributed to the plasma membrane and the endoplasmic reticulum. Staining of MOLT-4 cell plasma membranes but not nuclei was also observed by immunofluorescence microscopy by using an antibody specific to the N-terminal extension. Consistent with this, the antibody reacted with a major endogenous protein of ≈145 kDa present in nonnuclear but absent from nuclear fractions prepared from MOLT-4 cells. Because these data suggested that ATG-1-translated 4.1R isoforms were predominantly excluded from the nucleus, we fused the 209-aa domain to nuclear 4.1R isoforms encoded from ATG-2 and observed that this domain inhibited their nuclear targeting. All these results indicate that the N-terminal domain of ATG-1-translated 4.1R isoforms plays a pivotal role in differential targeting of proteins 4.1R.

Amino-terminal Polypeptides of Vimentin Are Responsible for the Changes in Nuclear Architecture Associated with Human Immunodeficiency Virus Type 1 Protease Activity in Tissue Culture Cells

Electron microscopy of human skin fibroblasts syringe-loaded with human immunodeficiency virus type 1 protease (HIV-1 PR) revealed several effects on nuclear architecture. The most dramatic is a change from a spherical nuclear morphology to one with multiple lobes or deep invaginations. The nuclear matrix collapses or remains only as a peripheral rudiment, with individual elements thicker than in control cells. Chromatin organization and distribution is also perturbed. Attempts to identify a major nuclear protein whose cleavage by the protease might be responsible for these alterations were unsuccessful. Similar changes were observed in SW 13 T3 M [vimentin+] cells, whereas no changes were observed in SW 13 [vimentin−] cells after microinjection of protease. Treatment of SW 13 [vimentin−] cells, preinjected with vimentin to establish an intermediate filament network, with HIV-1 PR resulted in alterations in chromatin staining and distribution, but not in nuclear shape. These same changes were produced in SW 13 [vimentin−] cells after the injection of a mixture of vimentin peptides, produced by the cleavage of vimentin to completion by HIV-1 PR in vitro. Similar experiments with 16 purified peptides derived from wild-type or mutant vimentin proteins and five synthetic peptides demonstrated that exclusively N-terminal peptides were capable of altering chromatin distribution. Furthermore, two separate regions of the N-terminal head domain are primarily responsible for perturbing nuclear architecture. The ability of HIV-1 to affect nuclear organization via the liberation of vimentin peptides may play an important role in HIV-1-associated cytopathogenesis and carcinogenesis.

CD8+ T cells control the TH phenotype of MBP-reactive CD4+ T cells in EAE mice

Trimolecular interactions between the T cell antigen receptor and MHC/peptide complexes, together with costimulatory molecules and cytokines, control the initial activation of naïve T cells and determine whether the helper precursor cell differentiates into either T helper (TH)1 or TH2 effector cells. We now present evidence that regulatory CD8+ T cells provide another level of control of TH phenotype during further evolution of immune responses. These regulatory CD8+ T cells are induced by antigen-triggered CD4+ TH1 cells during T cell vaccination and, in vitro, distinguish mature TH1 from TH2 cells in a T cell antigen receptor Vβ-specific and Qa-1-restricted manner. In vivo, protection from experimental autoimmune encephalomyelitis (EAE) induced by T cell vaccination depends on CD8+ T cells, and myelin basic protein-reactive TH1 Vβ8+ clones, but not TH2 Vβ8+ clones, used as vaccine T cells, protect animals from subsequent induction of EAE. Moreover, in vivo depletion of CD8+ T cells during the first episode of EAE results in skewing of the TH phenotype toward TH1 upon secondary myelin basic protein stimulation. These data provide evidence that CD8+ T cells control autoimmune responses, in part, by regulating the TH phenotype of self-reactive CD4+ T cells.

Kinetics and thermodynamics of T cell receptor– autoantigen interactions in murine experimental autoimmune encephalomyelitis

In the current study, cellular and molecular approaches have been used to analyze the biophysical nature of T cell receptor (TCR)–peptide MHC (pMHC) interactions for two autoreactive TCRs. These two TCRs recognize the N-terminal epitope of myelin basic protein (MBP1–11) bound to the MHC class II protein, I-Au, and are associated with murine experimental autoimmune encephalomyelitis. Mice transgenic for the TCRs have been generated and characterized in other laboratories. These analyses indicate that the mice either develop encephalomyelitis spontaneously (172.10 TCR) or only if immunized with autoantigen in adjuvant (1934.4 TCR). Here, we show that the 172.10 TCR binds MBP1–11:I-Au with a 4–5-fold higher affinity than the 1934.4 TCR. Consistent with the higher affinity, 172.10 T hybridoma cells are significantly more responsive to autoantigen than 1934.4 cells. The interaction of the 172.10 TCR with cognate ligand is more entropically unfavorable than that of the 1934.4 TCR, indicating that the 172.10 TCR undergoes greater conformational rearrangements upon ligand binding. The studies therefore suggest a correlation between the strength and plasticity of a TCR–pMHC interaction and the frequency of spontaneous disease in the corresponding TCR transgenic mice. The comparative analysis of these two TCRs has implications for understanding autoreactive T cell recognition and activation.

Differential Control of Xanthophylls and Light-Induced Stress Proteins, as Opposed to Light-Harvesting Chlorophyll a/b Proteins, during Photosynthetic Acclimation of Barley Leaves to Light Irradiance

Barley (Hordeum vulgare L.) plants were grown at different photon flux densities ranging from 100 to 1800 μmol m−2 s−1 in air and/or in atmospheres with reduced levels of O2 and CO2. Low O2 and CO2 partial pressures allowed plants to grow under high photosystem II (PSII) excitation pressure, estimated in vivo by chlorophyll fluorescence measurements, at moderate photon flux densities. The xanthophyll-cycle pigments, the early light-inducible proteins, and their mRNA accumulated with increasing PSII excitation pressure irrespective of the way high excitation pressure was obtained (high-light irradiance or decreased CO2 and O2 availability). These findings indicate that the reduction state of electron transport chain components could be involved in light sensing for the regulation of nuclear-encoded chloroplast gene expression. In contrast, no correlation was found between the reduction state of PSII and various indicators of the PSII light-harvesting system, such as the chlorophyll a-to-b ratio, the abundance of the major pigment-protein complex of PSII (LHCII), the mRNA level of LHCII, the light-saturation curve of O2 evolution, and the induced chlorophyll-fluorescence rise. We conclude that the chlorophyll antenna size of PSII is not governed by the redox state of PSII in higher plants and, consequently, regulation of early light-inducible protein synthesis is different from that of LHCII.