Immune Evasion Proteins of Murine Cytomegalovirus Preferentially Affect Cell Surface Display of Recently Generated Peptide Presentation Complexes

For recognition of infected cells by CD8 T cells, antigenic peptides are presented at the cell surface, bound to major histocompatibility complex class I (MHC-I) molecules. Downmodulation of cell surface MHC-I molecules is regarded as a hallmark function of cytomegalovirus-encoded immunoevasins. The molecular mechanisms by which immunoevasins interfere with the MHC-I pathway suggest, however, that this downmodulation may be secondary to an interruption of turnover replenishment and that hindrance of the vesicular transport of recently generated peptide-MHC (pMHC) complexes to the cell surface is the actual function of immunoevasins. Here we have used the model of murine cytomegalovirus (mCMV) infection to provide experimental evidence for this hypothesis. To quantitate pMHC complexes at the cell surface after infection in the presence and absence of immunoevasins, we generated the recombinant viruses mCMV-SIINFEKL and mCMV-Δm06m152-SIINFEKL, respectively, expressing the K^b-presented peptide SIINFEKL with early-phase kinetics in place of an immunodominant peptide of the viral carrier protein gp36.5/m164. The data revealed ∼10,000 K^b molecules presenting SIINFEKL in the absence of immunoevasins, which is an occupancy of ∼10% of all cell surface K^b molecules, whereas immunoevasins reduced this number to almost the detection limit. To selectively evaluate their effect on preexisting pMHC complexes, cells were exogenously loaded with SIINFEKL peptide shortly after infection with mCMV-SIINFEKA, in which endogenous presentation is prevented by an L174A mutation of the C-terminal MHC-I anchor residue. The data suggest that pMHC complexes present at the cell surface in advance of immunoevasin gene expression are downmodulated due to constitutive turnover in the absence of resupply.CD8 T cells recognize infected cells by interaction of their T-cell receptor (TCR) with a cell surface presentation complex composed of a cognate antigenic peptide bound to a presenting allelic form of a major histocompatibility complex class I (MHC-I) glycoprotein (77, 85, 97, 98). The number of such “peptide receptors” per cell has been estimated to be on the order of 10⁵ to 10⁶ for each MHC-I allomorph (for a review, see reference 82). Viral antigenic peptides are generated within infected cells by proteolytic processing of viral proteins, usually in the proteasome, and associate with nascent MHC-I proteins in the endoplasmic reticulum (ER) before the peptide-MHC (pMHC) complexes travel to the cell surface with the cellular vesicular flow (for reviews, see references 13, 87, 92, and 93). CD8 T cells have long been known to protect against cytomegalovirus (CMV) infection and disease in animal models (60, 72; reviewed in references 33 and 36) and in humans (9, 61, 67, 75, 76). As shown only recently in the murine CMV (mCMV) model of infection of immunocompromised mice by adoptive transfer of epitope-specific CD8 T cells, antiviral protection against CMV is indeed TCR mediated and epitope dependent. Specifically, memory cells purified by TCR-based epitope-specific cell sorting, as well as cells of a peptide-selected cytolytic T-lymphocyte line, protected against mCMV expressing the cognate antigenic peptide, the IE1 peptide 168-YPHFMPTNL-176 in this example, but failed to control infection with a recombinant mCMV expressing a peptide analogue in which the C-terminal MHC-I anchor residue leucine was replaced with alanine (3).Interference with the MHC-I pathway of antigen presentation has evolved as a viral immune evasion mechanism of CMVs and other viruses, mediated by virally encoded proteins that inhibit MHC-I trafficking to the cell surface (for reviews, see references 1, 24, 27, 29, 63, 70, 71, 84, and 95). These molecules are known as immunoevasins (50, 70, 89), as “viral proteins interfering with antigen presentation” (VIPRs) (95), or as negative “viral regulators of antigen presentation” (vRAPs) (34). Although the detailed molecular mechanisms differ between different CMV species in their respective hosts, the common biological outcome is the inhibition of antigen presentation. Accordingly, downmodulation of MHC-I cell surface expression is a hallmark of molecular immune evasion and actually led to the discovery of this class of molecules. Since CD8 T cells apparently protect against infection with wild-type CMV strains despite the expression of immunoevasins, the in vivo relevance of these molecules is an issue of current interest and investigation (for a review, see reference 14). As shown recently with the murine model, antigen presentation in infected host cells is not completely blocked for all epitopes, because pMHC complexes that are constitutively formed in sufficiently large amounts can exhaust the inhibitory capacity of the immunoevasins (40). Likewise, enhancing antigen processing conditionally with gamma interferon (IFN-γ) aids in peptide presentation in the presence of immunoevasins (18, 28). Thus, by raising the threshold of the amount of peptide required for presentation, immunoevasins determine whether a particular viral peptide can function as a protective epitope—an issue of relevance for rational vaccine design as well (94). Whereas deletion of immunoevasin genes gives only incremental improvement to the control of infection in immunocompetent mice (22, 51), expression of immunoevasins reduces the protective effect of adoptively transferred CD8 T cells in immunocompromised recipients (37, 40, 47, 48). In a bone marrow transplantation model, immunoevasins were recently found to contribute to enhanced and prolonged virus replication during hematopoietic reconstitution and, consequently, also to higher latent viral genome loads in the lungs and a higher incidence of virus recurrence (4). Notably, however, immunoevasins do not inhibit but, rather, enhance CD8 T-cell priming (5, 21, 22, 56), due to higher viral replication levels in draining lymph nodes associated with sustained antigen supply for the cross-priming of CD8 T cells by uninfected antigen-presenting cells (5).For mCMV, three molecules are proposed to function as vRAPs, only two of which are confirmed negative regulators that downmodulate cell surface MHC-I (34, 62, 89) and inhibit the presentation of antigenic peptides to CD8 T cells (34, 62). Immunoevasin gp40/m152 transiently interacts with MHC-I molecules and mediates their retention in a cis-Golgi compartment (96), whereas gp48/m06 stably binds to MHC-I molecules in the ER and mediates sorting of the complexes for lysosomal degradation by a mechanism that involves the cellular cargo sorting adaptor proteins AP1-A and AP3-A (73, 74). The third proposed immunoevasin of mCMV, gp34/m04 (46), also binds stably to MHC-I molecules. A function as a CD8 T-cell immunoevasin was predicted from some alleviation of immune evasion for certain epitopes and MHC-I molecules in cells infected with the deletion mutant mCMV-Δm04 (34, 42, 89), but gp34/m04 does not reduce the steady-state level of cell surface class I molecules and does not inhibit peptide presentation when expressed selectively after infection with mCMV-Δm06m152 (34, 62). The m04-MHC-I complexes are expressed on the cell surface (46) and appear to be involved in the modulation of natural killer cell activity (45).Here we give the first report on quantitating the efficacy of immunoevasins in terms of absolute numbers of pMHC complexes displayed at the cell surface. By comparing the fate of pMHC complexes already present at the cell surface in advance of immunoevasin gene expression with that of newly formed pMHC complexes, our data provide direct evidence to conclude that downmodulation of cell surface MHC-I molecules is secondary to an interruption of the flow of newly formed pMHC complexes to the cell surface.(Part of this work was presented at the 12th International CMV/Betaherpesvirus Workshop, 10 to 14 May 2009, Boston, MA.)     

A pH-dependent dimer lock in spider silk protein

Spider dragline silk, one of the strongest polymers in nature, is composed of proteins termed major ampullate spidroin (MaSp) 1 and MaSp2. The N-terminal (NT) domain of MaSp1 produced by the nursery web spider Euprosthenops australis acts as a pH-sensitive relay, mediating spidroin assembly at around pH 6.3. Using amide hydrogen/deuterium exchange combined with mass spectrometry (MS), we detected pH-dependent changes in deuterium incorporation into the core of the NT domain, indicating global structural stabilization at low pH. The stabilizing effects were diminished or abolished at high ionic strength, or when the surface-exposed residues Asp40 and Glu84 had been exchanged with the corresponding amides. Nondenaturing electrospray ionization MS revealed the presence of dimers in the gas phase at pH values below—but not above—6.4, indicating a tight electrostatic association that is dependent on Asp40 and Glu84 at low pH. Results from analytical ultracentrifugation support these findings. Together, the data suggest a mechanism whereby lowering the pH to < 6.4 results in structural changes and alteration of charge-mediated interactions between subunits, thereby locking the spidroin NT dimer into a tight entity important for aggregation and silk formation.     

Dynamin-like protein 1 at the Golgi complex: A novel component of the sorting/targeting machinery en route to the plasma membrane

The final step in the liberation of secretory vesicles from the trans-Golgi network (TGN) involves the mechanical action of the large GTPase dynamin as well as conserved dynamin-independent fission mechanisms, e.g. mediated by Brefeldin A-dependent ADP-ribosylated substrate (BARS). Another member of the dynamin family is the mammalian dynamin-like protein 1 (DLP1/Drp1) that is known to constrict and tubulate membranes, and to divide mitochondria and peroxisomes. Here, we examined a potential role for DLP1 at the Golgi complex. DLP1 localized to the Golgi complex in some but not all cell lines tested, thus explaining controversial reports on its cellular distribution. After silencing of DLP1, an accumulation of the apical reporter protein YFP-GL-GPI, but not the basolateral reporter VSVG-SP-GFP at the Golgi complex was observed. A reduction in the transport of YFP-GL-GPI to the plasma membrane was confirmed by surface immunoprecipitation and TGN-exit assays. In contrast, YFP-GL-GPI trafficking was not disturbed in cells silenced for BARS, which is involved in basolateral sorting and trafficking of VSVG-SP-GFP in COS-7 cells. Our data indicate a new role for DLP1 at the Golgi complex and thus a role for DLP1 as a novel component of the apical sorting machinery at the TGN is discussed.     

Rationally Evolving MCP-1/CCL2 into a Decoy Protein with Potent Anti-inflammatory Activity in Vivo

Leukocyte recruitment from the blood into injured tissues during inflammatory diseases is the result of sequential events involving chemokines binding to their GPC receptors as well as to their glycosaminoglycan (GAG) co-receptors. The induction and the crucial role of MCP-1/CCL2 in the course of diseases that feature monocyte-rich infiltrates have been validated in many animal models, and several MCP-1/CCL2 as well as CCR2 antagonists have since been generated. However, despite some of them being shown to be efficacious in a number of animal models, many failed in clinical trials, and therapeutically interfering with the activity of this chemokine is not yet possible. We have therefore generated novel MCP-1/CCL2 mutants with increased GAG binding affinity and knocked out CCR2 activity, which were designed to interrupt the MCP-1/CCL2-related signaling cascade. We provide evidence that our lead mutant MCP-1(Y13A/S21K/Q23R) exhibits a 4-fold higher affinity toward the natural MCP-1 GAG ligand heparan sulfate and that it shows a complete deficiency in activating CCR2 on THP-1 cells. Furthermore, a significantly longer residual time on GAG ligands was observed by surface plasmon resonance. Finally, we were able to show that MCP-1(Y13A/S21K/Q23R) had a mild ameliorating effect on experimental autoimmune uveitis and that a marginal effect on oral tolerance in the group co-fed with Met-MCP-1(Y13A/S21K/Q23R) plus immunogenic peptide PDSAg was observed. These results suggest that disrupting wild type chemokine-GAG interactions by a chemokine-based antagonist can result in anti-inflammatory activity that could have potential therapeutic implications.     

Proteins at the Polypeptide Tunnel Exit of the Yeast Mitochondrial Ribosome

Oxidative phosphorylation in mitochondria requires the synthesis of proteins encoded in the mitochondrial DNA. The mitochondrial translation machinery differs significantly from that of the bacterial ancestor of the organelle. This is especially evident from many mitochondria-specific ribosomal proteins. An important site of the ribosome is the polypeptide tunnel exit. Here, nascent chains are exposed to an aqueous environment for the first time. Many biogenesis factors interact with the tunnel exit of pro- and eukaryotic ribosomes to help the newly synthesized proteins to mature. To date, nothing is known about the organization of the tunnel exit of mitochondrial ribosomes. We therefore undertook a comprehensive approach to determine the composition of the yeast mitochondrial ribosomal tunnel exit. Mitochondria contain homologues of the ribosomal proteins located at this site in bacterial ribosomes. Here, we identified proteins located in their proximity by chemical cross-linking and mass spectrometry. Our analysis revealed a complex network of interacting proteins including proteins and protein domains specific to mitochondrial ribosomes. This network includes Mba1, the membrane-bound ribosome receptor of the inner membrane, as well as Mrpl3, Mrpl13, and Mrpl27, which constitute ribosomal proteins exclusively found in mitochondria. This unique architecture of the tunnel exit is presumably an adaptation of the translation system to the specific requirements of the organelle.     

Protein kinase C mediates erythrocyte "programmed cell death" following glucose depletion

Glucose depletion of erythrocytes leads to activation of Ca²⁺-permeable cation channels, Ca²⁺ entry, activation of a Ca²⁺-sensitive erythrocyte scramblase, and subsequent exposure of phosphatidylserine at the erythrocyte surface. Ca²⁺ entry into erythrocytes was previously shown to be stimulated by phorbol esters and to be inhibited by staurosporine and chelerythrine and is thus thought to be regulated by protein phosphorylation/dephosphorylation, presumably via protein kinase C (PKC) and the corresponding phosphoserine/threonine phosphatases. The present experiments explored whether PKC could contribute to effects of energy depletion on erythrocyte phosphatidylserine exposure and cell volume. Phosphatidylserine exposure was estimated from annexin binding and cell volume from forward scatter in fluorescence-activated cell sorter analysis. Removal of extracellular glucose led to depletion of cellular ATP, stimulated PKC activity, led to translocation of PKC, enhanced serine phosphorylation of membrane proteins, decreased cell volume, and increased annexin binding, the latter effect being blunted but not abolished in the presence of 1 µM staurosporine or 50 nM calphostin C. The PKC stimulator phorbol-12-myristate-13-acetate (3 µM) and the phosphatase inhibitor okadaic acid (1–10 µM) mimicked the effect of glucose depletion and similarly led to translocation of PKC and enhanced serine phosphorylation, increased annexin binding, and decreased forward scatter, the latter effects being abrogated by PKC inhibitor staurosporine (1 µM). Fluo-3 fluorescence measurements revealed that okadaic acid also enhanced erythrocyte Ca²⁺ activity. The present observations suggest that protein phosphorylation and dephosphorylation via PKC and the corresponding protein phosphatases contribute to phosphatidylserine exposure and cell shrinkage after energy depletion. cell volume; eryptosis; calcium; okadaic acid; staurosporine     

Monitoring Lactobacillus plantarum in grass silages with the aid of 16S rDNA-based quantitative real-time PCR assays

Ensiling plant material with the aid of lactic acid bacteria (LAB) is a common agricultural practice for conserving forages independently of the time point of harvest. Despite ensiling being a natural process, it can be improved by the treatment of the harvested forage with starter cultures before storage. Within this context, Lactobacillus plantarum (L. plantarum) is the most frequently used LAB in commercially available starter cultures. In order to enable the monitoring of the population dynamics of L. plantarum in silage, methods for species-specific detection based on the 16S ribosomal DNA (rDNA) sequence were developed by applying a quantitative real-time polymerase chain reaction (QRT-PCR) approach. The QRT-PCR assay was also applied to estimate the development of the L. plantarum population within experimental grass silages. In addition, a multiplex QRT-PCR assay was developed to estimate the amount of L. plantarum 16S rDNA in relation to total bacterial 16S rDNA. This multiplex QRT-PCR assay was applied to monitor the influence of different silage additives on the L. plantarum population.     

The Brichos domain-containing C-terminal part of pro-surfactant protein C binds to an unfolded poly-val transmembrane segment

Native lung surfactant protein C (SP-C) is a 4.2-kDa acylpeptide that associates with alveolar surfactant phospholipids via a transmembrane alpha-helix. This helix contains mainly Val, although poly-Val is inefficient in helix formation, and helical SP-C can spontaneously convert to beta-sheet aggregates and amyloid-like fibrils. SP-C is cleaved out from a 21-kDa integral membrane protein, proSP-C, in the alveolar type II cell. Recently several mutations localized in the endoplasmic reticulum-lumenal (C-terminal) part of proSP-C (CTproSP-C) have been associated with intracellular accumulation of toxic forms of proSP-C, low levels of mature SP-C, and development of interstitial lung disease. CTproSP-C contains a approximately 100-residue Brichos domain of unknown function that is also found in other membrane proteins associated with amyloid formation, dementia, and cancer. Here we find that recombinant CTproSP-C binds lipid-associated SP-C, which is in beta-strand conformation, and that this interaction results in an increased helical content. In contrast, CTproSP-C does not bind alpha-helical SP-C. Recombinant CTproSP-C(L188Q), a mutation associated with interstitial lung disease, shows secondary and quaternary structures similar to those of wild type CTproSP-C but is unable to bind lipid-associated beta-strand SP-C. Transfection of CTproSP-C into HEK293 cells that express proSP-C(L188Q) increases the amount of proSP-C protein, whereas no effect is seen on cells expressing wild type proSP-C. These findings suggest that CTproSP-C binds nonhelical SP-C and thereby prevents beta-sheet aggregation and that mutations in CTproSP-C can interfere with this function.     

Allergic airway inflammation inhibits pulmonary antibacterial host defense

The innate immune system of the lung is a multicomponent host defense system and in addition has an instructing role in regulating the quality and quantity of the adaptive immune response. When the interaction between innate and adaptive immunity is disturbed, pathological conditions such as asthma can develop. It was the aim of the study to investigate the effect of the allergic inflammation of the lung on the innate host defense during bacterial infection. Human bronchial epithelial cells were preincubated with Th2 cytokines and infected with Pseudomonas aeruginosa. The effect of the Th2 cytokines on the mRNA levels of antimicrobial peptides and the antimicrobial activity of HBEC was determined. To investigate the influence of an allergic inflammation on pulmonary host defense in vivo, mice sensitized and challenged with OVA were infected with P. aeruginosa, and the number of viable bacteria in the lungs was determined together with markers of inflammation like cytokines and antimicrobial peptides. Exposure of airway epithelial cells to Th2 cytokines resulted in a significantly decreased antimicrobial activity of the cells and in suppressed mRNA levels of the antimicrobial peptide human beta-defensin 2. Furthermore, mice with allergic airway inflammation had significantly more viable bacteria in their lungs after infection. This was consistent with reduced levels of proinflammatory cytokines and of the antimicrobial peptide cathelin-related antimicrobial peptide. These results show that an allergic airway inflammation suppresses the innate antimicrobial host defense. The adaptive immune system modulates the functions of the pulmonary innate immune system.     

Absorption of zinc and retention of calcium: Dose-dependent inhibition by phytate

The dose-dependent inhibitory effect of sodium phytate (myo-inositol-hexaphosphate) on absorption of zinc and retention of calcium was studied in man. No systematic study of this dose-response effect has been reported to this time. Forty subjects were served meals containing white wheat rolls without/with additions of phytate. Ten subjects were given test meals containing one or two of the studied levels of phytate and in addition all subjects were served meals to which no phytate was added. The zinc content was 3.1 mg (47 micromol) and the calcium content 266 mg (6.6 mmol). The rolls were labelled extrinsically with radioisotopes, 65Zn and 47Ca, and whole-body retention of both minerals was measured. Totally 105 meals were served, 36 meals in which no phytate was added and 9-10 meals on each level of phytate. The zinc absorption in meals to which either 0, 25, 50, 75, 100, 140, 175 or 250 mg of phytate-P (0, 134, 269, 403, 538, 753, 941 or 1344 micromol phytate) had been added was 22%, 16%, 14%, 11%, 7%, 7%, 7% and 6%, respectively (mean values). The addition of 50 mg phytate-P or more significantly decreased zinc absorption (p=0.01) as compared to absorption from the test meals with no added phytate. The calcium retention at day 7 in the same meals was 31%, 28%, 27%, 26%, 22%, 19%, 14% and 11% (mean values). The addition of 100 mg phytate-P or more significantly decreased calcium retention (p=0.03) compared to the test meals with no added phytate. It was concluded that the inhibitory effect of phytate on the absorption of zinc and the retention of calcium was dose dependent.