An Essential Role of the Cytoplasmic Tail of CXCR4 in G-Protein Signaling and Organogenesis

CXCR4 regulates cell proliferation, enhances cell survival and induces chemotaxis, yet molecular mechanisms underlying its signaling remain elusive. Like all other G-protein coupled receptors (GPCRs), CXCR4 delivers signals through G-protein-dependent and -independent pathways, the latter involving its serine-rich cytoplasmic tail. To evaluate the signaling and biological contribution of this G-protein-independent pathway, we generated mutant mice that express cytoplasmic tail-truncated CXCR4 (ΔT) by a gene knock-in approach. We found that ΔT mice exhibited multiple developmental defects, with not only G-protein-independent but also G-protein-dependent signaling events completely abolished, despite ΔT's ability to still associate with G-proteins. These results reveal an essential positive regulatory role of the cytoplasmic tail in CXCR4 signaling and suggest the tail is crucial for mediating G-protein activation and initiating crosstalk between G-protein-dependent and G-protein-independent pathways for correct GPCR signaling

The Stability and Formation of Native Proteins from Unfolded Monomers Is Increased through Interactions with Unrelated Proteins

The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins

Innate Killing of Leishmania donovani by Macrophages of the Splenic Marginal Zone Requires IRF-7

Highly phagocytic macrophages line the marginal zone (MZ) of the spleen and the lymph node subcapsular sinus. Although these macrophages have been attributed with a variety of functions, including the uptake and clearance of blood and lymph-borne pathogens, little is known about the effector mechanisms they employ after pathogen uptake. Here, we have combined gene expression profiling and RNAi using a stromal macrophage cell line with in situ analysis of the leishmanicidal activity of marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM) in wild type and gene targeted mice. Our data demonstrate a critical role for interferon regulatory factor-7 (IRF-7) in regulating the killing of intracellular Leishmania donovani by these specialised splenic macrophage sub-populations. This study, therefore, identifies a new role for IRF-7 as a regulator of innate microbicidal activity against this, and perhaps other, non-viral intracellular pathogens. This study also highlights the importance of selecting appropriate macrophage populations when studying pathogen interactions with this functionally diverse lineage of cells

Duffy antigen receptor for chemokines mediates chemokine endocytosis through a macropinocytosis-like process in endothelial cells

Background: The Duffy antigen receptor for chemokines (DARC) shows high affinity binding to multiple inflammatory CC and CXC chemokines and is expressed by erythrocytes and endothelial cells. Recent evidence suggests that endothelial DARC facilitates chemokine transcytosis to promote neutrophil recruitment. However, the mechanism of chemokine endocytosis by DARC remains unclear. Methodology/Principal Findings: We investigated the role of several endocytic pathways in DARC-mediated ligand internalization. Here we report that, although DARC co-localizes with caveolin-1 in endothelial cells, caveolin-1 is dispensable for DARC-mediated 125I-CXCL1 endocytosis as knockdown of caveolin-1 failed to inhibit ligand internalization. 125I-CXCL1 endocytosis by DARC was also independent of clathrin and flotillin-1 but required cholesterol and was, in part, inhibited by silencing Dynamin II expression. 125I-CXCL1 endocytosis was inhibited by amiloride, cytochalasin D, and the PKC inhibitor Gö6976 whereas Platelet Derived Growth Factor (PDGF) enhanced ligand internalization through DARC. The majority of DARC-ligand interactions occurred on the endothelial surface, with DARC identified along plasma membrane extensions with the appearance of ruffles, supporting the concept that DARC provides a high affinity scaffolding function for surface retention of chemokines on endothelial cells. Conclusions/Significance: These results show DARC-mediated chemokine endocytosis occurs through a macropinocytosis-like process in endothelial cells and caveolin-1 is dispensable for CXCL1 internalization. © 2011 Zhao et al

Can Non-lytic CD8+T Cells Drive HIV-1 Escape?

The CD8+ T cell effector mechanisms that mediate control of HIV-1 and SIV infections remain poorly understood. Recent work suggests that the mechanism may be primarily non-lytic. This is in apparent conflict with the observation that SIV and HIV-1 variants that escape CD8+ T cell surveillance are frequently selected. Whilst it is clear that a variant that has escaped a lytic response can have a fitness advantage compared to the wild-type, it is less obvious that this holds in the face of non-lytic control where both wild-type and variant infected cells would be affected by soluble factors. In particular, the high motility of T cells in lymphoid tissue would be expected to rapidly destroy local effects making selection of escape variants by non-lytic responses unlikely. The observation of frequent HIV-1 and SIV escape poses a number of questions. Most importantly, is the consistent observation of viral escape proof that HIV-1- and SIV-specific CD8+ T cells lyse infected cells or can this also be the result of non-lytic control? Additionally, the rate at which a variant strain escapes a lytic CD8+ T cell response is related to the strength of the response. Is the same relationship true for a non-lytic response? Finally, the potential anti-viral control mediated by non-lytic mechanisms compared to lytic mechanisms is unknown. These questions cannot be addressed with current experimental techniques nor with the standard mathematical models. Instead we have developed a 3D cellular automaton model of HIV-1 which captures spatial and temporal dynamics. The model reproduces in vivo HIV-1 dynamics at the cellular and population level. Using this model we demonstrate that non-lytic effector mechanisms can select for escape variants but that outgrowth of the variant is slower and less frequent than from a lytic response so that non-lytic responses can potentially offer more durable control

Comparison of wheat spindle streak mosaic virus (WSSMV) and barley yellow mosaic virus (BaYMV): 2 closely related bymoviruses

In order to better understand the evolutionary development that leads to different host ranges, we made a comparison of the closely related barley yellow mosaic bymovirus (BaYMV) and wheat spindle streak mosaic bymovirus (WSSMV). Using different methods both viruses were found to be closely related. Leaves of BaYMV-infected winter barley and WSSMV-infected wheat typically showed similar symptoms. Electron microscopy studies revealed that both viruses lead to formation of 2 types of cytoplasmic inclusions bodies (Cl, 'pinwheel' structures; crystal-like respectively membranous) in infected cells that showed structural differences. This close relationship is also reflected by the high degree of amino acid sequence homology (74% in CP-, 83% in Nlb-, 68% in Nla-region). In spite of the remarkably high similarities both viruses have separate hosts. We demonstrated that, independently of the host range, a reliable distinction between WSSMV and BaYMV is possible using Northern blot hybridisation, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. Antisera raised against BaYMV proteins also detected WSSMV proteins in Western blot analyses. Even though corresponding proteins of both viruses were usually similar in size, the BaYMV-Cl antiserum cross-reacted with an additional protein about 8 kDa larger in wheat extracts and the BaYMV 28 kDa antiserum detected a protein of about 30 kDa in wheat extracts.Comparaison du virus de la mosaïque striée du blé (WSSMV) et du virus de la mosaïque jaune de l'orge (BaYMV) : 2 bymovirus très proches. Dans le but de mieux comprendre l'évolution qui mène à différentes gammes d'hôtes de virus transmis par des champignons, nous avons comparé le virus de la mosaïque jaune de l'orge (B AYMV) et le virus de la mosaïque striée du blé (WSSMV). En utilisant différentes méthodes, nous avons remarqué que les 2 virus étaient très proches. Les feuilles de l'orge d'hiver infecté par le Ba YMV et du blé infecté par le WSSMV ont révélé des symptômes similaires. Des études au microscope électronique ont montré que les 2 virus entraînaient la formation d'inclusions cytoplasmiques cylindriques (Cl ; structure «pinwheel») dans les cellules infectées et ont souligné quelques différences structurelles. Ce lien de parenté est également révélé par la forte homologie des séquences d'acides aminés et de cADN (74% en CP, 83% en Nlb et 68% en Nla). Jusqu'à présent, le BaYMV et le WSSMV avaient été distingués sur le seul critère des plantes hôtes. Nous avons montré que, indépendamment de la gamme d'hôtes, des distinctions reproductibles entre WSSMV et BaYMV sont possibles en Northern RT-PCR (transcription reverse associée à la réaction de polymérase en chaîne) et Western blot. Bien que les protéines correspondantes des virus aient généralement une taille similaire, l'antisérum dirigé contre la protéine Cl du BaYMV a réagi chez le blé avec une protéine supplémentaire d'une taille de 8 kDa supérieure à celle de la protéine homologue, tandis que l'antisérum dirigé contre la protéine «28 kDa» du BaYMV reconnaît chez le blé une protéine de 30 kDa

Management of yellow dwarf disease in Europe in a post-neonicotinoid agriculture

Barley/cereal yellow dwarf viruses (YDVs) cause yellow dwarf disease (YDD), which is a continuous risk to cereals production worldwide. These viruses cause leaf yellowing and stunting, resulting in yield reductions of up to 80%. YDVs have been a consistent but low-level problem in European cereal cultivation for the last three decades, mostly due to the availability of several effective insecticides (largely pyrethroids and more recently neonicotinoids) against aphid vectors. However, this has changed recently, with many insecticides being lost, culminating in a recent European Union (EU) regulation prohibiting outdoor use of the neonicotinoid-insecticide compounds. This change is coupled with the growing challenge of insecticide-resistant aphids, the lack of genetic resources against YDVs, and a knowledge deficit around the parameters responsible for the emergence and spread of YDD. This means that economic sustainability of cereal cultivation in several European countries including France and United Kingdom is now again threatened by this aphid-vectored viral disease. In this review, we summarize the current knowledge on the YDV pathosystem, describe management options against YDD, analyse the impacts of the neonicotinoid ban in Europe, and consider future strategies to control YDV