Identification of Drosophila Yin and PEPT2 as Evolutionarily Conserved Phagosome-associated Muramyl Dipeptide Transporters

NOD2 (nucleotide-binding oligomerization domain containing 2) is an important cytosolic pattern recognition receptor that activates NF-κB and other immune effector pathways such as autophagy and antigen presentation. Despite its intracellular localization, NOD2 participates in sensing of extracellular microbes such as Staphylococcus aureus. NOD2 ligands similar to the minimal synthetic ligand muramyl dipeptide (MDP) are generated by internalization and processing of bacteria in hydrolytic phagolysosomes. However, how these derived ligands exit this organelle and access the cytosol to activate NOD2 is poorly understood. Here, we address how phagosome-derived NOD2 ligands access the cytosol in human phagocytes. Drawing on data from Drosophila phagosomes, we identify an evolutionarily conserved role of SLC15A transporters, Drosophila Yin and PEPT2, as MDP transporters in fly and human phagocytes, respectively. We show that PEPT2 is highly expressed by human myeloid cells. Ectopic expression of both Yin and PEPT2 increases the sensitivity of NOD2-dependent NF-κB activation. Additionally, we show that PEPT2 associates with phagosome membranes. Together, these data identify Drosophila Yin and PEPT2 as evolutionarily conserved phagosome-associated transporters that are likely to be of particular importance in delivery of bacteria-derived ligands generated in phagosomes to cytosolic sensors recruited to the vicinity of these organelles.     

Effects of inflammatory cytokine IL-27 on the activation of fibroblast-like synoviocytes in rheumatoid arthritis

Introduction  

Interleukin (IL)-27 is a novel member of the IL-6/IL-12 family cytokines that are produced early by antigen-presenting cells in T helper (Th)1-mediated inflammation. Elevated expression of IL-27 has been detected in the synovial membranes and fluid of rheumatoid arthritis (RA).     

Common requirements for melanocortin-4 receptor selectivity of structurally unrelated melanocortin agonist and endogenous antagonist, Agouti protein

The activity of melanocortin receptors (MCR) is regulated by melanocortin peptide agonists and by the endogenous antagonists, Agouti protein and AgRP (Agouti-related protein). To understand how the selectivity for these structurally unrelated agonists and antagonist is achieved, chimeric and mutants MC3R and MC4R were expressed in cell lines and pharmacologically analyzed. A region containing the third extracellular loop, EC3, of MC4R was essential for selective Agouti protein antagonism. In addition, this part of MC4R, when introduced in MC3R, conferred Agouti protein antagonism. Further mutational analysis of this region of MC4R demonstrated that Tyr(268) was required for the selective interaction with Agouti protein, because a profound loss of the ability of Agouti protein to inhibit (125)I-labeled [Nle(4),d-Phe(7)]alpha-melanocyte-stimulating hormone (MSH) binding was observed by the single mutation of Tyr(268) to Ile. This same residue conferred selectivity for the MC4R selective agonist, [d-Tyr(4)]MT-II, whereas it inhibited interaction with the MC3R-selective agonist, [Nle(4)]Lys-gamma(2)-MSH. Conversely, mutation of Ile(265) in MC3 (the corresponding residue of Tyr(268)) to Tyr displayed a gain of affinity for [d-Tyr(4)]MT-II, but not for Agouti protein, and a loss of affinity for [Nle(4)]Lys-gamma(2)-MSH as compared with wild-type MC3R. This single amino acid mutation thus confers the selectivity of MC3R toward a pharmacological profile like that observed for MC4R agonists but not for the antagonist, Agouti protein. Thus, selectivity for structurally unrelated ligands with opposite activities is achieved in a similar manner for MC4R but not for MC3R.     

Variability of the canonical loop conformations in serine proteinases inhibitors and other proteins

Canonical loops of protein inhibitors of serine proteinases occur in proteins having completely different folds. In this article, conformations of the loops have been analyzed for inhibitors belonging to 10 structurally different families. Using deviation in C_α-C_α distances as a criterion for loop similarity, we found that the P3-P3′ segment defines most properly the length of the loop. When conformational differences among loops of individual inhibitors were compared using root mean square deviation (rmsd) in atomic coordinates for all main chain atoms (Δr method) and rmsd operating in main chain torsion angles (Δt method), differences of up to 2.1 Å and 72.3°, respectively, were observed. Such large values indicate significant conformational differences among individual loops. Nevertheless, the overall geometry of the inhibitor-proteinase interaction is very well preserved, as judged from the similarity of C_α-C_α distances between C_α of catalytic Ser and C_α of P3-P3′ residues in various enzyme-inhibitor complexes. The mode of interaction is very well preserved both in the chymotrypsin and subtilisin families, as distances calculated for subtilisin-inhibitor complexes are almost always within the range of those for chymotrypsin-inhibitor complexes. Complex formation leads to conformational changes of up to 160° for χ₁ angle. Side chains of residue P1 and P2′ adopt in different complexes a similar orientation (χ₁ angle = −60° and −180°, respectively). To check whether the canonical conformation can be found among non–proteinase-inhibitor Brookhaven Protein Data Bank structures, two selection criteria—the allowed main chain dihedral angles and C_α-C_α distances for the P3-P3′ segment—were applied to all these structures. This procedure detected 132 unique hexapeptide segments in 121 structurally and functionally unrelated proteins. Partial preferences for certain amino acids occurring at particular positions in these hexapeptides could be noted. Further restriction of this set to hexapeptides with a highly exposed P1 residue side chain resulted in 40 segments. The possibility of complexes formation between these segments and serine proteinases was ruled out in molecular modeling due to steric clashes. Several structural features that determine the canonical conformation of the loop both in inhibitors and in other proteins can be distinguished. They include main chain hydrogen bonds both within the P3-P3′ segment and with the scaffold region, P3-P4 and P3′-P4′ hydrophobic interactions, and finally either hydrophobic or polar interactions involving the P1′ residue. Proteins 32:459–474, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of the active site of DNA polymerase beta by molecular dynamics and quantum chemical calculation

It is well established that the fully formed polymerase active site of the DNA repair enzyme, polymerase beta (pol beta), including two bound Mg2+ cations and the nucleoside triphosphate (dNTP) substrate, exists at only one point in the catalytic cycle just prior to the chemical nucleotidyl transfer step. The structure of the active conformation has been the subject of much interest as it relates to the mechanism of the chemical step and also to the question of fidelity assurance. Although crystal structures of ternary pol beta-(primer-template) DNA-dNTP complexes have provided the main structural features of the active site, they are necessarily incomplete due to intentional alterations (e.g., removal of the 3'OH groups from primer and substrate) needed to obtain a structure from midcycle. Working from the crystal structure closest to the fully formed active site [Protein Data Bank (PDB) code: 1bpy], two molecular dynamics (MD) simulations of the solvated ternary complex were performed: one with the missing 3'OHs restored, via modeling, to the primer and substrate, and the other without restoration of the 3'OHs. The results of the simulations, together with ab initio optimizations on simplified active-site models, indicate that the missing primer 3'OH in the crystal structure is responsible for a significant perturbation in the coordination sphere of the catalytic cation and allow us to suggest several corrections and additions to the active-site structure as observed by crystallography. In addition, the calculations help to resolve questions raised regarding the protonation states of coordinating ligands.     

Azithromycin and cough-specific health status in patients with chronic obstructive pulmonary disease and chronic cough: a randomised controlled trial

Background

Macrolides reduce exacerbations in patients with COPD. Their effects on health status has not been assessed as primary outcome and is less clear. This study assessed the effects of prophylactic azithromycin on cough-specific health status in COPD-patients with chronic productive cough.

Methods

In this randomised controlled trial 84 patients met the eligibility criteria: age of ≥40 years, COPD GOLD stage ≥2 and chronic productive cough. The intervention-group (n = 42) received azithromycin 250 mg 3 times a week and the control-group (n = 42) received a placebo. Primary outcome was cough-specific health status at 12 weeks, measured with the Leicester Cough Questionnaire (LCQ). Secondary outcomes included generic and COPD-specific health status and exacerbations. Changes in adverse events and microbiology were monitored.

Results

Mean age of participants was 68 ± 10 years and mean FEV1 was 1.36 ± 0.47 L. The improvement in LCQ total score at 12 weeks was significantly greater with azithromycin (difference 1.3 ± 0.5, 95% CI 0.3;2.3, p = 0.01) and met the minimal clinically important difference. Similar results were found for the domain scores, and COPD-specific and generic health status questionnaires. Other secondary endpoints were non-significant. No imbalances in adverse events were found.

Conclusions

Prophylactic azithromycin improved cough-specific health status in COPD-patients with chronic productive cough to a clinically relevant degree.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01071161","term_id":"NCT01071161"}}NCT01071161     

Large oncosomes mediate intercellular transfer of functional microRNA

Prostate cancer cells release atypically large extracellular vesicles (EVs), termed large oncosomes, which may play a role in the tumor microenvironment by transporting bioactive molecules across tissue spaces and through the blood stream. In this study, we applied a novel method for selective isolation of large oncosomes applicable to human platelet-poor plasma, where the presence of caveolin-1-positive large oncosomes identified patients with metastatic disease. This procedure was also used to validate results of a miRNA array performed on heterogeneous populations of EVs isolated from tumorigenic RWPE-2 prostate cells and from isogenic non-tumorigenic RWPE-1 cells. The results showed that distinct classes of miRNAs are expressed at higher levels in EVs derived from the tumorigenic cells in comparison to their non-tumorigenic counterpart. Large oncosomes enhanced migration of cancer-associated fibroblasts (CAFs), an effect that was increased by miR-1227, a miRNA abundant in large oncosomes produced by RWPE-2 cells. Our findings suggest that large oncosomes in the circulation report metastatic disease in patients with prostate cancer, and that this class of EV harbors functional molecules that may play a role in conditioning the tumor microenvironment.     

Zoospore interspecific signaling promotes plant infection by <Emphasis Type="Italic">Phytophthora</Emphasis>

Background  

Oomycetes attack a huge variety of economically and ecologically important plants. These pathogens release, detect and respond to signal molecules to coordinate their communal behaviors including the infection process. When signal molecules are present at or above threshold level, single zoospores can infect plants. However, at the beginning of a growing season population densities of individual species are likely below those required to reach a quorum and produce threshold levels of signal molecules to trigger infection. It is unclear whether these molecules are shared among related species and what their chemistries are.