Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library

Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.     

A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements.

We have identified and characterized a new orphan member of the nuclear hormone receptor superfamily, called MB67, which is predominantly expressed in liver. MB67 binds and transactivates the retinoic acid response elements that control expression of the retinoic acid receptor beta 2 and alcohol dehydrogenase 3 genes, both of which consist of a direct repeat hexamers related to the consensus AGGTCA, separated by 5 bp. MB67 binds these elements as a heterodimer with the 9-cis-retinoic acid receptor, RXR. However, MB67 does not bind or activate other retinoic acid response elements with alternative hexamer arrangements or any of several other wild-type and synthetic hormone response elements examined. The transactivation of retinoic acid response elements by MB67 is weaker than that conferred by the retinoic acid receptors but does not require the presence of all-trans retinoic acid, 9-cis-retinoic acid, or any exogenously added ligand. We propose that MB67 plays an important role in the complex network of proteins that govern response to retinoic acid and its metabolites.     

Prostaglandin E2 augments IL-10 signaling and function

In inflamed joints of rheumatoid arthritis, PGE(2) is highly expressed, and IL-10 and IL-6 are also abundant. PGE(2) is a well-known activator of the cAMP signaling pathway, and there is functional cross-talk between cAMP signaling and the Jak-STAT signaling pathway. In this study, we evaluated the modulating effect of PGE(2) on STAT signaling and its biological function induced by IL-10 and IL-6, and elucidated its mechanism in THP-1 cells. STAT phosphorylation was determined by Western blot, and gene expression was analyzed using real-time PCR. Pretreatment with PGE(2) significantly augmented IL-10-induced STAT3 and STAT1 phosphorylation, as well as suppressors of cytokine signaling 3 (SOCS3) and IL-1R antagonist gene expression. In contrast, PGE(2) suppressed IL-6-induced phosphorylation of STAT3 and STAT1. These PGE(2)-induced modulating effects were largely reversed by actinomycin D. Pretreatment with dibutyryl cAMP augmented IL-10-induced, but did not change IL-6-induced STAT3 phosphorylation. Misoprostol, an EP2/3/4 agonist, and butaprost, an EP2 agonist, augmented IL-10-induced STAT3 phosphorylation and SOCS3 gene expression, but sulprostone, an EP1/3 agonist, had no effect. H89, a protein kinase A inhibitor, and LY294002, a PI3K inhibitor, diminished PGE(2)-mediated augmentation of IL-10-induced STAT3 phosphorylation. In this study, we found that PGE(2) selectively regulates cytokine signaling via increased intracellular cAMP levels and de novo gene expression, and these modulating effects may be mediated through EP2 or EP4 receptors. PGE(2) may modulate immune responses by alteration of cytokine signaling in THP-1 cells.     

Relationship of the lung microbiome with PD-L1 expression and immunotherapy response in lung cancer

IL-35 subunit EBI3 is up-regulated in pulmonary fibrosis tissues. In this study, we investigated the pathological role of EBI3 in pulmonary fibrosis and dissected the underlying molecular mechanism. Bleomycin-induced pulmonary fibrosis mouse model was established, and samples were performed gene expression analyses through RNAseq, qRT-PCR and Western blot. Wild type and EBI3 knockout mice were exposed to bleomycin to investigate the pathological role of IL-35, via lung function and gene expression analyses. Primary lung epithelial cells were used to dissect the regulatory mechanism of EBI3 on STAT1/STAT4 and STAT3. IL-35 was elevated in both human and mouse with pulmonary fibrosis. EBI3 knockdown aggravated the symptoms of pulmonary fibrosis in mice. EBI3 deficiency enhanced the expressions of fibrotic and extracellular matrix-associated genes. Mechanistically, IL-35 activated STAT1 and STAT4, which in turn suppressed DNA enrichment of STAT3 and inhibited the fibrosis process. IL-35 might be one of the potential therapeutic targets for bleomycin-induced pulmonary fibrosis.     

Programmable in situ amplification for multiplexed imaging of mRNA expression

In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos, representing a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. Here, we report a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reactions (HCR). With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample. Robust performance is achieved when imaging five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos. HCR amplifiers exhibit deep sample penetration, high signal-to-background ratios and sharp signal localization.     

Kaposi's sarcoma-associated herpesvirus OX2 glycoprotein activates myeloid-lineage cells to induce inflammatory cytokine production

Kaposi's sarcoma is an inflammatory cytokine-mediated angioproliferative disease which is triggered by infection by Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV contains an open reading frame, K14, that has significant homology with cellular OX2, designated viral OX2 (vOX2). In this report, we demonstrate that vOX2 encodes a glycosylated cell surface protein with an apparent molecular mass of 55 kDa. Purified glycosylated vOX2 protein dramatically stimulated primary monocytes, macrophages, and dendritic cells to produce the inflammatory cytokines interleukin 1beta (IL-1beta), IL-6, monocyte chemoattractant protein 1, and TNF-alpha. Furthermore, expression of vOX2 on B lymphocytes stimulated monocytes to produce inflammatory cytokines in mixed culture. These results demonstrate that like its cellular counterpart, vOX2 targets myeloid-lineage cells, but unlike cellular OX2, which delivers a restrictive signal, KSHV vOX2 provides an activating signal, resulting in the production of inflammatory cytokines. Thus, this is a novel viral strategy where KSHV has acquired the cellular OX2 gene to induce inflammatory cytokine production, which potentially promotes the cytokine-mediated angiogenic proliferation of KSHV-infected cells.     

Minimalistic Predictor of Protein Binding Energy: Contribution of Solvation Factor to Protein Binding

It has long been known that solvation plays an important role in protein-protein interactions. Here, we use a minimalistic solvation-based model for predicting protein binding energy to estimate quantitatively the contribution of the solvation factor in protein binding. The factor is described by a simple linear combination of buried surface areas according to amino-acid types. Even without structural optimization, our minimalistic model demonstrates a predictive power comparable to more complex methods, making the proposed approach the basis for high throughput applications. Application of the model to a proteomic database shows that receptor-substrate complexes involved in signaling have lower affinities than enzyme-inhibitor and antibody-antigen complexes, and they differ by chemical compositions on interfaces. Also, we found that protein complexes with components that come from the same genes generally have lower affinities than complexes formed by proteins from different genes, but in this case the difference originates from different interface areas. The model was implemented in the software PYTHON, and the source code can be found on the Shakhnovich group webpage: http://faculty.chemistry.harvard.edu/shakhnovich/software.     

Organization and assembly of the TRAPPII complex

Current models suggest that TRAPP tethering complexes exist in two forms. Whereas the seven-subunit TRAPPI complex mediates ER-to-Golgi transport, TRAPPII contains three additional subunits (Trs65, Trs120 and Trs130) and is required for distinct tethering events at Golgi membranes. It is not clear how TRAPPII assembly is regulated. Here, we show that Tca17 is a fourth TRAPPII-specific component, and that Trs65 and Tca17 interact with distinct domains of Trs130 and make different contributions to complex assembly. Whereas Tca17 promotes the stable association of TRAPPII-specific subunits with the core complex, Trs65 stabilizes TRAPPII in an oligomeric form. We show that Trs85, which was previously reported to be a subunit of both TRAPPI and TRAPPII, is not associated with the TRAPPII complex in yeast. However, we find that proteins related to Trs85, Trs65 and Tca17 are part of the same TRAPP complex in mammalian cells. These findings have implications for models of TRAPP complex formation and suggest that TRAPP complexes may be organized differently in yeast and mammals.