Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells

Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein–coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase–dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal–regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal–regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction.     

Pan-Caspase Inhibitor zVAD Induces Necroptotic and Autophagic Cell Death in TLR3/4-Stimulated Macrophages

In addition to inducing apoptosis, caspase inhibition contributes to necroptosis and/or autophagy depending on the cell type and cellular context. In macrophages, necroptosis can be induced by co-treatment with Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS] for TLR4 and polyinosinic-polycytidylic acid [poly I:C] for TLR3) and a cell-permeable pan-caspase inhibitor zVAD. Here, we elucidated the signaling pathways and molecular mechanisms of cell death. We showed that LPS/zVAD- and poly I:C/zVAD-induced cell death in bone marrow-derived macrophages (BMDMs) was inhibited by receptor-interacting protein kinase 1 (RIP1) inhibitor necrostatin-1 and autophagy inhibitor 3-methyladenine. Electron microscopic images displayed autophagosome/autolysosomes, and immunoblotting data revealed increased LC3II expression. Although zVAD did not affect LPS- or poly I:C-induced activation of IKK, JNK, and p38, it enhanced IRF3 and STAT1 activation as well as type I interferon (IFN) expression. In addition, zVAD inhibited ERK and Akt phosphorylation induced by LPS and poly I:C. Of note, zVAD-induced enhancement of the IRF3/IFN/STAT1 axis was abolished by necrostatin-1, while zVAD-induced inhibition of ERK and Akt was not. Our data further support the involvement of autocrine IFNs action in reactive oxygen species (ROS)-dependent necroptosis, LPS/zVAD-elicited ROS production was inhibited by necrostatin-1, neutralizing antibody of IFN receptor (IFNR) and JAK inhibitor AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. We conclude that enhanced TRIF-RIP1-dependent autocrine action of IFNβ, rather than inhibition of ERK or Akt, is involved in TLRs/zVAD-induced autophagic and necroptotic cell death via the JAK/STAT1/ROS pathway.     

Modulation of lncRNA H19 enhances resveratrol‐inhibited cancer cell proliferation and migration by regulating endoplasmic reticulum stress

The phytoalexin resveratrol exhibits anti‐tumour activity in many types of cancer. In this study, we showed that resveratrol suppressed the survival of gastric tumour cells both in vivo and in vitro. Resveratrol promoted apoptosis, autophagy and endoplasmic reticulum (ER) stress in a dose‐dependent manner. RNA‐seq analysis showed that multiple cell death signalling pathways were activated after resveratrol treatment, while the use of ER stress activators (tunicamycin and thapsigargin) in combinatorial with resveratrol led to further inhibition of cancer cell survival. Results also showed that resveratrol altered the expression of several long non‐coding RNAs (lncRNAs), including MEG3, PTTG3P, GAS5, BISPR, MALAT1 and H19. Knockdown of H19 in resveratrol‐treated cells further enhanced the effects of resveratrol on apoptosis, ER stress and cell cycle S‐phase arrest. Furthermore, the migratory ability of resveratrol‐treated cells was dramatically decreased after H19 knockdown. In conclusion, resveratrol inhibited cancer cell survival, while knockdown of lncRNA H19 resulted in increased sensitivity to resveratrol therapy.     

Why did only one genus of insects,Halobates, take to the high seas?

Oceans cover more than 70% of the Earth’s surface and house a dizzying array of organisms. Mammals, birds, and all manner of fish can be commonly sighted at sea, but insects, the world’s most common animals, seem to be completely absent. Appearances can deceive, however, as 5 species of the ocean skater Halobates live exclusively at the ocean surface. Discovered 200 years ago, these peppercorn-sized insects remain rather mysterious. How do they cope with life at the ocean surface, and why are they the only genus of insects to have taken to the high seas?

Oceans cover over 70% of the earth’s surface and house a dizzying array of organisms, including five species of the peppercorn-sized ocean-skater Halobates, which live exclusively at the ocean surface. How do they cope with life at the ocean surface and why are they the only genus of insects able to conquer the high seas?     

四种昼行性动物取食和贮藏红松种子的行为比较

于2005年10月1~7日,采用全时焦点观察法研究了黑龙江凉水国家级自然保护区19林班原始红松林内松鼠(Sciurus vulgaris)、星鸦(Nucifraga caryocatactes)、花鼠(Eutamias sibiricus)和普通(Sitta europaea)4种昼行性动物取食和贮藏红松种子的行为。结果表明①4种动物的取食和贮藏过程包括找寻、松塔的处理与种子剥食、松塔和种子的搬运、确定贮藏点、掩藏和警戒等6个环节,但它们在6个环节所耗费的时间均存在较大差异;②4种动物的种子消耗量亦存在较大差异(χ2=144.67,df=3,P<0.05),松鼠、星鸦、花鼠和普通每次平均取食红松种子的数量分别为40.0粒、20.9粒、16.7粒和1.3粒。松鼠与星鸦能够在地面和树冠层取食或收获红松松塔及种子,而花鼠与普通则主要在地面取食;③4种动物贮藏种子的能力亦有较大差异(χ2=68.76,df=3,P<0.05Kruskal-Wallis),松鼠、星鸦和普通主要分散贮藏红松种子,松鼠和星鸦的贮藏量最多,而花鼠很少分散贮藏种子,但能将许多红松种子集中贮藏在少数的贮藏点;④取食松籽的种间竞争强烈,在红松林的垂直高度上,松鼠和星鸦具有高度重叠的取食生态位,花鼠和普通因为种群数量大,对地面种子库的消耗量较大。分析结果显示,松鼠与星鸦是红松种子扩散的传播者,普通是种子消耗者兼传播者,而花鼠则近乎是完全的捕食者。     

Cancer-associated adipocytes promote the invasion and metastasis in breast cancer through LIF/CXCLs positive feedback loop

Cancer-associated adipocytes (CAAs), which are adipocytes transformed by cancer cells, are of great importance in promoting the progression of breast cancer. However, the underlying mechanisms involved in the crosstalk between cancer cells and adipocytes are still unknown. Here we report that CAAs and breast cancer cells communicate with each other by secreting the cytokines leukemia inhibitory factor (LIF) and C-X-C subfamily chemokines (CXCLs), respectively. LIF is a pro-inflammatory cytokine secreted by CAAs, which promotes migration and invasion of breast cancer cells via the Stat3 signaling pathway. The activation of Stat3 induced the secretion of glutamic acid-leucine-arginine (ELR) motif CXCLs (CXCL1, CXCL2, CXCL3 and CXCL8) in tumor cells. Interestingly, CXCLs in turn activated the ERK1/2/NF-κB/Stat3 signaling cascade to promote the expression of LIF in CAAs. In clinical breast cancer pathology samples, the up-regulation of LIF in paracancerous adipose tissue was positively correlated with the activation of Stat3 in breast cancer. Furthermore, we verified that adipocytes enhanced lung metastasis of breast cancer cells, and the combination of EC330 (targeting LIF) and SB225002 (targeting C-X-C motility chemokine receptor 2 (CXCR2)) significantly reduced lung metastasis of breast cancer cells in vivo. Our findings reveal that the interaction of adipocytes with breast cancer cells depends on a positive feedback loop between the cytokines LIF and CXCLs, which promotes breast cancer invasion and metastasis.     

Draft Genome Assembly and Annotation for Cutaneotrichosporon dermatis NICC30027, an Oleaginous Yeast Capable of Simultaneous Glucose and Xylose Assimilation

The identification of oleaginous yeast species capable of simultaneously utilizing xylose and glucose as substrates to generate value-added biological products is an area of key economic interest. We have previously demonstrated that the Cutaneotrichosporon dermatis NICC30027 yeast strain is capable of simultaneously assimilating both xylose and glucose, resulting in considerable lipid accumulation. However, as no high-quality genome sequencing data or associated annotations for this strain are available at present, it remains challenging to study the metabolic mechanisms underlying this phenotype. Herein, we report a 39,305,439 bp draft genome assembly for C. dermatis NICC30027 comprised of 37 scaffolds, with 60.15% GC content. Within this genome, we identified 524 tRNAs, 142 sRNAs, 53 miRNAs, 28 snRNAs, and eight rRNA clusters. Moreover, repeat sequences totaling 1,032,129 bp in length were identified (2.63% of the genome), as were 14,238 unigenes that were 1,789.35 bp in length on average (64.82% of the genome). The NCBI non-redundant protein sequences (NR) database was employed to successfully annotate 11,795 of these unigenes, while 3,621 and 11,902 were annotated with the Swiss-Prot and TrEMBL databases, respectively. Unigenes were additionally subjected to pathway enrichment analyses using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Cluster of Orthologous Groups of proteins (COG), Clusters of orthologous groups for eukaryotic complete genomes (KOG), and Non-supervised Orthologous Groups (eggNOG) databases. Together, these results provide a foundation for future studies aimed at clarifying the mechanistic basis for the ability of C. dermatis NICC30027 to simultaneously utilize glucose and xylose to synthesize lipids.     

Amino acid starvation‐induced LDLR trafficking accelerates lipoprotein endocytosis and LDL clearance

Mammalian cells utilize Akt‐dependent signaling to deploy intracellular Glut4 toward cell surface to facilitate glucose uptake. Low‐density lipoprotein receptor (LDLR) is the cargo receptor mediating endocytosis of apolipoprotein B‐containing lipoproteins. However, signaling‐controlled regulation of intracellular LDLR trafficking remains elusive. Here, we describe a unique amino acid stress response, which directs the deployment of intracellular LDLRs, causing enhanced LDL endocytosis, likely via Ca²⁺ and calcium/calmodulin‐dependent protein kinase II‐mediated signalings. This response is independent of induction of autophagy. Amino acid stress‐induced increase in LDL uptake in vitro is comparable to that by pravastatin. In vivo, acute AAS challenge for up to 72 h enhanced the rate of hepatic LDL uptake without changing the total expression level of LDLR. Reducing dietary amino acids by 50% for 2 to 4 weeks ameliorated high fat diet‐induced hypercholesterolemia in heterozygous LDLR‐deficient mice, with reductions in both LDL and VLDL fractions. We suggest that identification of signaling‐controlled regulation of intracellular LDLR trafficking has advanced our understanding of the LDLR biology, and may benefit future development of additional therapeutic strategies for treating hypercholesterolemia.     

How does a small molecule bind at a cryptic binding site?

Protein-protein interactions (PPIs) are ubiquitous biomolecular processes that are central to virtually all aspects of cellular function. Identifying small molecules that modulate specific disease-related PPIs is a strategy with enormous promise for drug discovery. The design of drugs to disrupt PPIs is challenging, however, because many potential drug-binding sites at PPI interfaces are “cryptic”: When unoccupied by a ligand, cryptic sites are often flat and featureless, and thus not readily recognizable in crystal structures, with the geometric and chemical characteristics of typical small-molecule binding sites only emerging upon ligand binding. The rational design of small molecules to inhibit specific PPIs would benefit from a better understanding of how such molecules bind at PPI interfaces. To this end, we have conducted unbiased, all-atom MD simulations of the binding of four small-molecule inhibitors (SP4206 and three SP4206 analogs) to interleukin 2 (IL2)—which performs its function by forming a PPI with its receptor—without incorporating any prior structural information about the ligands’ binding. In multiple binding events, a small molecule settled into a stable binding pose at the PPI interface of IL2, resulting in a protein–small-molecule binding site and pose virtually identical to that observed in an existing crystal structure of the IL2-SP4206 complex. Binding of the small molecule stabilized the IL2 binding groove, which when the small molecule was not bound emerged only transiently and incompletely. Moreover, free energy perturbation (FEP) calculations successfully distinguished between the native and non-native IL2–small-molecule binding poses found in the simulations, suggesting that binding simulations in combination with FEP may provide an effective tool for identifying cryptic binding sites and determining the binding poses of small molecules designed to disrupt PPI interfaces by binding to such sites.