Showing papers in "Cell Research in 2015"

Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis.

Abstract: Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis

Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion

Abstract: Inflammasome is an intracellular signaling complex of the innate immune system. Activation of inflammasomes promotes the secretion of interleukin 1β (IL-1β) and IL-18 and triggers pyroptosis. Caspase-1 and -11 (or -4/5 in human) in the canonical and non-canonical inflammasome pathways, respectively, are crucial for inflammasome-mediated inflammatory responses. Here we report that gasdermin D (GSDMD) is another crucial component of inflammasomes. We discovered the presence of GSDMD protein in nigericin-induced NLRP3 inflammasomes by a quantitative mass spectrometry-based analysis. Gene deletion of GSDMD demonstrated that GSDMD is required for pyroptosis and for the secretion but not proteolytic maturation of IL-1β in both canonical and non-canonical inflammasome responses. It was known that GSDMD is a substrate of caspase-1 and we showed its cleavage at the predicted site during inflammasome activation and that this cleavage was required for pyroptosis and IL-1β secretion. Expression of the N-terminal proteolytic fragment of GSDMD can trigger cell death and N-terminal modification such as tagging with Flag sequence disrupted the function of GSDMD. We also found that pro-caspase-1 is capable of processing GSDMD and ASC is not essential for GSDMD to function. Further analyses of LPS plus nigericin- or Salmonella typhimurium-treated macrophage cell lines and primary cells showed that apoptosis became apparent in Gsdmd(-/-) cells, indicating a suppression of apoptosis by pyroptosis. The induction of apoptosis required NLRP3 or other inflammasome receptors and ASC, and caspase-1 may partially contribute to the activation of apoptotic caspases in Gsdmd(-/-) cells. These data provide new insights into the molecular mechanisms of pyroptosis and reveal an unexpected interplay between apoptosis and pyroptosis.

Metabolic reprogramming in macrophages and dendritic cells in innate immunity

Abstract: Activation of macrophages and dendritic cells (DCs) by pro-inflammatory stimuli causes them to undergo a metabolic switch towards glycolysis and away from oxidative phosphorylation (OXPHOS), similar to the Warburg effect in tumors. However, it is only recently that the mechanisms responsible for this metabolic reprogramming have been elucidated in more detail. The transcription factor hypoxia-inducible factor-1α (HIF-1α) plays an important role under conditions of both hypoxia and normoxia. The withdrawal of citrate from the tricarboxylic acid (TCA) cycle has been shown to be critical for lipid biosynthesis in both macrophages and DCs. Interference with this process actually abolishes the ability of DCs to activate T cells. Another TCA cycle intermediate, succinate, activates HIF-1α and promotes inflammatory gene expression. These new insights are providing us with a deeper understanding of the role of metabolic reprogramming in innate immunity.

Use of double-stranded dna in exosomes: a novel biomarker in cancer detection

Abstract: The present invention is directed to methods of prognosing, treating, or managing treatment of cancer in a subject. These methods involve selecting a subject having cancer, obtaining, from the selected subject, a sample containing exosomes, recovering the exosomes from the sample, and isolating the double-stranded DNA from within the exosomes. The isolated double-stranded DNA is then used to detect the presence or absence of one or more genetic mutations associated with cancer, quantify the amount of isolated double-stranded DNA from the recovered exosomes in the sample, detect the methylation status of the isolated double- stranded DNA, or quantify the amount isolated double-stranded DNA able to enter a recipient cell. The prognosing, treating, or managing treatment is carried out based on this information.

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response.

Abstract: The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.

Methylation-dependent loss of RIP3 expression in cancer represses programmed necrosis in response to chemotherapeutics

Abstract: Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is an essential part of the cellular machinery that executes “programmed” or “regulated” necrosis. Here we show that programmed necrosis is activated in response to many chemotherapeutic agents and contributes to chemotherapy-induced cell death. However, we show that RIP3 expression is often silenced in cancer cells due to genomic methylation near its transcriptional start site, thus RIP3-dependent activation of MLKL and downstream programmed necrosis during chemotherapeutic death is largely repressed. Nevertheless, treatment with hypomethylating agents restores RIP3 expression, and thereby promotes sensitivity to chemotherapeutics in a RIP3-dependent manner. RIP3 expression is reduced in tumors compared to normal tissue in 85% of breast cancer patients, suggesting that RIP3 deficiency is positively selected during tumor growth/development. Since hypomethylating agents are reasonably well-tolerated in patients, we propose that RIP3-deficient cancer patients may benefit from receiving hypomethylating agents to induce RIP3 expression prior to treatment with conventional chemotherapeutics.

Honeysuckle-encoded atypical microRNA2911 directly targets influenza A viruses

Abstract: Influenza A viruses (IAVs), particularly H1N1, H5N1 and H7N9, pose a substantial threat to public health worldwide. Here, we report that MIR2911, a honeysuckle (HS)-encoded atypical microRNA, directly targets IAVs with a broad spectrum. MIR2911 is highly stable in HS decoction, and continuous drinking or gavage feeding of HS decoction leads to a significant elevation of the MIR2911 level in mouse peripheral blood and lung. Bioinformatics prediction and a luciferase reporter assay showed that MIR2911 could target various IAVs, including H1N1, H5N1 and H7N9. Synthetic MIR2911 significantly inhibited H1N1-encoded PB2 and NS1 protein expression, but did not affect mutants in which the MIR2911-binding nucleotide sequences were altered. Synthetic MIR2911, extracted RNA from HS decoction and HS decoction all significantly inhibited H1N1 viral replication and rescued viral infection-induced mouse weight loss, but did not affect infection with a mutant virus in which the MIR2911-binding nucleotide sequences of PB2 and NS1 were altered. Importantly, the inhibitory effect of HS decoction on viral replication was abolished by an anti-MIR2911 antagomir, indicating that the physiological concentration of MIR2911 in HS decoction could directly and sufficiently suppress H1N1 viral replication. MIR2911 also inhibited H5N1 and H7N9 viral replication in vitro and in vivo. Strikingly, administration of MIR2911 or HS decoction dramatically reduced mouse mortality caused by H5N1 infection. Our results demonstrate that MIR2911 is the first active component identified in Traditional Chinese Medicine to directly target various IAVs and may represent a novel type of natural product that effectively suppresses viral infection.

MIWI and piRNA-mediated cleavage of messenger RNAs in mouse testes.

Abstract: The piRNA machinery is known for its role in mediating epigenetic silencing of transposons. Recent studies suggest that this function also involves piRNA-guided cleavage of transposon-derived transcripts. As many piRNAs also appear to have the capacity to target diverse mRNAs, this raises the intriguing possibility that piRNAs may act extensively as siRNAs to degrade specific mRNAs. To directly test this hypothesis, we compared mouse PIWI (MIWI)-associated piRNAs with experimentally identified cleaved mRNA fragments from mouse testes, and observed cleavage sites that predominantly occur at position 10 from the 5' end of putative targeting piRNAs. We also noted strong biases for U and A residues at nucleotide positions 1 and 10, respectively, in both piRNAs and mRNA fragments, features that resemble the pattern of piRNA amplification by the 'ping-pong' cycle. Through mapping of MIWI-RNA interactions by CLIP-seq and gene expression profiling, we found that many potential piRNA-targeted mRNAs directly interact with MIWI and show elevated expression levels in the testes of Miwi catalytic mutant mice. Reporter-based assays further revealed the importance of base pairing between piRNAs and mRNA targets and the requirement for both the slicer activity and piRNA-loading ability of MIWI in piRNA-mediated target repression. Importantly, we demonstrated that proper turnover of certain key piRNA targets is essential for sperm formation. Together, these findings reveal the siRNA-like function of the piRNA machinery in mouse testes and its central requirement for male germ cell development and maturation.

Heparanase activates the syndecan-syntenin-ALIX exosome pathway

Abstract: Exosomes are secreted vesicles of endosomal origin involved in signaling processes. We recently showed that the syndecan heparan sulfate proteoglycans control the biogenesis of exosomes through their interaction with syntenin-1 and the endosomal-sorting complex required for transport accessory component ALIX. Here we investigated the role of heparanase, the only mammalian enzyme able to cleave heparan sulfate internally, in the syndecan-syntenin-ALIX exosome biogenesis pathway. We show that heparanase stimulates the exosomal secretion of syntenin-1, syndecan and certain other exosomal cargo, such as CD63, in a concentration-dependent manner. In contrast, exosomal CD9, CD81 and flotillin-1 are not affected. Conversely, reduction of endogenous heparanase reduces the secretion of syntenin-1-containing exosomes. The ability of heparanase to stimulate exosome production depends on syntenin-1 and ALIX. Syndecans, but not glypicans, support exosome biogenesis in heparanase-exposed cells. Finally, heparanase stimulates intraluminal budding of syndecan and syntenin-1 in endosomes, depending on the syntenin-ALIX interaction. Taken together, our findings identify heparanase as a modulator of the syndecan-syntenin-ALIX pathway, fostering endosomal membrane budding and the biogenesis of exosomes by trimming the heparan sulfate chains on syndecans. In addition, our data suggest that this mechanism controls the selection of specific cargo to exosomes.

Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration.

Abstract: Cells communicate with each other through secreting and releasing proteins and vesicles. Many cells can migrate. In this study, we report the discovery of migracytosis, a cell migration-dependent mechanism for releasing cellular contents, and migrasomes, the vesicular structures that mediate migracytosis. As migrating cells move, they leave long tubular strands, called retraction fibers, behind them. Large vesicles, which contain numerous smaller vesicles, grow on the tips and intersections of retraction fibers. These fibers, which connect the vesicles with the main cell body, eventually break, and the vesicles are released into the extracellular space or directly taken up by surrounding cells. Since the formation of these vesicles is migration-dependent, we named them “migrasomes”. We also found that cytosolic contents can be transported into migrasomes and released from the cell through migrasomes. We named this migration-dependent release mechanism “migracytosis”.

Recurrent gain-of-function USP8 mutations in Cushing's disease

Abstract: Cushing's disease, also known as adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas (PAs) that cause excess cortisol production, accounts for up to 85% of corticotrophin-dependent Cushing's syndrome cases. However, the genetic alterations in this disease are unclear. Here, we performed whole-exome sequencing of DNA derived from 12 ACTH-secreting PAs and matched blood samples, which revealed three types of somatic mutations in a candidate gene, USP8 (encoding ubiquitin-specific protease 8), exclusively in exon 14 in 8 of 12 ACTH-secreting PAs. We further evaluated somatic USP8 mutations in additional 258 PAs by Sanger sequencing. Targeted sequencing further identified a total of 17 types of USP8 variants in 67 of 108 ACTH-secreting PAs (62.04%). However, none of these mutations was detected in other types of PAs (n = 150). These mutations aggregate within the 14-3-3 binding motif of USP8 and disrupt the interaction between USP8 and 14-3-3 protein, resulting in an elevated capacity to protect EGFR from lysosomal degradation. Accordingly, PAs with mutated USP8 display a higher incidence of EGFR expression, elevated EGFR protein abundance and mRNA expression levels of POMC, which encodes the precursor of ACTH. PAs with mutated USP8 are significantly smaller in size and have higher ACTH production than wild-type PAs. In surgically resected primary USP8-mutated tumor cells, USP8 knockdown or blocking EGFR effectively attenuates ACTH secretion. Taken together, somatic gain-of-function USP8 mutations are common and contribute to ACTH overproduction in Cushing's disease. Inhibition of USP8 or EGFR is promising for treating USP8-mutated corticotrophin adenoma. Our study highlights the potentially functional mutated gene in Cushing's disease and provides insights into the therapeutics of this disease.

cMyc-mediated activation of serine biosynthesis pathway is critical for cancer progression under nutrient deprivation conditions

Abstract: Cancer cells are known to undergo metabolic reprogramming to sustain survival and rapid proliferation, however, it remains to be fully elucidated how oncogenic lesions coordinate the metabolic switch under various stressed conditions. Here we show that deprivation of glucose or glutamine, two major nutrition sources for cancer cells, dramatically activated serine biosynthesis pathway (SSP) that was accompanied by elevated cMyc expression. We further identified that cMyc stimulated SSP activation by transcriptionally upregulating expression of multiple SSP enzymes. Moreover, we demonstrated that SSP activation facilitated by cMyc led to elevated glutathione (GSH) production, cell cycle progression and nucleic acid synthesis, which are essential for cell survival and proliferation especially under nutrient-deprived conditions. We further uncovered that phosphoserine phosphatase (PSPH), the final rate-limiting enzyme of the SSP pathway, is critical for cMyc-driven cancer progression both in vitro and in vivo, and importantly, aberrant expression of PSPH is highly correlated with mortality in hepatocellular carcinoma (HCC) patients, suggesting a potential causal relation between this cMyc-regulated enzyme, or SSP activation in general, and cancer development. Taken together, our results reveal that aberrant expression of cMyc leads to the enhanced SSP activation, an essential part of metabolic switch, to facilitate cancer progression under nutrient-deprived conditions.

LncRNA Dum interacts with Dnmts to regulate Dppa2 expression during myogenic differentiation and muscle regeneration.

Abstract: Emerging studies document the roles of long non-coding RNAs (LncRNAs) in regulating gene expression at chromatin level but relatively less is known how they regulate DNA methylation. Here we identify an lncRNA, Dum (developmental pluripotency-associated 2 (Dppa2) Upstream binding Muscle lncRNA) in skeletal myoblast cells. The expression of Dum is dynamically regulated during myogenesis in vitro and in vivo. It is also transcriptionally induced by MyoD binding upon myoblast differentiation. Functional analyses show that it promotes myoblast differentiation and damage-induced muscle regeneration. Mechanistically, Dum was found to silence its neighboring gene, Dppa2, in cis through recruiting Dnmt1, Dnmt3a and Dnmt3b. Furthermore, intrachromosomal looping between Dum locus and Dppa2 promoter is necessary for Dum/Dppa2 interaction. Collectively, we have identified a novel lncRNA that interacts with Dnmts to regulate myogenesis.

Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells

Abstract: Spermatogonial stem cells (SSCs) can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc (Crygc(-/-)) that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs.

DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination

Abstract: DNMT1 is recruited by PCNA and UHRF1 to maintain DNA methylation after replication. UHRF1 recognizes hemimethylated DNA substrates via the SRA domain, but also repressive H3K9me3 histone marks with its TTD. With systematic mutagenesis and functional assays, we could show that chromatin binding further involved UHRF1 PHD binding to unmodified H3R2. These complementation assays clearly demonstrated that the ubiquitin ligase activity of the UHRF1 RING domain is required for maintenance DNA methylation. Mass spectrometry of UHRF1-deficient cells revealed H3K18 as a novel ubiquitination target of UHRF1 in mammalian cells. With bioinformatics and mutational analyses, we identified a ubiquitin interacting motif (UIM) in the N-terminal regulatory domain of DNMT1 that binds to ubiquitinated H3 tails and is essential for DNA methylation in vivo. H3 ubiquitination and subsequent DNA methylation required UHRF1 PHD binding to H3R2. These results show the manifold regulatory mechanisms controlling DNMT1 activity that require the reading and writing of epigenetic marks by UHRF1 and illustrate the multifaceted interplay between DNA and histone modifications. The identification and functional characterization of the DNMT1 UIM suggests a novel regulatory principle and we speculate that histone H2AK119 ubiquitination might also lead to UIM-dependent recruitment of DNMT1 and DNA methylation beyond classic maintenance.

Cationic nanocarriers induce cell necrosis through impairment of Na(+)/K(+)-ATPase and cause subsequent inflammatory response.

Abstract: Nanocarriers with positive surface charges are known for their toxicity which has limited their clinical applications. The mechanism underlying their toxicity, such as the induction of inflammatory response, remains largely unknown. In the present study we found that injection of cationic nanocarriers, including cationic liposomes, PEI, and chitosan, led to the rapid appearance of necrotic cells. Cell necrosis induced by cationic nanocarriers is dependent on their positive surface charges, but does not require RIP1 and Mlkl. Instead, intracellular Na+ overload was found to accompany the cell death. Depletion of Na+ in culture medium or pretreatment of cells with the Na+/K+-ATPase cation-binding site inhibitor ouabain, protected cells from cell necrosis. Moreover, treatment with cationic nanocarriers inhibited Na+/K+-ATPase activity both in vitro and in vivo. The computational simulation showed that cationic carriers could interact with cation-binding site of Na+/K+-ATPase. Mice pretreated with a small dose of ouabain showed improved survival after injection of a lethal dose of cationic nanocarriers. Further analyses suggest that cell necrosis induced by cationic nanocarriers and the resulting leakage of mitochondrial DNA could trigger severe inflammation in vivo, which is mediated by a pathway involving TLR9 and MyD88 signaling. Taken together, our results reveal a novel mechanism whereby cationic nanocarriers induce acute cell necrosis through the interaction with Na+/K+-ATPase, with the subsequent exposure of mitochondrial damage-associated molecular patterns as a key event that mediates the inflammatory responses. Our study has important implications for evaluating the biocompatibility of nanocarriers and designing better and safer ones for drug delivery.

Direct reprogramming of mouse fibroblasts into cardiomyocytes with chemical cocktails

Abstract: The direct conversion, or transdifferentiation, of non-cardiac cells into cardiomyocytes by forced expression of transcription factors and microRNAs provides promising approaches for cardiac regeneration. However, genetic manipulations raise safety concerns and are thus not desirable in most clinical applications. The discovery of full chemically induced pluripotent stem cells suggest the possibility of replacing transcription factors with chemical cocktails. Here, we report the generation of automatically beating cardiomyocyte-like cells from mouse fibroblasts using only chemical cocktails. These chemical-induced cardiomyocyte-like cells (CiCMs) express cardiomyocyte-specific markers, exhibit sarcomeric organization, and possess typical cardiac calcium flux and electrophysiological features. Genetic lineage tracing confirms the fibroblast origin of these CiCMs. Further studies show the generation of CiCMs passes through a cardiac progenitor stage instead of a pluripotent stage. Bypassing the use of viral-derived factors, this proof of concept study lays a foundation for in vivo cardiac transdifferentiation with pharmacological agents and possibly safer treatment of heart failure.

High salt primes a specific activation state of macrophages, M(Na)

Abstract: High salt is positively associated with the risk of many diseases. However, little is known about the mechanisms. Here we showed that high salt increased proinflammatory molecules, while decreased anti-inflammatory and proendocytic molecules in both human and mouse macrophages. High salt also potentiated lipopolysaccharide-induced macrophage activation and suppressed interleukin 4-induced macrophage activation. High salt induced the proinflammatory aspects by activating p38/cFos and/or Erk1/2/cFos pathways, while inhibited the anti-inflammatory and proendocytic aspects by Erk1/2/signal transducer and activator of transcription 6 pathway. Consistent with the in vitro results, high-salt diet increased proinflammatory gene expression of mouse alveolar macrophages. In mouse models of acute lung injury, high-salt diet aggravated lipopolysaccharide-induced pulmonary macrophage activation and inflammation in lungs. These results identify a novel macrophage activation state, M(Na), and high salt as a potential environmental risk factor for lung inflammation through the induction of M(Na).

The p53-induced lincRNA-p21 derails somatic cell reprogramming by sustaining H3K9me3 and CpG methylation at pluripotency gene promoters.

Abstract: Recent studies have boosted our understanding of long noncoding RNAs (lncRNAs) in numerous biological processes, but few have examined their roles in somatic cell reprogramming. Through expression profiling and functional screening, we have identified that the large intergenic noncoding RNA p21 (lincRNA-p21) impairs reprogramming. Notably, lincRNA-p21 is induced by p53 but does not promote apoptosis or cell senescence in reprogramming. Instead, lincRNA-p21 associates with the H3K9 methyltransferase SETDB1 and the maintenance DNA methyltransferase DNMT1, which is facilitated by the RNA-binding protein HNRNPK. Consequently, lincRNA-p21 prevents reprogramming by sustaining H3K9me3 and/or CpG methylation at pluripotency gene promoters. Our results provide insight into the role of lncRNAs in reprogramming and establish a novel link between p53 and heterochromatin regulation.

The Hippo pathway effectors YAP and TAZ promote cell growth by modulating amino acid signaling to mTORC1.

Abstract: YAP and TAZ are transcriptional co-activators and function as the major effectors of the Hippo tumor suppressor pathway, which controls cell growth, tissue homeostasis, and organ size. Here we show that YAP/TAZ play an essential role in amino acid-induced mTORC1 activation, particularly under nutrient-limiting conditions. Mechanistically, YAP/TAZ act via the TEAD transcription factors to induce expression of the high-affinity leucine transporter LAT1, which is a heterodimeric complex of SLC7A5 and SLC3A2. Deletion of YAP/TAZ abolishes expression of LAT1 and reduces leucine uptake. Re-expression of SLC7A5 in YAP/TAZ knockout cells restores leucine uptake and mTORC1 activation. Moreover, SLC7A5 knockout cells phenocopies YAP/TAZ knockout cells which exhibit defective mTORC1 activation in response to amino acids. We further demonstrate that YAP/TAZ act through SLC7A5 to provide cells with a competitive growth advantage. Our study provides molecular insight into the mechanism of YAP/TAZ target genes in cell growth regulation.

Structural basis of AMPK regulation by adenine nucleotides and glycogen

Abstract: AMP-activated protein kinase (AMPK) is a central cellular energy sensor and regulator of energy homeostasis, and a promising drug target for the treatment of diabetes, obesity, and cancer. Here we present low-resolution crystal structures of the human α1β2γ1 holo-AMPK complex bound to its allosteric modulators AMP and the glycogen-mimic cyclodextrin, both in the phosphorylated (4.05 A) and non-phosphorylated (4.60 A) state. In addition, we have solved a 2.95 A structure of the human kinase domain (KD) bound to the adjacent autoinhibitory domain (AID) and have performed extensive biochemical and mutational studies. Together, these studies illustrate an underlying mechanism of allosteric AMPK modulation by AMP and glycogen, whose binding changes the equilibria between alternate AID (AMP) and carbohydrate-binding module (glycogen) interactions.

Destabilization of strigolactone receptor DWARF14 by binding of ligand and E3-ligase signaling effector DWARF3.

Abstract: Strigolactones (SLs) are endogenous hormones and exuded signaling molecules in plant responses to low levels of mineral nutrients. Key mediators of the SL signaling pathway in rice include the α/β-fold hydrolase DWARF 14 (D14) and the F-box component DWARF 3 (D3) of the ubiquitin ligase SCFD3 that mediate ligand-dependent degradation of downstream signaling repressors. One perplexing feature is that D14 not only functions as the SL receptor but is also an active enzyme that slowly hydrolyzes diverse natural and synthetic SLs including GR24, preventing the crystallization of a binary complex of D14 with an intact SL as well as the ternary D14/SL/D3 complex. Here we overcome these barriers to derive a structural model of D14 bound to intact GR24 and identify the interface that is required for GR24-mediated D14-D3 interaction. The mode of GR24-mediated signaling, including ligand recognition, hydrolysis by D14, and ligand-mediated D14-D3 interaction, is conserved in structurally diverse SLs. More importantly, D14 is destabilized upon the binding of ligands and D3, thus revealing an unusual mechanism of SL recognition and signaling, in which the hormone, the receptor, and the downstream effectors are systematically destabilized during the signal transduction process.

Epigenetic silencing of microRNA-149 in cancer-associated fibroblasts mediates prostaglandin E2/interleukin-6 signaling in the tumor microenvironment.

Abstract: Tumor initiation and growth depend on its microenvironment in which cancer-associated fibroblasts (CAFs) in tumor stroma play an important role. Prostaglandin E2 (PGE2) and interleukin (IL)-6 signal pathways are involved in the crosstalk between tumor and stromal cells. However, how PGE2-mediated signaling modulates this crosstalk remains unclear. Here, we show that microRNA (miR)-149 links PGE2 and IL-6 signaling in mediating the crosstalk between tumor cells and CAFs in gastric cancer (GC). miR-149 inhibited fibroblast activation by targeting IL-6 and miR-149 expression was substantially suppressed in the CAFs of GC. miR-149 negatively regulated CAFs and their effect on GC development both in vitro and in vivo. CAFs enhanced epithelial-to-mesenchymal transition (EMT) and the stem-like properties of GC cells in a miR-149-IL-6-dependent manner. In addition to IL-6, PGE2 receptor 2 (PTGER2/EP2) was revealed as another potential target of miR-149 in fibroblasts. Furthermore, H. pylori infection, a leading cause of human GC, was able to induce cyclooxygenase-2 (COX-2)/PGE2 signaling and to enhance PGE2 production, resulting in the hypermethylation of miR-149 in CAFs and increased IL-6 secretion. Our findings indicate that miR-149 mediates the crosstalk between tumor cells and CAFs in GC and highlight the potential of interfering miRNAs in stromal cells to improve cancer therapy.

Anticancer immunotherapy by CTLA-4 blockade: obligatory contribution of IL-2 receptors and negative prognostic impact of soluble CD25

Abstract: The cytotoxic T lymphocyte antigen-4 (CTLA-4)-blocking antibody ipilimumab induces immune-mediated long-term control of metastatic melanoma in a fraction of patients. Although ipilimumab undoubtedly exerts its therapeutic effects via immunostimulation, thus far clinically useful, immunologically relevant biomarkers that predict treatment efficiency have been elusive. Here, we show that neutralization of IL-2 or blocking the α and β subunits of the IL-2 receptor (CD25 and CD122, respectively) abolished the antitumor effects and the accompanying improvement of the ratio of intratumoral T effector versus regulatory cells (Tregs), which were otherwise induced by CTLA-4 blockade in preclinical mouse models. CTLA-4 blockade led to the reduction of a suppressive CD4+ T cell subset expressing Lag3, ICOS, IL-10 and Egr2 with a concomitant rise in IL-2-producing effector cells that lost FoxP3 expression and accumulated in regressing tumors. While recombinant IL-2 improved the therapeutic efficacy of CTLA-4 blockade, the decoy IL-2 receptor α (IL-2Rα, sCD25) inhibited the anticancer effects of CTLA-4 blockade. In 262 metastatic melanoma patients receiving ipilimumab, baseline serum concentrations of sCD25 represented an independent indicator of overall survival, with high levels predicting resistance to therapy. Altogether, these results unravel a role for IL-2 and IL-2 receptors in the anticancer activity of CTLA-4 blockade. Importantly, our study provides the first immunologically relevant biomarker, namely elevated serum sCD25, that predicts resistance to CTLA-4 blockade in patients with melanoma.

A humanized neutralizing antibody against MERS-CoV targeting the receptor-binding domain of the spike protein

Abstract: The newly-emerging Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans. Despite global efforts, the potential for an associated pandemic in the future cannot be excluded. The development of effective counter-measures is urgent. MERS-CoV-specific anti-viral drugs or vaccines are not yet available. Using the spike receptor-binding domain of MERS-CoV (MERS-RBD) to immunize mice, we identified two neutralizing monoclonal antibodies (mAbs) 4C2 and 2E6. Both mAbs potently bind to MERS-RBD and block virus entry in vitro with high efficacy. We further investigated their mechanisms of neutralization by crystallizing the complex between the Fab fragments and the RBD, and solved the structure of the 4C2 Fab/MERS-RBD complex. The structure showed that 4C2 recognizes an epitope that partially overlaps the receptor-binding footprint in MERS-RBD, thereby interfering with the virus/receptor interactions by both steric hindrance and interface-residue competition. 2E6 also blocks receptor binding, and competes with 4C2 for binding to MERS-RBD. Based on the structure, we further humanized 4C2 by preserving only the paratope residues and substituting the remaining amino acids with the counterparts from human immunoglobulins. The humanized 4C2 (4C2h) antibody sustained similar neutralizing activity and biochemical characteristics to the parental mouse antibody. Finally, we showed that 4C2h can significantly abate the virus titers in lungs of Ad5-hCD26-transduced mice infected with MERS-CoV, therefore representing a promising agent for prophylaxis and therapy in clinical settings.

Substrate-bound structure of the E. coli multidrug resistance transporter MdfA

Abstract: Multidrug resistance is a serious threat to public health. Proton motive force-driven antiporters from the major facilitator superfamily (MFS) constitute a major group of multidrug-resistance transporters. Currently, no reports on crystal structures of MFS antiporters in complex with their substrates exist. The E. coli MdfA transporter is a well-studied model system for biochemical analyses of multidrug-resistance MFS antiporters. Here, we report three crystal structures of MdfA-ligand complexes at resolutions up to 2.0 A, all in the inward-facing conformation. The substrate-binding site sits proximal to the conserved acidic residue, D34. Our mutagenesis studies support the structural observations of the substrate-binding mode and the notion that D34 responds to substrate binding by adjusting its protonation status. Taken together, our data unveil the substrate-binding mode of MFS antiporters and suggest a mechanism of transport via this group of transporters.

Chondroitin sulfate proteoglycan 4 functions as the cellular receptor for Clostridium difficile toxin B

Abstract: As a gram-positive, spore-forming anaerobic bacillus, Clostridium difficile (C. difficile) is responsible for severe and fatal pseudomembranous colitis, and poses the most urgent antibiotic resistance threat worldwide. Epidemic C. difficile is the leading cause of antibiotic-associated diarrhoea globally, especially diarrhoea due to the emergence of hypervirulent strains associated with high mortality and morbidity. TcdB, one of the key virulence factors secreted by this bacterium, enters host cells through a poorly understood mechanism to elicit its pathogenic effect. Here we report the first identification of the TcdB cellular receptor, chondroitin sulfate proteoglycan 4 (CSPG4). CSPG4 was initially isolated from a whole-genome human shRNAmir library screening, and its role was confirmed by both TALEN- and CRISPR/Cas9-mediated gene knockout in human cells. CSPG4 is critical for TcdB binding to the cell surface, inducing cytoskeleton disruption and cell death. A direct interaction between the N-terminus of CSPG4 and the C-terminus of TcdB was confirmed, and the soluble peptide of the toxin-binding domain of CSPG4 could protect cells from the action of TcdB. Notably, the complete loss of CSPG4/NG2 decreased TcdB-triggered interleukin-8 induction in mice without significantly affecting animal mortality. Based on both the in vitro and in vivo studies, we propose a dual-receptor model for TcdB endocytosis. The discovery of the first TcdB receptor reveals a previously unsuspected role for CSPG4 and provides a new therapeutic target for the treatment of C. difficile infection.

Structural basis for recognition of an endogenous peptide by the plant receptor kinase PEPR1

Abstract: The endogenous peptides AtPep1-8 in Arabidopsis mature from the conserved C-terminal portions of their precursor proteins PROPEP1-8, respectively The two homologous leucine-rich repeat-receptor kinases (LRR-RKs) PEPR1 and PEPR2 act as receptors of AtPeps AtPep binding leads to stable association of PEPR1,2 with the shared receptor LRR-RK BAK1, eliciting immune responses similar to those induced by pathogens Here we report a crystal structure of the extracellular LRR domain of PEPR1 (PEPR1LRR) in complex with AtPep1 The structure reveals that AtPep1 adopts a fully extended conformation and binds to the inner surface of the superhelical PEPR1LRR Biochemical assays showed that AtPep1 is capable of inducing PEPR1LRR-BAK1LRR heterodimerization The conserved C-terminal portion of AtPep1 dominates AtPep1 binding to PEPR1LRR, with the last amino acid of AtPep1 Asn23 forming extensive interactions with PEPR1LRR Deletion of the last residue of AtPep1 significantly compromised AtPep1 interaction with PEPR1LRR Together, our data reveal a conserved structural mechanism of AtPep1 recognition by PEPR1, providing significant insight into prediction of recognition of other peptides by their cognate LRR-RKs

Siglec1 suppresses antiviral innate immune response by inducing TBK1 degradation via the ubiquitin ligase TRIM27.

Abstract: Type I interferon (IFN) production plays pivotal roles in host antiviral innate immune responses, but an excessive production of type I IFN leads to the development of immunopathological conditions. Investigations on the regulatory mechanisms underlying host type I IFN production are currently of great interest. Here, we found that the expression of lectin family member Siglec1 was upregulated by viral infection in macrophages, which was dependent on the IFN/JAK/STAT1 signaling pathway. Siglec1 was found to negatively regulate viral infection-triggered type I IFN production. Mechanistically, Siglec1 associates with DAP12 to recruit and activate the scaffolding function of SHP2; SHP2 then recruits E3 ubiquitin ligase TRIM27, which induces TBK1 degradation via K48-linked ubiquitination at Lys251 and Lys372. Therefore, viral infection-induced upregulation of Siglec1 feedback loop inhibits type I IFN production and suppresses antiviral innate immune responses. Our study outlines a novel mechanism of negative regulation of type I IFN production, which may help virus to escape immune elimination.

Acute inflammation stimulates a regenerative response in the neonatal mouse heart

Abstract: Cardiac injury in neonatal 1-day-old mice stimulates a regenerative response characterized by reactive cardiomyocyte proliferation, which is distinguished from the fibrotic repair process in adults. Acute inflammation occurs immediately after heart injury and has generally been believed to exert a negative effect on heart regeneration by promoting scar formation in adults; however, little is known about the role of acute inflammation in the cardiac regenerative response in neonatal mice. Here, we show that acute inflammation induced cardiomyocyte proliferation after apical intramyocardial microinjection of immunogenic zymosan A particles into the neonatal mouse heart. We also found that cardiac injury-induced regenerative response was suspended after immunosuppression in neonatal mice, and that cardiomyocytes could not be reactivated to proliferate after neonatal heart injury in the absence of interleukin-6 (IL-6). Furthermore, cardiomyocyte-specific deletion of signal transducer and activator of transcription 3 (STAT3), the major downstream effector of IL-6 signaling, decreased reactive cardiomyocyte proliferation after apical resection. Our results indicate that acute inflammation stimulates the regenerative response in neonatal mouse heart, and suggest that modulation of inflammatory signals might have important implications in cardiac regenerative medicine.