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Sample records for adaptor protein myeloid

  1. XB130: A novel adaptor protein in cancer signal transduction

    PubMed Central

    ZHANG, RUIYAO; ZHANG, JINGYAO; WU, QIFEI; MENG, FANDI; LIU, CHANG

    2016-01-01

    Adaptor proteins are functional proteins that contain two or more protein-binding modules to link signaling proteins together, which affect cell growth and shape and have no enzymatic activity. The actin filament-associated protein (AFAP) family is an important member of the adaptor proteins, including AFAP1, AFAP1L1 and AFAP1L2/XB130. AFAP1 and AFAP1L1 share certain common characteristics and function as an actin-binding protein and a cSrc-activating protein. XB130 exhibits certain unique features in structure and function. The mRNA of XB130 is expressed in human spleen, thyroid, kidney, brain, lung, pancreas, liver, colon and stomach, and the most prominent disease associated with XB130 is cancer. XB130 has a controversial effect on cancer. Studies have shown that XB130 can promote cancer progression and downregulation of XB130-reduced growth of tumors derived from certain cell lines. A higher mRNA level of XB130 was shown to be associated with a better survival in non-small cell lung cancer. Previous studies have shown that XB130 can regulate cell growth, migration and invasion and possibly has the effect through the cAMP-cSrc-phosphoinositide 3-kinase/Akt pathway. Except for cancer, XB130 is also associated with other pathological or physiological procedures, such as airway repair and regeneration. PMID:26998266

  2. The effect of smoking on myeloid-related protein-8 and myeloid-related protein-14.

    PubMed

    Ertugrul, Abdullah Seckin; Sahin, Hacer

    2016-05-20

    The aim of this study was to determine the myeloid-related protein-8 and myeloid-related protein-14 levels in the gingival crevicular fluid of smoker patients with generalized aggressive periodontitis (SAgP), smoker patients with chronic periodontitis (SCP), smoker patients with gingivitis (SG-smoker control), non-smoker patients with generalized aggressive periodontitis (AgP), non-smoker patients with chronic periodontitis (CP), and non-smoker patients with gingivitis (G-non-smoker control). The periodontal statuses of the patients were determined by periodontal clinical measurements and radiographical evaluations. The levels of myeloid-related protein-8 and myeloid-related protein-14 in the gingival crevicular fluid were assessed using enzyme-linked immuno sorbent assay. The myeloid-related protein-8 and myeloid-related protein-14 levels in the gingival crevicular fluid of patients with generalized aggressive periodontitis (non-smoker and smoker) were found to be statistically higher than patients with chronic periodontitis (non-smoker and smoker) and patients with gingivitis (non-smoker and smoker). Myeloid-related protein-8 and myeloid-related protein-14 levels of non-smokers were significantly higher than smokers in all types of periodontitis and gingivitis. The decreased myeloid-related protein-8 and myeloid-related protein-14 level could have prevented the haemostasis of calcium which plays a significant role in the migration of neutrophiles. Smoking affects myeloid-related protein-8 and myeloid-related protein-14 levels and may inhibit the antimicrobial efficiency against microorganisms. Due to these reasons smoker generalized aggressive periodontitis patients need to be treated in detail and their maintenance durations should be shortened. PMID:27223132

  3. PHF6 Degrees of Separation: The Multifaceted Roles of a Chromatin Adaptor Protein.

    PubMed

    Todd, Matthew A M; Ivanochko, Danton; Picketts, David J

    2015-01-01

    The importance of chromatin regulation to human disease is highlighted by the growing number of mutations identified in genes encoding chromatin remodeling proteins. While such mutations were first identified in severe developmental disorders, or in specific cancers, several genes have been implicated in both, including the plant homeodomain finger protein 6 (PHF6) gene. Indeed, germline mutations in PHF6 are the cause of the Börjeson-Forssman-Lehmann X-linked intellectual disability syndrome (BFLS), while somatic PHF6 mutations have been identified in T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Studies from different groups over the last few years have made a significant impact towards a functional understanding of PHF6 protein function. In this review, we summarize the current knowledge of PHF6 with particular emphasis on how it interfaces with a distinct set of interacting partners and its functional roles in the nucleoplasm and nucleolus. Overall, PHF6 is emerging as a key chromatin adaptor protein critical to the regulation of neurogenesis and hematopoiesis.

  4. PHF6 Degrees of Separation: The Multifaceted Roles of a Chromatin Adaptor Protein

    PubMed Central

    Todd, Matthew A.M.; Ivanochko, Danton; Picketts, David J.

    2015-01-01

    The importance of chromatin regulation to human disease is highlighted by the growing number of mutations identified in genes encoding chromatin remodeling proteins. While such mutations were first identified in severe developmental disorders, or in specific cancers, several genes have been implicated in both, including the plant homeodomain finger protein 6 (PHF6) gene. Indeed, germline mutations in PHF6 are the cause of the Börjeson–Forssman–Lehmann X-linked intellectual disability syndrome (BFLS), while somatic PHF6 mutations have been identified in T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Studies from different groups over the last few years have made a significant impact towards a functional understanding of PHF6 protein function. In this review, we summarize the current knowledge of PHF6 with particular emphasis on how it interfaces with a distinct set of interacting partners and its functional roles in the nucleoplasm and nucleolus. Overall, PHF6 is emerging as a key chromatin adaptor protein critical to the regulation of neurogenesis and hematopoiesis. PMID:26103525

  5. Negative regulation of lymphocyte activation by the adaptor protein LAX.

    PubMed

    Zhu, Minghua; Granillo, Olivia; Wen, Renren; Yang, Kaiyong; Dai, Xuezhi; Wang, Demin; Zhang, Weiguo

    2005-05-01

    The membrane-associated adaptor protein LAX is a linker for activation of T cells (LAT)-like molecule that is expressed in lymphoid tissues. Upon stimulation of T or B cells, it is phosphorylated and interacts with Grb2 and the p85 subunit of PI3K. LAX, however, is not capable of replacing LAT in the TCR signaling pathway. In this study we report that upon T or B cell activation, the LAX protein was up-regulated dramatically. Although disruption of the LAX gene by homologous recombination had no major impact on lymphocyte development, it caused a significant reduction in CD23 expression on mature B cells. Interestingly, naive LAX(-/-) mice had spontaneous germinal center formation. Compared with normal T and B cells, LAX(-/-) T and B cells were hyperresponsive and had enhanced calcium flux, protein tyrosine phosphorylation, MAPK and Akt activation, and cell survival upon engagement of the T or B AgRs. Our data demonstrate that LAX functions as a negative regulator in lymphocyte signaling.

  6. Alternative Splicing in CaV2.2 Regulates Neuronal Trafficking via Adaptor Protein Complex-1 Adaptor Protein Motifs

    PubMed Central

    Macabuag, Natsuko

    2015-01-01

    N-type voltage-gated calcium (CaV2.2) channels are expressed in neurons and targeted to the plasma membrane of presynaptic terminals, facilitating neurotransmitter release. Here, we find that the adaptor protein complex-1 (AP-1) mediates trafficking of CaV2.2 from the trans-Golgi network to the cell surface. Examination of splice variants of CaV2.2, containing either exon 37a (selectively expressed in nociceptors) or 37b in the proximal C terminus, reveal that canonical AP-1 binding motifs, YxxΦ and [DE]xxxL[LI], present only in exon 37a, enhance intracellular trafficking of exon 37a-containing CaV2.2 to the axons and plasma membrane of rat DRG neurons. Finally, we identify differential effects of dopamine-2 receptor (D2R) and its agonist-induced activation on trafficking of CaV2.2 isoforms. D2R slowed the endocytosis of CaV2.2 containing exon 37b, but not exon 37a, and activation by the agonist quinpirole reversed the effect of the D2R. Our work thus reveals key mechanisms involved in the trafficking of N-type calcium channels. SIGNIFICANCE STATEMENT CaV2.2 channels are important for neurotransmitter release, but how they are trafficked is still poorly understood. Here, we describe a novel mechanism for trafficking of CaV2.2 from the trans-Golgi network to the cell surface which is mediated by the adaptor protein AP-1. Alternative splicing of exon 37 produces CaV2.2-exon 37a, selectively expressed in nociceptors, or CaV2.2-exon 37b, which is the major splice isoform. Our study reveals that canonical AP-1 binding motifs (YxxΦ and [DE]xxxL[LI]), present in exon 37a, but not 37b, enhance intracellular trafficking of exon 37a-containing CaV2.2 to axons and plasma membrane of DRG neurons. Interaction of APs with CaV2.2 channels may also be key underlying mechanisms for differential effects of the dopamine D2 receptor on trafficking of CaV2.2 splice variants. PMID:26511252

  7. The Exosome Is Recruited to RNA Substrates through Specific Adaptor Proteins.

    PubMed

    Thoms, Matthias; Thomson, Emma; Baßler, Jochen; Gnädig, Marén; Griesel, Sabine; Hurt, Ed

    2015-08-27

    The exosome regulates the processing, degradation, and surveillance of a plethora of RNA species. However, little is known about how the exosome recognizes and is recruited to its diverse substrates. We report the identification of adaptor proteins that recruit the exosome-associated helicase, Mtr4, to unique RNA substrates. Nop53, the yeast homolog of the tumor suppressor PICT1, targets Mtr4 to pre-ribosomal particles for exosome-mediated processing, while a second adaptor Utp18 recruits Mtr4 to cleaved rRNA fragments destined for degradation by the exosome. Both Nop53 and Utp18 contain the same consensus motif, through which they dock to the "arch" domain of Mtr4 and target it to specific substrates. These findings show that the exosome employs a general mechanism of recruitment to defined substrates and that this process is regulated through adaptor proteins.

  8. SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export

    PubMed Central

    Müller-McNicoll, Michaela; Botti, Valentina; de Jesus Domingues, Antonio M.; Brandl, Holger; Schwich, Oliver D.; Steiner, Michaela C.; Curk, Tomaz; Poser, Ina; Zarnack, Kathi; Neugebauer, Karla M.

    2016-01-01

    Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1–7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1–7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3′ untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3′ ends. PMID:26944680

  9. SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export.

    PubMed

    Müller-McNicoll, Michaela; Botti, Valentina; de Jesus Domingues, Antonio M; Brandl, Holger; Schwich, Oliver D; Steiner, Michaela C; Curk, Tomaz; Poser, Ina; Zarnack, Kathi; Neugebauer, Karla M

    2016-03-01

    Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends. PMID:26944680

  10. Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCN5.

    PubMed Central

    Candau, R; Moore, P A; Wang, L; Barlev, N; Ying, C Y; Rosen, C A; Berger, S L

    1996-01-01

    Transcriptional adaptor proteins are required for full function of higher eukaryotic acidic activators in the yeast Saccharomyces cerevisiae, suggesting that this pathway of activation is evolutionarily conserved. Consistent with this view, we have identified possible human homologs of yeast ADA2 (yADA2) and yeast GCN5 (yGCN5), components of a putative adaptor complex. While there is overall sequence similarity between the yeast and human proteins, perhaps more significant is conservation of key sequence features with other known adaptors. We show several functional similarities between the human and yeast adaptors. First, as shown for yADA2 and yGCN5, human ADA2 (hADA2) and human GCN5 (hGCN5) interacted in vivo in a yeast two-hybrid assay. Moreover, hGCN5 interacted with yADA2 in this assay, suggesting that the human proteins form similar complexes. Second, both yADA2 and hADA2 contain cryptic activation domains. Third, hGCN5 and yGCN5 had similar stabilizing effects on yADA2 in vivo. Furthermore, the region of yADA2 that interacted with yGCN5 mapped to the amino terminus of yADA2, which is highly conserved in hADA2. Most striking, is the behavior of the human proteins in human cells. First, GAL4-hADA2 activated transcription in HeLa cells, and second, either hADA2 or hGCN5 augmented GAL4-VP16 activation. These data indicated that the human proteins correspond to functional homologs of the yeast adaptors, suggesting that these cofactors play a key role in transcriptional activation. PMID:8552087

  11. Insulin Receptor Substrate Adaptor Proteins Mediate Prognostic Gene Expression Profiles in Breast Cancer

    PubMed Central

    Becker, Marc A.; Ibrahim, Yasir H.; Oh, Annabell S.; Fagan, Dedra H.; Byron, Sara A.; Sarver, Aaron L.; Lee, Adrian V.; Shaw, Leslie M.; Fan, Cheng; Perou, Charles M.; Yee, Douglas

    2016-01-01

    Therapies targeting the type I insulin-like growth factor receptor (IGF-1R) have not been developed with predictive biomarkers to identify tumors with receptor activation. We have previously shown that the insulin receptor substrate (IRS) adaptor proteins are necessary for linking IGF1R to downstream signaling pathways and the malignant phenotype in breast cancer cells. The purpose of this study was to identify gene expression profiles downstream of IGF1R and its two adaptor proteins. IRS-null breast cancer cells (T47D-YA) were engineered to express IRS-1 or IRS-2 alone and their ability to mediate IGF ligand-induced proliferation, motility, and gene expression determined. Global gene expression signatures reflecting IRS adaptor specific and primary vs. secondary ligand response were derived (Early IRS-1, Late IRS-1, Early IRS-2 and Late IRS-2) and functional pathway analysis examined. IRS isoforms mediated distinct gene expression profiles, functional pathways, and breast cancer subtype association. For example, IRS-1/2-induced TGFb2 expression and blockade of TGFb2 abrogated IGF-induced cell migration. In addition, the prognostic value of IRS proteins was significant in the luminal B breast tumor subtype. Univariate and multivariate analyses confirmed that IRS adaptor signatures correlated with poor outcome as measured by recurrence-free and overall survival. Thus, IRS adaptor protein expression is required for IGF ligand responses in breast cancer cells. IRS-specific gene signatures represent accurate surrogates of IGF activity and could predict response to anti-IGF therapy in breast cancer. PMID:26991655

  12. Exploring structure and interactions of the bacterial adaptor protein YjbH by crosslinking mass spectrometry.

    PubMed

    Al-Eryani, Yusra; Ib Rasmussen, Morten; Kjellström, Sven; Højrup, Peter; Emanuelsson, Cecilia; von Wachenfeldt, Claes

    2016-09-01

    Adaptor proteins assist proteases in degrading specific proteins under appropriate conditions. The adaptor protein YjbH promotes the degradation of an important global transcriptional regulator Spx, which controls the expression of hundreds of genes and operons in response to thiol-specific oxidative stress in Bacillus subtilis. Under normal growth conditions, the transcription factor is bound to the adaptor protein and therefore degraded by the AAA+ protease ClpXP. If this binding is alleviated during stress, the transcription factor accumulates and turns on genes encoding stress-alleviating proteins. The adaptor protein YjbH is thus a key player involved in these interactions but its structure is unknown. To gain insight into its structure and interactions we have used chemical crosslinking mass spectrometry. Distance constraints obtained from the crosslinked monomer were used to select and validate a structure model of YjbH and then to probe its interactions with other proteins. The core structure of YjbH is reminiscent of DsbA family proteins. One lysine residue in YjbH (K177), located in one of the α-helices outside the thioredoxin fold, crosslinked to both Spx K99 and Spx K117, thereby suggesting one side of the YjbH for the interaction with Spx. Another lysine residue that crosslinked to Spx was YjbH K5, located in the long and presumably very flexible N-terminal arm of YjbH. Our crosslinking data lend support to a model proposed based on site-directed mutagenesis where the YjbH interaction with Spx can stabilize and present the C-terminal region of Spx for protease recognition and proteolysis. Proteins 2016; 84:1234-1245. © 2016 Wiley Periodicals, Inc. PMID:27191337

  13. Adapting the machine: adaptor proteins for Hsp100/Clp and AAA+ proteases.

    PubMed

    Kirstein, Janine; Molière, Noël; Dougan, David A; Turgay, Kürşad

    2009-08-01

    Members of the AAA+ protein superfamily contribute to many diverse aspects of protein homeostasis in prokaryotic cells. As a fundamental component of numerous proteolytic machines in bacteria, AAA+ proteins play a crucial part not only in general protein quality control but also in the regulation of developmental programmes, through the controlled turnover of key proteins such as transcription factors. To manage these many, varied tasks, Hsp100/Clp and AAA+ proteases use specific adaptor proteins to enhance or expand the substrate recognition abilities of their cognate protease. Here, we review our current knowledge of the modulation of bacterial AAA+ proteases by these cellular arbitrators.

  14. Role of SRC-like adaptor protein (SLAP) in immune and malignant cell signaling.

    PubMed

    Kazi, Julhash U; Kabir, Nuzhat N; Rönnstrand, Lars

    2015-07-01

    SRC-like adaptor protein (SLAP) is an adaptor protein structurally similar to the SRC family protein kinases. Like SRC, SLAP contains an SH3 domain followed by an SH2 domain but the kinase domain has been replaced by a unique C-terminal region. SLAP is expressed in a variety of cell types. Current studies suggest that it regulates signaling of various cell surface receptors including the B cell receptor, the T cell receptor, cytokine receptors and receptor tyrosine kinases which are important regulator of immune and cancer cell signaling. SLAP targets receptors, or its associated components, by recruiting the ubiquitin machinery and thereby destabilizing signaling. SLAP directs receptors to ubiquitination-mediated degradation and controls receptors turnover as well as signaling. Thus, SLAP appears to be an important component in regulating signal transduction required for immune and malignant cells.

  15. Nuclear Translocation of Crk Adaptor Proteins by the Influenza A Virus NS1 Protein

    PubMed Central

    Ylösmäki, Leena; Fagerlund, Riku; Kuisma, Inka; Julkunen, Ilkka; Saksela, Kalle

    2016-01-01

    The non-structural protein-1 (NS1) of many influenza A strains, especially those of avian origin, contains an SH3 ligand motif, which binds tightly to the cellular adaptor proteins Crk (Chicken tumor virus number 10 (CT10) regulator of kinase) and Crk-like adapter protein (CrkL). This interaction has been shown to potentiate NS1-induced activation of the phosphatidylinositol 3-kinase (PI3K), but additional effects on the host cell physiology may exist. Here we show that NS1 can induce an efficient translocation of Crk proteins from the cytoplasm into the nucleus, which results in an altered pattern of nuclear protein tyrosine phosphorylation. This was not observed using NS1 proteins deficient in SH3 binding or engineered to be exclusively cytoplasmic, indicating a physical role for NS1 as a carrier in the nuclear translocation of Crk. These data further emphasize the role of Crk proteins as host cell interaction partners of NS1, and highlight the potential for host cell manipulation gained by a viral protein simply via acquiring a short SH3 binding motif. PMID:27092521

  16. The Lnk adaptor protein: a key regulator of normal and pathological hematopoiesis.

    PubMed

    Velazquez, Laura

    2012-12-01

    The development and function of blood cells are regulated by specific growth factors/cytokines and their receptors' signaling pathways. In this way, these factors influence cell survival, proliferation and differentiation of hematopoietic cells. Central to this positive and/or negative control are the adaptor proteins. Since their identification 10 years ago, members of the Lnk adaptor protein family have proved to be important activators and/or inhibitors in the hematopoietic, immune and vascular system. In particular, the generation of animal and cellular models for the Lnk and APS proteins has helped establish the physiological role of these molecules through the identification of their specific signaling pathways and the characterization of their binding partners. Moreover, the recent identification of mutations in the LNK gene in myeloproliferative disorders, as well as the correlation of a single nucleotide polymorphism on LNK with hematological, immune and vascular diseases have suggested its involvement in the pathophysiology of these malignancies. The latter findings have thus raised the possibility of addressing Lnk signaling for the treatment of certain human diseases. This review therefore describes the pathophysiological role of this adaptor protein in hematological malignancies and the potential benefits of Lnk therapeutic targeting.

  17. Binding of AP-2 adaptor complex to brain membrane is regulated by phosphorylation of proteins

    SciTech Connect

    Alberdi, A. . E-mail: aalberdi@fcm.uncu.edu.ar; Sartor, T.; Sosa, M.A.

    2005-05-13

    Phosphorylation of proteins appears as a key process in early steps of clathrin coated vesicle formation. Here, we report that treatment of post-nuclear fraction with alkaline phosphatase induced redistribution of {alpha} subunits of AP-2 adaptor complex to cytosol and this effect was higher in the {alpha}2 subunit. A high serine phosphorylation status of {alpha} subunits correlated with the higher affinity of AP-2 to membranes. Using a simple binding assay, where membranes were incubated with either purified adaptors or cytosols, we observed an inhibitory effect of tyrphostin, a tyrosine kinase inhibitor, on the binding of AP-2 to membranes, but also an unexpected decrease induced by the phosphatase inhibitor cyclosporine. We also show an inhibitory effect of ATP mediated by cytosolic proteins, although it could not be related to the phosphorylation of AP-2, suggesting an action upstream a cascade of phosphorylations that participate in the regulation of the assembly of AP-2 to membranes.

  18. Differential Regulation of Clathrin and Its Adaptor Proteins during Membrane Recruitment for Endocytosis1[OPEN

    PubMed Central

    Wang, Chao; Hu, Tianwei; Yan, Xu; Meng, Tingting; Wang, Yutong; Wang, Qingmei; Zhang, Xiaoyue; Gu, Ying; Sánchez-Rodríguez, Clara; Gadeyne, Astrid; Lin, Jinxing

    2016-01-01

    In plants, clathrin-mediated endocytosis (CME) is dependent on the function of clathrin and its accessory heterooligomeric adaptor protein complexes, ADAPTOR PROTEIN2 (AP-2) and the TPLATE complex (TPC), and is negatively regulated by the hormones auxin and salicylic acid (SA). The details for how clathrin and its adaptor complexes are recruited to the plasma membrane (PM) to regulate CME, however, are poorly understood. We found that SA and the pharmacological CME inhibitor tyrphostin A23 reduce the membrane association of clathrin and AP-2, but not that of the TPC, whereas auxin solely affected clathrin membrane association, in Arabidopsis (Arabidopsis thaliana). Genetic and pharmacological experiments revealed that loss of AP2μ or AP2σ partially affected the membrane association of other AP-2 subunits and that the AP-2 subunit AP2σ, but not AP2μ, was required for SA- and tyrphostin A23-dependent inhibition of CME. Furthermore, we show that although AP-2 and the TPC are both required for the PM recruitment of clathrin in wild-type cells, the TPC is necessary for clathrin PM association in AP-2-deficient cells. These results indicate that developmental signals may differentially modulate the membrane recruitment of clathrin and its core accessory complexes to regulate the process of CME in plant cells. PMID:26945051

  19. Your personalized protein structure: Andrei N. Lupas fused to GCN4 adaptors.

    PubMed

    Deiss, Silvia; Hernandez Alvarez, Birte; Bär, Kerstin; Ewers, Carolin P; Coles, Murray; Albrecht, Reinhard; Hartmann, Marcus D

    2014-06-01

    This work presents a protein structure that has been designed purely for aesthetic reasons, symbolizing decades of coiled-coil research and praising its most fundamental model system, the GCN4 leucine zipper. The GCN4 leucine zipper is a highly stable coiled coil which can be tuned to adopt different oligomeric states via mutation of its core residues. For these reasons it is used in structural studies as a stabilizing fusion adaptor. On the occasion of the 50th birthday of Andrei N. Lupas, we used it to create the first personalized protein structure: we fused the sequence ANDREI-N-LVPAS in heptad register to trimeric GCN4 adaptors and determined its structure by X-ray crystallography. The structure demonstrates the robustness and versatility of GCN4 as a fusion adaptor. We learn how proline can be accommodated in trimeric coiled coils, and put the structure into the context of the other GCN4-fusion structures known to date. PMID:24486584

  20. Intersectin adaptor proteins are associated with actin-regulating protein WIP in invadopodia.

    PubMed

    Gryaznova, Tetyana; Kropyvko, Sergii; Burdyniuk, Mariia; Gubar, Olga; Kryklyva, Valentyna; Tsyba, Liudmyla; Rynditch, Alla

    2015-07-01

    Invasive cancer cells form actin-rich membrane protrusions called invadopodia that degrade extracellular matrix and facilitate cell invasion and metastasis. WIP (WASP-interacting protein) together with N-WASP (neural Wiskott-Aldrich syndrome protein) are localized in invadopodia and play a crucial role in their formation. Here we show that WIP interacts with endocytic adaptor proteins of the intersectin (ITSN) family, ITSN1 and ITSN2. The interaction is mediated by the SH3 domains of ITSNs and the middle part of the WIP proline-rich motifs. We have also demonstrated that ITSN1, WIP and N-WASP can form a complex in cells. Endogenous ITSN1 and ITSN2 are located in invasive protrusions of MDA-MB-231 breast cancer cell line. Moreover, data from immunofluorescent analysis revealed co-localization of ITSN1 and WIP at sites of invadopodia formation and in clathrin-coated pits. Together, these findings provide insights into the molecular mechanisms of invadopodia formation and identify ITSNs as scaffold proteins involved in this process.

  1. A Role for the Adaptor Proteins TRAM and TRIF in Toll-like Receptor 2 Signaling*

    PubMed Central

    Nilsen, Nadra J.; Vladimer, Gregory I.; Stenvik, Jørgen; Orning, M. Pontus A.; Zeid-Kilani, Maria V.; Bugge, Marit; Bergstroem, Bjarte; Conlon, Joseph; Husebye, Harald; Hise, Amy G.; Fitzgerald, Katherine A.; Espevik, Terje; Lien, Egil

    2015-01-01

    Toll-like receptors (TLRs) are involved in sensing invading microbes by host innate immunity. TLR2 recognizes bacterial lipoproteins/lipopeptides, and lipopolysaccharide activates TLR4. TLR2 and TLR4 signal via the Toll/interleukin-1 receptor adaptors MyD88 and MAL, leading to NF-κB activation. TLR4 also utilizes the adaptors TRAM and TRIF, resulting in activation of interferon regulatory factor (IRF) 3. Here, we report a new role for TRAM and TRIF in TLR2 regulation and signaling. Interestingly, we observed that TLR2-mediated induction of the chemokine Ccl5 was impaired in TRAM or TRIF deficient macrophages. Inhibition of endocytosis reduced Ccl5 release, and the data also suggested that TRAM and TLR2 co-localize in early endosomes, supporting the hypothesis that signaling may occur from an intracellular compartment. Ccl5 release following lipoprotein challenge additionally involved the kinase Tbk-1 and Irf3, as well as MyD88 and Irf1. Induction of Interferon-β and Ccl4 by lipoproteins was also partially impaired in cells lacking TRIF cells. Our results show a novel function of TRAM and TRIF in TLR2-mediated signal transduction, and the findings broaden our understanding of how Toll/interleukin-1 receptor adaptor proteins may participate in signaling downstream from TLR2. PMID:25505250

  2. Structural basis for concerted recruitment and activation of IRF-3 by innate immune adaptor proteins.

    PubMed

    Zhao, Baoyu; Shu, Chang; Gao, Xinsheng; Sankaran, Banumathi; Du, Fenglei; Shelton, Catherine L; Herr, Andrew B; Ji, Jun-Yuan; Li, Pingwei

    2016-06-14

    Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)-like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-β) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses. PMID:27302953

  3. A novel role of Shc adaptor proteins in steroid hormone-regulated cancers

    PubMed Central

    Alam, Syed Mahfuzul; Rajendran, Mythilypriya; Ouyang, Shouqiang; Veeramani, Suresh; Zhang, Li; Lin, Ming-Fong

    2009-01-01

    Tyrosine phosphorylation plays a critical role in growth regulation, and its aberrant regulation can be involved in carcinogenesis. The association of Shc (Src homolog and collagen homolog) adaptor protein family members in tyrosine phosphorylation signaling pathway is well recognized. Shc adaptor proteins transmit activated tyrosine phosphorylation signaling that suggest their plausible role in growth regulation including carcinogenesis and metastasis. In parallel, by sharing a similar mechanism of carcinogenesis, the steroids are involved in the early stage of carcinogenesis as well as the regulation of cancer progression and metastatic processes. Recent evidence indicates a cross-talk between tyrosine phosphorylation signaling and steroid hormone action in epithelial cells, including prostate and breast cancer cells. Therefore, the members of Shc proteins may function as mediators between tyrosine phosphorylation and steroid signaling in steroid-regulated cell proliferation and carcinogenesis. In this communication, we discuss the novel roles of Shc proteins, specifically p52Shc and p66Shc, in steroid hormone-regulated cancers and a novel molecular mechanism by which redox signaling induced by p66Shc mediates steroid action via a non-genomic pathway. The p66Shc protein may serve as an effective biomarker for predicting cancer prognosis as well as a useful target for treatment. PMID:19001530

  4. The role of small adaptor proteins in the control of oncogenic signaling driven by tyrosine kinases in human cancer

    PubMed Central

    Naudin, Cécile; Chevalier, Clément; Roche, Serge

    2016-01-01

    Protein phosphorylation on tyrosine (Tyr) residues has evolved as an important mechanism to coordinate cell communication in multicellular organisms. The importance of this process has been revealed by the discovery of the prominent oncogenic properties of tyrosine kinases (TK) upon deregulation of their physiological activities, often due to protein overexpression and/or somatic mutation. Recent reports suggest that TK oncogenic signaling is also under the control of small adaptor proteins. These cytosolic proteins lack intrinsic catalytic activity and signal by linking two functional members of a catalytic pathway. While most adaptors display positive regulatory functions, a small group of this family exerts negative regulatory functions by targeting several components of the TK signaling cascade. Here, we review how these less studied adaptor proteins negatively control TK activities and how their loss of function induces abnormal TK signaling, promoting tumor formation. We also discuss the therapeutic consequences of this novel regulatory mechanism in human oncology. PMID:26788993

  5. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

    NASA Astrophysics Data System (ADS)

    Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

    2014-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

  6. Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae.

    PubMed

    Unterweger, Daniel; Kostiuk, Benjamin; Ötjengerdes, Rina; Wilton, Ashley; Diaz-Satizabal, Laura; Pukatzki, Stefan

    2015-08-13

    Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines.

  7. The Cytoplasmic Adaptor Protein Dok7 Activates the Receptor Tyrosine Kinase MuSK via Dimerization

    SciTech Connect

    Bergamin, E.; Hallock, P; Burden, S; Hubbard, S

    2010-01-01

    Formation of the vertebrate neuromuscular junction requires, among others proteins, Agrin, a neuronally derived ligand, and the following muscle proteins: LRP4, the receptor for Agrin; MuSK, a receptor tyrosine kinase (RTK); and Dok7 (or Dok-7), a cytoplasmic adaptor protein. Dok7 comprises a pleckstrin-homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and C-terminal sites of tyrosine phosphorylation. Unique among adaptor proteins recruited to RTKs, Dok7 is not only a substrate of MuSK, but also an activator of MuSK's kinase activity. Here, we present the crystal structure of the Dok7 PH-PTB domains in complex with a phosphopeptide representing the Dok7-binding site on MuSK. The structure and biochemical data reveal a dimeric arrangement of Dok7 PH-PTB that facilitates trans-autophosphorylation of the kinase activation loop. The structure provides the molecular basis for MuSK activation by Dok7 and for rationalizing several Dok7 loss-of-function mutations found in patients with congenital myasthenic syndromes.

  8. Nck adaptor proteins link Tks5 to invadopodia actin regulation and ECM degradation

    PubMed Central

    Stylli, Stanley S.; I, Stacey T. T.; Verhagen, Anne M.; Xu, San San; Pass, Ian; Courtneidge, Sara A.; Lock, Peter

    2009-01-01

    Summary Invadopodia are actin-based projections enriched with proteases, which invasive cancer cells use to degrade the extracellular matrix (ECM). The Phox homology (PX)-Src homology (SH)3 domain adaptor protein Tks5 (also known as SH3PXD2A) cooperates with Src tyrosine kinase to promote invadopodia formation but the underlying pathway is not clear. Here we show that Src phosphorylates Tks5 at Y557, inducing it to associate directly with the SH3-SH2 domain adaptor proteins Nck1 and Nck2 in invadopodia. Tks5 mutants unable to bind Nck show reduced matrix degradation-promoting activity and recruit actin to invadopodia inefficiently. Conversely, Src- and Tks5-driven matrix proteolysis and actin assembly in invadopodia are enhanced by Nck1 or Nck2 overexpression and inhibited by Nck1 depletion. We show that clustering at the plasma membrane of the Tks5 inter-SH3 region containing Y557 triggers phosphorylation at this site, facilitating Nck recruitment and F-actin assembly. These results identify a Src-Tks5-Nck pathway in ECM-degrading invadopodia that shows parallels with pathways linking several mammalian and pathogen-derived proteins to local actin regulation. PMID:19596797

  9. Src-like-adaptor protein (SLAP) differentially regulates normal and oncogenic c-Kit signaling.

    PubMed

    Kazi, Julhash U; Agarwal, Shruti; Sun, Jianmin; Bracco, Enrico; Rönnstrand, Lars

    2014-02-01

    The Src-like-adaptor protein (SLAP) is an adaptor protein sharing considerable structural homology with Src. SLAP is expressed in a variety of cells and regulates receptor tyrosine kinase signaling by direct association. In this report, we show that SLAP associates with both wild-type and oncogenic c-Kit (c-Kit-D816V). The association involves the SLAP SH2 domain and receptor phosphotyrosine residues different from those mediating Src interaction. Association of SLAP triggers c-Kit ubiquitylation which, in turn, is followed by receptor degradation. Although SLAP depletion potentiates c-Kit downstream signaling by stabilizing the receptor, it remains non-functional in c-Kit-D816V signaling. Ligand-stimulated c-Kit or c-Kit-D816V did not alter membrane localization of SLAP. Interestingly oncogenic c-Kit-D816V, but not wild-type c-Kit, phosphorylates SLAP on residues Y120, Y258 and Y273. Physical interaction between c-Kit-D816V and SLAP is mandatory for the phosphorylation to take place. Although tyrosine-phosphorylated SLAP does not affect c-Kit-D816V signaling, mutation of these tyrosine sites to phenylalanine can restore SLAP activity. Taken together the data demonstrate that SLAP negatively regulates wild-type c-Kit signaling, but not its oncogenic counterpart, indicating a possible mechanism by which the oncogenic c-Kit bypasses the normal cellular negative feedback control.

  10. Synthetic Protein Scaffolds Based on Peptide Motifs and Cognate Adaptor Domains for Improving Metabolic Productivity.

    PubMed

    Horn, Anselm H C; Sticht, Heinrich

    2015-01-01

    The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity. PMID:26636078

  11. Synthetic Protein Scaffolds Based on Peptide Motifs and Cognate Adaptor Domains for Improving Metabolic Productivity

    PubMed Central

    Horn, Anselm H. C.; Sticht, Heinrich

    2015-01-01

    The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity. PMID:26636078

  12. Synthetic Protein Scaffolds Based on Peptide Motifs and Cognate Adaptor Domains for Improving Metabolic Productivity.

    PubMed

    Horn, Anselm H C; Sticht, Heinrich

    2015-01-01

    The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity.

  13. A role for adaptor protein-3 complex in the organization of the endocytic pathway in Dictyostelium.

    PubMed

    Charette, Steve J; Mercanti, Valentina; Letourneur, François; Bennett, Nelly; Cosson, Pierre

    2006-11-01

    Dictyostelium discoideum cells continuously internalize extracellular material, which accumulates in well-characterized endocytic vacuoles. In this study, we describe a new endocytic compartment identified by the presence of a specific marker, the p25 protein. This compartment presents features reminiscent of mammalian recycling endosomes: it is localized in the pericentrosomal region but distinct from the Golgi apparatus. It specifically contains surface proteins that are continuously endocytosed but rapidly recycled to the cell surface and thus absent from maturing endocytic compartments. We evaluated the importance of each clathrin-associated adaptor complex in establishing a compartmentalized endocytic system by studying the phenotype of the corresponding mutants. In knockout cells for mu3, a subunit of the AP-3 clathrin-associated complex, membrane proteins normally restricted to p25-positive endosomes were mislocalized to late endocytic compartments. Our results suggest that AP-3 plays an essential role in the compartmentalization of the endocytic pathway in Dictyostelium. PMID:17010123

  14. Phosphorylation-Dependent Regulation of the DNA Damage Response of Adaptor Protein KIBRA in Cancer Cells

    PubMed Central

    Mavuluri, Jayadev; Beesetti, Swarnalatha; Surabhi, Rohan; Kremerskothen, Joachim

    2016-01-01

    Multifunctional adaptor proteins encompassing various protein-protein interaction domains play a central role in the DNA damage response pathway. In this report, we show that KIBRA is a physiologically interacting reversible substrate of ataxia telangiectasia mutated (ATM) kinase. We identified the site of phosphorylation in KIBRA as threonine 1006, which is embedded within the serine/threonine (S/T) Q consensus motif, by site-directed mutagenesis, and we further confirmed the same with a phospho-(S/T) Q motif-specific antibody. Results from DNA repair functional assays such as the γ-H2AX assay, pulsed-field gel electrophoresis (PFGE), Comet assay, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and clonogenic cell survival assay using stable overexpression clones of wild-type (wt.) KIBRA and active (T1006E) and inactive (T1006A) KIBRA phosphorylation mutants showed that T1006 phosphorylation on KIBRA is essential for optimal DNA double-strand break repair in cancer cells. Further, results from stable retroviral short hairpin RNA-mediated knockdown (KD) clones of KIBRA and KIBRA knockout (KO) model cells generated by a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that depleting KIBRA levels compromised the DNA repair functions in cancer cells upon inducing DNA damage. All these phenotypic events were reversed upon reconstitution of KIBRA into cells lacking KIBRA knock-in (KI) model cells. All these results point to the fact that phosphorylated KIBRA might be functioning as a scaffolding protein/adaptor protein facilitating the platform for further recruitment of other DNA damage response factors. In summary, these data demonstrate the imperative functional role of KIBRA per se (KIBRA phosphorylation at T1006 site as a molecular switch that regulates the DNA damage response, possibly via the nonhomologous end joining [NHEJ] pathway), suggesting that KIBRA could be a potential

  15. Structural and motional contributions of the Bacillus subtilis ClpC N-domain in adaptor protein interactions

    PubMed Central

    Kojetin, Douglas J.; McLaughlin, Patrick D.; Thompson, Richele J.; Dubnau, David; Prepiak, Peter; Rance, Mark; Cavanagh, John

    2009-01-01

    Summary The AAA+ superfamily protein ClpC is a key regulator of cell development in Bacillus subtilis. As part of a large oligomeric complex, ClpC controls an array of cellular processes by recognizing, unfolding, and providing misfolded and aggregated proteins as substrates for the ClpP peptidase. ClpC is unique compared to other HSP100/Clp proteins, as it requires an adaptor protein for all fundamental activities. The NMR solution structure of the N-terminal repeat domain of ClpC (N-ClpCR) comprises two structural repeats of a four-helix motif. NMR experiments used to map the MecA adaptor protein interaction surface of N-ClpCR reveal that regions involved in the interaction possess conformational flexibility, as well as conformational exchange on the μs-ms time-scale. The electrostatic surface of N-ClpCR differs substantially compared to the N-domain of Escherichia coli ClpA and ClpB, suggesting that the electrostatic surface characteristics of HSP100/Clp N-domains may play a role in adaptor protein and substrate interaction specificity, and perhaps contribute to the unique adaptor protein requirement of ClpC. PMID:19361434

  16. Control of MAPK specificity by feedback phosphorylation of shared adaptor protein Ste50.

    PubMed

    Hao, Nan; Zeng, Yaxue; Elston, Timothy C; Dohlman, Henrik G

    2008-12-01

    Many different signaling pathways share common components but nevertheless invoke distinct physiological responses. In yeast, the adaptor protein Ste50 functions in multiple mitogen-activated protein (MAP) kinase pathways, each with unique dynamical and developmental properties. Although Kss1 activity is sustained and promotes invasive growth, Hog1 activity is transient and promotes cell adaptation to osmotic stress. Here we show that osmotic stress activates Kss1 as well as Hog1. We show further that Hog1 phosphorylates Ste50 and that phosphorylation of Ste50 limits the duration of Kss1 activation and prevents invasive growth under high osmolarity growth conditions. Thus feedback regulation of a shared component can restrict the activity of a competing MAP kinase to ensure signal fidelity. PMID:18854322

  17. Autosomal recessive hypercholesterolemia caused by mutations in a putative LDL receptor adaptor protein.

    PubMed

    Garcia, C K; Wilund, K; Arca, M; Zuliani, G; Fellin, R; Maioli, M; Calandra, S; Bertolini, S; Cossu, F; Grishin, N; Barnes, R; Cohen, J C; Hobbs, H H

    2001-05-18

    Atherogenic low density lipoproteins are cleared from the circulation by hepatic low density lipoprotein receptors (LDLR). Two inherited forms of hypercholesterolemia result from loss of LDLR activity: autosomal dominant familial hypercholesterolemia (FH), caused by mutations in the LDLR gene, and autosomal recessive hypercholesterolemia (ARH), of unknown etiology. Here we map the ARH locus to an approximately 1-centimorgan interval on chromosome 1p35 and identify six mutations in a gene encoding a putative adaptor protein (ARH). ARH contains a phosphotyrosine binding (PTB) domain, which in other proteins binds NPXY motifs in the cytoplasmic tails of cell-surface receptors, including the LDLR. ARH appears to have a tissue-specific role in LDLR function, as it is required in liver but not in fibroblasts. PMID:11326085

  18. The Role of Crk Adaptor Proteins in T-Cell Adhesion and Migration

    PubMed Central

    Braiman, Alex; Isakov, Noah

    2015-01-01

    Crk adaptor proteins are key players in signal transduction from a variety of cell surface receptors. They are involved in early steps of lymphocyte activation through their SH2-mediated transient interaction with signal transducing effector molecules, such as Cbl, ZAP-70, CasL, and STAT5. In addition, they constitutively associate, via their SH3 domain, with effector molecules, such as C3G, that mediate cell adhesion and regulate lymphocyte extravasation and recruitment to sites of inflammation. Recent studies demonstrated that the conformation and function of CrkII is subjected to a regulation by immunophilins, which also affect CrkII-dependent T-cell adhesion to fibronectin and migration toward chemokines. This article addresses mechanisms that regulate CrkII conformation and function, in general, and emphasizes the role of Crk proteins in receptor-coupled signaling pathways that control T-lymphocyte adhesion and migration to inflammatory sites. PMID:26500649

  19. Molecular basis of substrate selection by the N-end rule adaptor protein ClpS

    SciTech Connect

    Román-Hernández, Giselle; Grant, Robert A.; Sauer, Robert T.; Baker, Tania A.

    2009-06-19

    The N-end rule is a conserved degradation pathway that relates the stability of a protein to its N-terminal amino acid. Here, we present crystal structures of ClpS, the bacterial N-end rule adaptor, alone and engaged with peptides containing N-terminal phenylalanine, leucine, and tryptophan. These structures, together with a previous structure of ClpS bound to an N-terminal tyrosine, illustrate the molecular basis of recognition of the complete set of primary N-end rule amino acids. In each case, the alpha-amino group and side chain of the N-terminal residue are the major determinants of recognition. The binding pocket for the N-end residue is preformed in the free adaptor, and only small adjustments are needed to accommodate N-end rule residues having substantially different sizes and shapes. M53A ClpS is known to mediate degradation of an expanded repertoire of substrates, including those with N-terminal valine or isoleucine. A structure of Met53A ClpS engaged with an N-end rule tryptophan reveals an essentially wild-type mechanism of recognition, indicating that the Met(53) side chain directly enforces specificity by clashing with and excluding beta-branched side chains. Finally, experimental and structural data suggest mechanisms that make proteins with N-terminal methionine bind very poorly to ClpS, explaining why these high-abundance proteins are not degraded via the N-end rule pathway in the cell.

  20. Conserved interdomain linker promotes phase separation of the multivalent adaptor protein Nck

    PubMed Central

    Banjade, Sudeep; Wu, Qiong; Mittal, Anuradha; Peeples, William B.; Pappu, Rohit V.; Rosen, Michael K.

    2015-01-01

    The organization of membranes, the cytosol, and the nucleus of eukaryotic cells can be controlled through phase separation of lipids, proteins, and nucleic acids. Collective interactions of multivalent molecules mediated by modular binding domains can induce gelation and phase separation in several cytosolic and membrane-associated systems. The adaptor protein Nck has three SRC-homology 3 (SH3) domains that bind multiple proline-rich segments in the actin regulatory protein neuronal Wiskott-Aldrich syndrome protein (N-WASP) and an SH2 domain that binds to multiple phosphotyrosine sites in the adhesion protein nephrin, leading to phase separation. Here, we show that the 50-residue linker between the first two SH3 domains of Nck enhances phase separation of Nck/N-WASP/nephrin assemblies. Two linear motifs within this element, as well as its overall positively charged character, are important for this effect. The linker increases the driving force for self-assembly of Nck, likely through weak interactions with the second SH3 domain, and this effect appears to promote phase separation. The linker sequence is highly conserved, suggesting that the sequence determinants of the driving forces for phase separation may be generally important to Nck functions. Our studies demonstrate that linker regions between modular domains can contribute to the driving forces for self-assembly and phase separation of multivalent proteins. PMID:26553976

  1. Calmodulin has the Potential to Function as a Ca2+-Dependent Adaptor Protein

    PubMed Central

    Yamniuk, Aaron P; Rainaldi, Mario

    2007-01-01

    Calmodulin (CaM) is a versatile Ca2+-binding protein that regulates the activity of numerous effector proteins in response to Ca2+ signals. Several CaM-dependent regulatory mechanisms have been identified, including autoinhibitory domain displacement, sequestration of a ligand-binding site, active site reorganization, and target protein dimerization. We recently showed that the N- and C-lobes of animal and plant CaM isoforms could independently and sequentially bind to target peptides derived from the CaM-binding domain of Nicotiana tabacum mitogen-activated protein kinase phosphatase (NtMKP1), to form a 2:1 peptide:CaM complex. This suggests that CaM might facilitate the dimerization of NtMKP1, although the dimerization mechanism is distinct from the previously described simultaneous binding of other target peptides to CaM. The independent and sequential binding of the NtMKP1 peptides to CaM also suggests an alternative plausible scenario in which the C-lobe of CaM remains tethered to NtMKP1, and the N-lobe is free to recruit a second target protein to the complex, such as an NtMKP1 target. Thus, we hypothesize that CaM may be capable of functioning as a Ca2+-dependent adaptor or recruiter protein. PMID:19704657

  2. The tyrosine kinase McsB is a regulated adaptor protein for ClpCP.

    PubMed

    Kirstein, Janine; Dougan, David A; Gerth, Ulf; Hecker, Michael; Turgay, Kürşad

    2007-04-18

    Cells of the soil bacterium Bacillus subtilis have to adapt to fast environmental changes in their natural habitat. Here, we characterized a novel system in which cells respond to heat shock by regulatory proteolysis of a transcriptional repressor CtsR. In B. subtilis, CtsR controls the synthesis of itself, the tyrosine kinase McsB, its activator McsA and the Hsp100/Clp proteins ClpC, ClpE and their cognate peptidase ClpP. The AAA+ protein family members ClpC and ClpE can form an ATP-dependent protease complex with ClpP and are part of the B. subtilis protein quality control system. The regulatory response is mediated by a proteolytic switch, which is formed by these proteins under heat-shock conditions, where the tyrosine kinase McsB acts as a regulated adaptor protein, which in its phosphorylated form activates the Hsp100/Clp protein ClpC and targets the repressor CtsR for degradation by the general protease ClpCP.

  3. Transcriptional repression of Kruppel like factor-2 by the adaptor protein p66shc

    PubMed Central

    Kumar, Ajay; Hoffman, Timothy A.; DeRicco, Jeremy; Naqvi, Asma; Jain, Mukesh K.; Irani, Kaikobad

    2009-01-01

    The adaptor protein p66shc promotes cellular oxidative stress and apoptosis. Here, we demonstrate a novel mechanistic relationship between p66shc and the kruppel like factor-2 (KLF2) transcription factor and show that this relationship has biological relevance to p66shc-regulated cellular oxidant level, as well as KLF2-induced target gene expression. Genetic knockout of p66shc in mouse embryonic fibroblasts (MEFs) stimulates activity of the core KLF2 promoter and increases KLF2 mRNA and protein expression. Similarly, shRNA-induced knockdown of p66shc increases KLF2-promoter activity in HeLa cells. The increase in KLF2-promoter activity in p66shc-knockout MEFs is dependent on a myocyte enhancing factor-2A (MEF2A)-binding sequence in the core KLF2 promoter. Short-hairpin RNA-induced knockdown of p66shc in endothelial cells also stimulates KLF2 mRNA and protein expression, as well as expression of the endothelial KLF2 target gene thrombomodulin. MEF2A protein and mRNA are more abundant in p66shc-knockout MEFs, resulting in greater occupancy of the KLF2 promoter by MEF2A. In endothelial cells, the increase in KLF2 and thrombomodulin protein by shRNA-induced decrease in p66shc expression is partly abrogated by knockdown of MEF2A. Finally, knockdown of KLF2 abolishes the decrease in the cellular reactive oxygen species hydrogen peroxide observed with knockdown of p66shc, and KLF2 overexpression suppresses cellular hydrogen peroxide levels, independent of p66shc expression. These findings illustrate a novel mechanism by which p66shc promotes cellular oxidative stress, through suppression of MEF2A expression and consequent repression of KLF2 transcription.—Kumar, A., Hoffman, T. A., DeRicco, J., Naqvi, A., Jain, M. K., Irani, K. Transcriptional repression of Kruppel like factor-2 by the adaptor protein p66shc. PMID:19696221

  4. A Novel Nuclear Function for the Interleukin-17 Signaling Adaptor Protein Act1

    PubMed Central

    Velichko, Sharlene; Zhou, Xu; Zhu, Lingxiang; Anderson, Johnathon David; Wu, Reen; Chen, Yin

    2016-01-01

    In the context of the human airway, interleukin-17A (IL-17A) signaling is associated with severe inflammation, as well as protection against pathogenic infection, particularly at mucosal surfaces such as the airway. The intracellular molecule Act1 has been demonstrated to be an essential mediator of IL-17A signaling. In the cytoplasm, it serves as an adaptor protein, binding to both the intracellular domain of the IL-17 receptor as well as members of the canonical nuclear factor kappa B (NF-κB) pathway. It also has enzymatic activity, and serves as an E3 ubiquitin ligase. In the context of airway epithelial cells, we demonstrate for the first time that Act1 is also present in the nucleus, especially after IL-17A stimulation. Ectopic Act1 expression can also increase the nuclear localization of Act1. Act1 can up-regulate the expression and promoter activity of a subset of IL-17A target genes in the absence of IL-17A signaling in a manner that is dependent on its N- and C-terminal domains, but is NF-κB independent. Finally, we show that nuclear Act1 can bind to both distal and proximal promoter regions of DEFB4, one of the IL-17A responsive genes. This transcriptional regulatory activity represents a novel function for Act1. Taken together, this is the first report to describe a non-adaptor function of Act1 by directly binding to the promoter region of IL-17A responsive genes and directly regulate their transcription. PMID:27723765

  5. The proteolysis adaptor, NblA, initiates protein pigment degradation by interacting with the cyanobacterial light-harvesting complexes.

    PubMed

    Sendersky, Eleonora; Kozer, Noga; Levi, Mali; Garini, Yuval; Shav-Tal, Yaron; Schwarz, Rakefet

    2014-07-01

    Degradation of the cyanobacterial protein pigment complexes, the phycobilisomes, is a central acclimation response that controls light energy capture. The small protein, NblA, is essential for proteolysis of these large complexes, which may reach a molecular mass of up to 4 MDa. Interactions of NblA in vitro supported the suggestion that NblA is a proteolysis adaptor that labels the pigment proteins for degradation. The mode of operation of NblA in situ, however, remained unresolved. Particularly, it was unclear whether NblA interacts with phycobilisome proteins while part of the large complex, or alternatively interaction with NblA, necessitates dissociation of pigment subunits from the assembly. Fluorescence intensity profiles demonstrated the preferential presence of NblA::GFP (green fluorescent protein) at the photosynthetic membranes, indicating co-localization with phycobilisomes. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for interaction of NblA with phycobilisome protein pigments. Additionally, we demonstrated the role of NblA in vivo as a proteolysis tag based on the rapid degradation of the fusion protein NblA::GFP compared with free GFP. Taken together, these observations demonstrated in vivo the role of NblA as a proteolysis adaptor. Additionally, the interaction of NblA with phycobilisomes indicates that the dissociation of protein pigment subunits from the large complex is not a prerequisite for interaction with this adaptor and, furthermore, implicates NblA in the disassembly of the protein pigment complex. Thus, we suggest that, in the case of proteolysis of the phycobilisome, the adaptor serves a dual function: undermining the complex stability and designating the dissociated pigments for degradation.

  6. Losses, Expansions, and Novel Subunit Discovery of Adaptor Protein Complexes in Haptophyte Algae.

    PubMed

    Lee, Laura J Y; Klute, Mary J; Herman, Emily K; Read, Betsy; Dacks, Joel B

    2015-11-01

    The phylum Haptophyta (Diaphoratickes) contains marine algae that perform biomineralization, extruding large, distinctive calcium carbonate scales (coccoliths) that completely cover the cell. Coccolith production is an important part of global carbon cycling; however, the membrane trafficking pathway by which they are secreted has not yet been elucidated. In most eukaryotes, post-Golgi membrane trafficking involves five heterotetrameric adaptor protein (AP) complexes, which impart cargo selection specificity. To better understand coccolith secretion, we performed comparative genomic, phylogenetic, and transcriptomic analyses of the AP complexes in Emiliania huxleyi strains 92A, Van556, EH2, and CCMP1516, and related haptophytes Gephyrocapsa oceanica and Isochrysis galbana; the latter has lost the ability to biomineralize. We show that haptophytes have a modified membrane trafficking system (MTS), as we found both AP subunit losses and duplications. Additionally, we identified a single conserved subunit of the AP-related TSET complex, whose expression suggests a functional role in membrane trafficking. Finally, we detected novel alpha adaptin ear and gamma adaptin ear proteins, the first of their kind to be described outside of opisthokonts. These novel ear proteins and the sculpting of the MTS may support the capacity for biomineralization in haptophytes, enhancing their ability to perform this highly specialized form of secretion. PMID:26519625

  7. Adaptor Protein 2 Regulates Receptor-Mediated Endocytosis and Cyst Formation in Giardia lamblia

    PubMed Central

    Rivero, Maria R.; Vranych, Cecilia V.; Bisbal, Mariano; Maletto, Belkys A.; Ropolo, Andrea S.; Touz, Maria C.

    2010-01-01

    Synopsis The parasite Giardia lamblia possesses peripheral vacuoles (PVs) that function as both endosomes and lysosomes and are implicated in the adaptation, differentiation, and survival of the parasite in different environments. The mechanisms by which Giardia traffics essential proteins to these organelles and regulates their secretion have important implications in the control of parasite dissemination. In this study, we describe the participation of the heterotetrameric clathrin-adaptor protein gAP2 complex in lysosomal protein trafficking. A specific monoclonal antibody against the medium subunit (gμ2) of gAP2 showed localization of this complex to the PVs, cytoplasm, and plasma membrane in the growing trophozoites. gAP2 also colocalized with clathrin in the PVs, suggesting its involvement in endocytosis. Uptake experiments using standard molecules for the study of endocytosis revealed that gAP2 specifically participated in the endocytosis of LDL. Targeted downregulation of the gene encoding gμ2 in growing and encysting trophozoites resulted in a large decrease in the amount of cell growth and cyst wall formation, suggesting a distinct mechanism in which gAP2 is directly involved in both endocytosis and vesicular trafficking. PMID:20199400

  8. Src-like Adaptor Protein (Slap) Is a Negative Regulator of T Cell Receptor Signaling

    PubMed Central

    Sosinowski, Tomasz; Pandey, Akhilesh; Dixit, Vishva M.; Weiss, Arthur

    2000-01-01

    Initiation of T cell antigen receptor (TCR) signaling is dependent on Lck, a Src family kinase. The Src-like adaptor protein (SLAP) contains Src homology (SH)3 and SH2 domains, which are highly homologous to those of Lck and other Src family members. Because of the structural similarity between Lck and SLAP, we studied its potential role in TCR signaling. Here, we show that SLAP is expressed in T cells, and that when expressed in Jurkat T cells it can specifically inhibit TCR signaling leading to nuclear factor of activated T cells (NFAT)-, activator protein 1 (AP-1)–, and interleukin 2–dependent transcription. The SH3 and SH2 domains of SLAP are required for maximal attenuation of TCR signaling. This inhibitory activity can be bypassed by the combination of phorbol myristate acetate (PMA) and ionomycin, suggesting that SLAP acts proximally in the TCR signaling pathway. SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents. In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3ζ, ZAP-70, SH2 domain–containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT). These results suggest that SLAP is a negative regulator of TCR signaling. PMID:10662792

  9. Src-like adaptor protein (SLAP) is a negative regulator of T cell receptor signaling.

    PubMed

    Sosinowski, T; Pandey, A; Dixit, V M; Weiss, A

    2000-02-01

    Initiation of T cell antigen receptor (TCR) signaling is dependent on Lck, a Src family kinase. The Src-like adaptor protein (SLAP) contains Src homology (SH)3 and SH2 domains, which are highly homologous to those of Lck and other Src family members. Because of the structural similarity between Lck and SLAP, we studied its potential role in TCR signaling. Here, we show that SLAP is expressed in T cells, and that when expressed in Jurkat T cells it can specifically inhibit TCR signaling leading to nuclear factor of activated T cells (NFAT)-, activator protein 1 (AP-1)-, and interleukin 2-dependent transcription. The SH3 and SH2 domains of SLAP are required for maximal attenuation of TCR signaling. This inhibitory activity can be bypassed by the combination of phorbol myristate acetate (PMA) and ionomycin, suggesting that SLAP acts proximally in the TCR signaling pathway. SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents. In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3zeta, ZAP-70, SH2 domain-containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT). These results suggest that SLAP is a negative regulator of TCR signaling.

  10. Losses, Expansions, and Novel Subunit Discovery of Adaptor Protein Complexes in Haptophyte Algae.

    PubMed

    Lee, Laura J Y; Klute, Mary J; Herman, Emily K; Read, Betsy; Dacks, Joel B

    2015-11-01

    The phylum Haptophyta (Diaphoratickes) contains marine algae that perform biomineralization, extruding large, distinctive calcium carbonate scales (coccoliths) that completely cover the cell. Coccolith production is an important part of global carbon cycling; however, the membrane trafficking pathway by which they are secreted has not yet been elucidated. In most eukaryotes, post-Golgi membrane trafficking involves five heterotetrameric adaptor protein (AP) complexes, which impart cargo selection specificity. To better understand coccolith secretion, we performed comparative genomic, phylogenetic, and transcriptomic analyses of the AP complexes in Emiliania huxleyi strains 92A, Van556, EH2, and CCMP1516, and related haptophytes Gephyrocapsa oceanica and Isochrysis galbana; the latter has lost the ability to biomineralize. We show that haptophytes have a modified membrane trafficking system (MTS), as we found both AP subunit losses and duplications. Additionally, we identified a single conserved subunit of the AP-related TSET complex, whose expression suggests a functional role in membrane trafficking. Finally, we detected novel alpha adaptin ear and gamma adaptin ear proteins, the first of their kind to be described outside of opisthokonts. These novel ear proteins and the sculpting of the MTS may support the capacity for biomineralization in haptophytes, enhancing their ability to perform this highly specialized form of secretion.

  11. Reconstitution of Rad53 Activation by Mec1 through Adaptor Protein Mrc1*

    PubMed Central

    Chen, Sheng-hong; Zhou, Huilin

    2009-01-01

    Upon DNA replication stress, stalled DNA replication forks serve as a platform to recruit many signaling proteins, leading to the activation of the DNA replication checkpoint. Activation of Rad53, a key effector kinase in the budding yeast Saccharomyces cerevisiae, is essential for stabilizing DNA replication forks during replication stress. Using an activity-based assay for Rad53, we found that Mrc1, a replication fork-associated protein, cooperates with Mec1 to activate Rad53 directly. Reconstitution of Rad53 activation using purified Mec1 and Mrc1 showed that the addition of Mrc1 stimulated a more than 70-fold increase in the ability of Mec1 to activate Rad53. Instead of increasing the catalytic activity of Mec1, Mrc1 was found to facilitate the phosphorylation of Rad53 by Mec1 via promotion of a stronger enzyme-substrate interaction between them. Further, the conserved C-terminal domain of Mrc1 was found to be required for Rad53 activation. These results thus provide insights into the role of the adaptor protein Mrc1 in activating Rad53 in the DNA replication checkpoint. PMID:19457865

  12. Merkel Cell Polyomavirus Small T Antigen Targets the NEMO Adaptor Protein To Disrupt Inflammatory Signaling

    PubMed Central

    Griffiths, David A.; Abdul-Sada, Hussein; Knight, Laura M.; Jackson, Brian R.; Richards, Kathryn; Prescott, Emma L.; Peach, A. Howard S.; Blair, G. Eric

    2013-01-01

    Merkel cell carcinoma (MCC) is a highly aggressive nonmelanoma skin cancer arising from epidermal mechanoreceptor Merkel cells. In 2008, a novel human polyomavirus, Merkel cell polyomavirus (MCPyV), was identified and is strongly implicated in MCC pathogenesis. Currently, little is known regarding the virus-host cell interactions which support virus replication and virus-induced mechanisms in cellular transformation and metastasis. Here we identify a new function of MCPyV small T antigen (ST) as an inhibitor of NF-κB-mediated transcription. This effect is due to an interaction between MCPyV ST and the NF-κB essential modulator (NEMO) adaptor protein. MCPyV ST expression inhibits IκB kinase α (IKKα)/IKKβ-mediated IκB phosphorylation, which limits translocation of the NF-κB heterodimer to the nucleus. Regulation of this process involves a previously undescribed interaction between MCPyV ST and the cellular phosphatase subunits, protein phosphatase 4C (PP4C) and/or protein phosphatase 2A (PP2A) Aβ, but not PP2A Aα. Together, these results highlight a novel function of MCPyV ST to subvert the innate immune response, allowing establishment of early or persistent infection within the host cell. PMID:24109239

  13. Activity-Regulated Cytoskeleton-Associated Protein Controls AMPAR Endocytosis through a Direct Interaction with Clathrin-Adaptor Protein 2.

    PubMed

    DaSilva, Luis L P; Wall, Mark J; P de Almeida, Luciana; Wauters, Sandrine C; Januário, Yunan C; Müller, Jürgen; Corrêa, Sonia A L

    2016-01-01

    The activity-regulated cytoskeleton-associated (Arc) protein controls synaptic strength by facilitating AMPA receptor (AMPAR) endocytosis. Here we demonstrate that Arc targets AMPAR to be internalized through a direct interaction with the clathrin-adaptor protein 2 (AP-2). We show that Arc overexpression in dissociated hippocampal neurons obtained from C57BL/6 mouse reduces the density of AMPAR GluA1 subunits at the cell surface and reduces the amplitude and rectification of AMPAR-mediated miniature-EPSCs (mEPSCs). Mutations of Arc, that prevent the AP-2 interaction reduce Arc-mediated endocytosis of GluA1 and abolish the reduction in AMPAR-mediated mEPSC amplitude and rectification. Depletion of the AP-2 subunit µ2 blocks the Arc-mediated reduction in mEPSC amplitude, an effect that is restored by reintroducing µ2. The Arc-AP-2 interaction plays an important role in homeostatic synaptic scaling as the Arc-dependent decrease in mEPSC amplitude, induced by a chronic increase in neuronal activity, is inhibited by AP-2 depletion. These data provide a mechanism to explain how activity-dependent expression of Arc decisively controls the fate of AMPAR at the cell surface and modulates synaptic strength, via the direct interaction with the endocytic clathrin adaptor AP-2. PMID:27257628

  14. Activity-Regulated Cytoskeleton-Associated Protein Controls AMPAR Endocytosis through a Direct Interaction with Clathrin-Adaptor Protein 2123

    PubMed Central

    Wall, Mark J.; P. de Almeida, Luciana; Wauters, Sandrine C.; Januário, Yunan C.; Müller, Jürgen

    2016-01-01

    Abstract The activity-regulated cytoskeleton-associated (Arc) protein controls synaptic strength by facilitating AMPA receptor (AMPAR) endocytosis. Here we demonstrate that Arc targets AMPAR to be internalized through a direct interaction with the clathrin-adaptor protein 2 (AP-2). We show that Arc overexpression in dissociated hippocampal neurons obtained from C57BL/6 mouse reduces the density of AMPAR GluA1 subunits at the cell surface and reduces the amplitude and rectification of AMPAR-mediated miniature-EPSCs (mEPSCs). Mutations of Arc, that prevent the AP-2 interaction reduce Arc-mediated endocytosis of GluA1 and abolish the reduction in AMPAR-mediated mEPSC amplitude and rectification. Depletion of the AP-2 subunit µ2 blocks the Arc-mediated reduction in mEPSC amplitude, an effect that is restored by reintroducing µ2. The Arc–AP-2 interaction plays an important role in homeostatic synaptic scaling as the Arc-dependent decrease in mEPSC amplitude, induced by a chronic increase in neuronal activity, is inhibited by AP-2 depletion. These data provide a mechanism to explain how activity-dependent expression of Arc decisively controls the fate of AMPAR at the cell surface and modulates synaptic strength, via the direct interaction with the endocytic clathrin adaptor AP-2. PMID:27257628

  15. Highly pathogenic avian influenza virus nucleoprotein interacts with TREX complex adaptor protein Aly/REF.

    PubMed

    Balasubramaniam, Vinod R M T; Hong Wai, Tham; Ario Tejo, Bimo; Omar, Abdul Rahman; Syed Hassan, Sharifah

    2013-01-01

    We constructed a novel chicken (Gallus gallus) lung cDNA library fused inside yeast acting domain vector (pGADT7). Using yeast two-hybrid screening with highly pathogenic avian influenza (HPAI) nucleoprotein (NP) from the strain (A/chicken/Malaysia/5858/2004(H5N1)) as bait, and the Gallus gallus lung cDNA library as prey, a novel interaction between the Gallus gallus cellular RNA export adaptor protein Aly/REF and the viral NP was identified. This interaction was confirmed and validated with mammalian two hybrid studies and co-immunoprecipitation assay. Cellular localization studies using confocal microscopy showed that NP and Aly/REF co-localize primarily in the nucleus. Further investigations by mammalian two hybrid studies into the binding of NP of other subtypes of influenza virus such as the swine A/New Jersey/1976/H1N1 and pandemic A/Malaysia/854/2009(H1N1) to human Aly/REF, also showed that the NP of these viruses interacts with human Aly/REF. Our findings are also supported by docking studies which showed tight and favorable binding between H5N1 NP and human Aly/REF, using crystal structures from Protein Data Bank. siRNA knockdown of Aly/REF had little effect on the export of HPAI NP and other viral RNA as it showed no significant reduction in virus titer. However, UAP56, another component of the TREX complex, which recruits Aly/REF to mRNA was found to interact even better with H5N1 NP through molecular docking studies. Both these proteins also co-localizes in the nucleus at early infection similar to Aly/REF. Intriguingly, knockdown of UAP56 in A549 infected cells shows significant reduction in viral titer (close to 10 fold reduction). Conclusively, our study have opened new avenues for research of other cellular RNA export adaptors crucial in aiding viral RNA export such as the SRSF3, 9G8 and ASF/SF2 that may play role in influenza virus RNA nucleocytoplasmic transport.

  16. The Adaptor Protein Myd88 Is a Key Signaling Molecule in the Pathogenesis of Irinotecan-Induced Intestinal Mucositis.

    PubMed

    Wong, Deysi V T; Lima-Júnior, Roberto C P; Carvalho, Cibele B M; Borges, Vanessa F; Wanderley, Carlos W S; Bem, Amanda X C; Leite, Caio A V G; Teixeira, Maraiza A; Batista, Gabriela L P; Silva, Rangel L; Cunha, Thiago M; Brito, Gerly A C; Almeida, Paulo R C; Cunha, Fernando Q; Ribeiro, Ronaldo A

    2015-01-01

    Intestinal mucositis is a common side effect of irinotecan-based anticancer regimens. Mucositis causes cell damage, bacterial/endotoxin translocation and production of cytokines including IL-1 and IL-18. These molecules and toll-like receptors (TLRs) activate a common signaling pathway that involves the Myeloid Differentiation adaptor protein, MyD88, whose role in intestinal mucositis is unknown. Then, we evaluated the involvement of TLRs and MyD88 in the pathogenesis of irinotecan-induced intestinal mucositis. MyD88-, TLR2- or TLR9-knockout mice and C57BL/6 (WT) mice were given either saline or irinotecan (75 mg/kg, i.p. for 4 days). On day 7, animal survival, diarrhea and bacteremia were assessed, and following euthanasia, samples of the ileum were obtained for morphometric analysis, myeloperoxidase (MPO) assay and measurement of pro-inflammatory markers. Irinotecan reduced the animal survival (50%) and induced a pronounced diarrhea, increased bacteremia, neutrophil accumulation in the intestinal tissue, intestinal damage and more than twofold increased expression of MyD88 (200%), TLR9 (400%), TRAF6 (236%), IL-1β (405%), IL-18 (365%), COX-2 (2,777%) and NF-κB (245%) in the WT animals when compared with saline-injected group (P<0.05). Genetic deletion of MyD88, TLR2 or TLR9 effectively controlled the signs of intestinal injury when compared with irinotecan-administered WT controls (P<0.05). In contrast to the MyD88-/- and TLR2-/- mice, the irinotecan-injected TLR9-/- mice showed a reduced survival, a marked diarrhea and an enhanced expression of IL-18 versus irinotecan-injected WT controls. Additionally, the expression of MyD88 was reduced in the TLR2-/- or TLR9-/- mice. This study shows a critical role of the MyD88-mediated TLR2 and TLR9 signaling in the pathogenesis of irinotecan-induced intestinal mucositis.

  17. The Adaptor Protein Myd88 Is a Key Signaling Molecule in the Pathogenesis of Irinotecan-Induced Intestinal Mucositis

    PubMed Central

    Wong, Deysi V. T.; Lima-Júnior, Roberto C. P.; Carvalho, Cibele B. M.; Borges, Vanessa F.; Wanderley, Carlos W. S.; Bem, Amanda X. C.; Leite, Caio A. V. G.; Teixeira, Maraiza A.; Batista, Gabriela L. P.; Silva, Rangel L.; Cunha, Thiago M.; Brito, Gerly A. C.; Almeida, Paulo R. C.; Cunha, Fernando Q.; Ribeiro, Ronaldo A.

    2015-01-01

    Intestinal mucositis is a common side effect of irinotecan-based anticancer regimens. Mucositis causes cell damage, bacterial/endotoxin translocation and production of cytokines including IL–1 and IL–18. These molecules and toll-like receptors (TLRs) activate a common signaling pathway that involves the Myeloid Differentiation adaptor protein, MyD88, whose role in intestinal mucositis is unknown. Then, we evaluated the involvement of TLRs and MyD88 in the pathogenesis of irinotecan-induced intestinal mucositis. MyD88-, TLR2- or TLR9-knockout mice and C57BL/6 (WT) mice were given either saline or irinotecan (75 mg/kg, i.p. for 4 days). On day 7, animal survival, diarrhea and bacteremia were assessed, and following euthanasia, samples of the ileum were obtained for morphometric analysis, myeloperoxidase (MPO) assay and measurement of pro-inflammatory markers. Irinotecan reduced the animal survival (50%) and induced a pronounced diarrhea, increased bacteremia, neutrophil accumulation in the intestinal tissue, intestinal damage and more than twofold increased expression of MyD88 (200%), TLR9 (400%), TRAF6 (236%), IL–1β (405%), IL–18 (365%), COX–2 (2,777%) and NF-κB (245%) in the WT animals when compared with saline-injected group (P<0.05). Genetic deletion of MyD88, TLR2 or TLR9 effectively controlled the signs of intestinal injury when compared with irinotecan-administered WT controls (P<0.05). In contrast to the MyD88-/- and TLR2-/- mice, the irinotecan-injected TLR9-/- mice showed a reduced survival, a marked diarrhea and an enhanced expression of IL–18 versus irinotecan-injected WT controls. Additionally, the expression of MyD88 was reduced in the TLR2-/- or TLR9-/- mice. This study shows a critical role of the MyD88-mediated TLR2 and TLR9 signaling in the pathogenesis of irinotecan-induced intestinal mucositis. PMID:26440613

  18. The crucial role of the MyD88 adaptor protein in the inflammatory response induced by Bothrops atrox venom.

    PubMed

    Moreira, Vanessa; Teixeira, Catarina; Borges da Silva, Henrique; D'Império Lima, Maria Regina; Dos-Santos, Maria Cristina

    2013-06-01

    Most snake accidents in North Brazil are attributed to Bothrops atrox, a snake species of the Viperidae family whose venom simultaneously induces local and systemic effects in the victims. The former are clinically more important than the latter, as they cause severe tissue lesions associated with strong inflammatory responses. Although several studies have shown that inflammatory mediators are produced in response to B. atrox venom (BaV), there is little information concerning the molecular pathways involved in innate immune system signaling. Myeloid differentiation factor 88 (MyD88) is an adaptor molecule responsible for transmitting intracellular signals from most toll-like receptors (TLRs) after they interact with pathogen-associated molecular patterns (PAMPs) or other stimuli such as endogenous damage-associated molecular patterns (DAMPs). The MyD88-dependent pathway leads to activation of transcription factors, which in turn induce synthesis of inflammatory mediators such as eicosanoids, cytokines and chemokines. The aim of this study was to investigate the involvement of MyD88 on the acute inflammatory response induced by BaV. Wild-type (WT) C57BL/6 mice and MyD88 knockout (MyD88(-/-)) mice were intraperitoneally injected with BaV. Compared to WT mice, MyD88(-/-) animals showed an impaired inflammatory response to BaV, with lower influx of polymorphonuclear and mononuclear cells to the peritoneal cavity. Furthermore, peritoneal leukocytes from BaV-injected MyD88(-/-) mice did not induce COX-2 or LTB4 protein expression and released low concentrations of PGE2. These mice also failed to produce Th1 and Th17 cytokines and CCL-2, but IL-10 levels were similar to those of BaV-injected WT mice. Our results indicate that MyD88 signaling is required for activation of the inflammatory response elicited by BaV, raising the possibility of developing new therapeutic targets to treat Bothrops sp. poisoning. PMID:23474268

  19. Architecture and roles of periplasmic adaptor proteins in tripartite efflux assemblies.

    PubMed

    Symmons, Martyn F; Marshall, Robert L; Bavro, Vassiliy N

    2015-01-01

    Recent years have seen major advances in the structural understanding of the different components of tripartite efflux assemblies, which encompass the multidrug efflux (MDR) pumps and type I secretion systems. The majority of these investigations have focused on the role played by the inner membrane transporters and the outer membrane factor (OMF), leaving the third component of the system - the Periplasmic Adaptor Proteins (PAPs) - relatively understudied. Here we review the current state of knowledge of these versatile proteins which, far from being passive linkers between the OMF and the transporter, emerge as active architects of tripartite assemblies, and play diverse roles in the transport process. Recognition between the PAPs and OMFs is essential for pump assembly and function, and targeting this interaction may provide a novel avenue for combating multidrug resistance. With the recent advances elucidating the drug efflux and energetics of the tripartite assemblies, the understanding of the interaction between the OMFs and PAPs is the last piece remaining in the complete structure of the tripartite pump assembly puzzle.

  20. Stargazin regulates AMPA receptor trafficking through adaptor protein complexes during long-term depression

    NASA Astrophysics Data System (ADS)

    Matsuda, Shinji; Kakegawa, Wataru; Budisantoso, Timotheus; Nomura, Toshihiro; Kohda, Kazuhisa; Yuzaki, Michisuke

    2013-11-01

    Long-term depression (LTD) underlies learning and memory in various brain regions. Although postsynaptic AMPA receptor trafficking mediates LTD, its underlying molecular mechanisms remain largely unclear. Here we show that stargazin, a transmembrane AMPA receptor regulatory protein, forms a ternary complex with adaptor proteins AP-2 and AP-3A in hippocampal neurons, depending on its phosphorylation state. Inhibiting the stargazin-AP-2 interaction disrupts NMDA-induced AMPA receptor endocytosis, and inhibiting that of stargazin-AP-3A abrogates the late endosomal/lysosomal trafficking of AMPA receptors, thereby upregulating receptor recycling to the cell surface. Similarly, stargazin’s interaction with AP-2 or AP-3A is necessary for low-frequency stimulus-evoked LTD in CA1 hippocampal neurons. Thus, stargazin has a crucial role in NMDA-dependent LTD by regulating two trafficking pathways of AMPA receptors—transport from the cell surface to early endosomes and from early endosomes to late endosomes/lysosomes—through its sequential binding to AP-2 and AP-3A.

  1. Impairment of dendritic cell functions in patients with adaptor protein-3 complex deficiency.

    PubMed

    Prandini, Alberto; Salvi, Valentina; Colombo, Francesca; Moratto, Daniele; Lorenzi, Luisa; Vermi, William; De Francesco, Maria Antonia; Notarangelo, Lucia Dora; Porta, Fulvio; Plebani, Alessandro; Facchetti, Fabio; Sozzani, Silvano; Badolato, Raffaele

    2016-06-30

    Hermansky-Pudlak syndrome type 2 (HPS2) is a primary immunodeficiency due to adaptor protein-3 (AP-3) complex deficiency. HPS2 patients present neutropenia, partial albinism, and impaired lysosomal vesicles formation in hematopoietic cells. Given the role of dendritic cells (DCs) in the immune response, we studied monocyte-derived DCs (moDCs) and plasmacytoid DCs (pDCs) in two HPS2 siblings. Mature HPS2 moDCs showed impaired expression of CD83 and DC-lysosome-associated membrane protein (LAMP), low levels of MIP1-β/CCL4, MIG/CXCL9, and severe defect of interleukin-12 (IL-12) secretion. DCs in lymph-node biopsies from the same patients showed a diffuse cytoplasm reactivity in a large fraction of DC-LAMP(+) cells, instead of the classical dot-like stain. In addition, analysis of pDC-related functions of blood-circulating mononuclear cells revealed reduced interferon-α secretion in response to herpes simplex virus-1 (HSV-1), whereas granzyme-B induction upon IL-3/IL-10 stimulation was normal. Finally, T-cell costimulatory activity, as measured by mixed lymphocyte reaction assay, was lower in patients, suggesting that function and maturation of DCs is abnormal in patients with HPS2. PMID:27207797

  2. Impairment of dendritic cell functions in patients with adaptor protein-3 complex deficiency.

    PubMed

    Prandini, Alberto; Salvi, Valentina; Colombo, Francesca; Moratto, Daniele; Lorenzi, Luisa; Vermi, William; De Francesco, Maria Antonia; Notarangelo, Lucia Dora; Porta, Fulvio; Plebani, Alessandro; Facchetti, Fabio; Sozzani, Silvano; Badolato, Raffaele

    2016-06-30

    Hermansky-Pudlak syndrome type 2 (HPS2) is a primary immunodeficiency due to adaptor protein-3 (AP-3) complex deficiency. HPS2 patients present neutropenia, partial albinism, and impaired lysosomal vesicles formation in hematopoietic cells. Given the role of dendritic cells (DCs) in the immune response, we studied monocyte-derived DCs (moDCs) and plasmacytoid DCs (pDCs) in two HPS2 siblings. Mature HPS2 moDCs showed impaired expression of CD83 and DC-lysosome-associated membrane protein (LAMP), low levels of MIP1-β/CCL4, MIG/CXCL9, and severe defect of interleukin-12 (IL-12) secretion. DCs in lymph-node biopsies from the same patients showed a diffuse cytoplasm reactivity in a large fraction of DC-LAMP(+) cells, instead of the classical dot-like stain. In addition, analysis of pDC-related functions of blood-circulating mononuclear cells revealed reduced interferon-α secretion in response to herpes simplex virus-1 (HSV-1), whereas granzyme-B induction upon IL-3/IL-10 stimulation was normal. Finally, T-cell costimulatory activity, as measured by mixed lymphocyte reaction assay, was lower in patients, suggesting that function and maturation of DCs is abnormal in patients with HPS2.

  3. Architecture and roles of periplasmic adaptor proteins in tripartite efflux assemblies

    PubMed Central

    Symmons, Martyn F.; Marshall, Robert L.

    2015-01-01

    Recent years have seen major advances in the structural understanding of the different components of tripartite efflux assemblies, which encompass the multidrug efflux (MDR) pumps and type I secretion systems. The majority of these investigations have focused on the role played by the inner membrane transporters and the outer membrane factor (OMF), leaving the third component of the system – the Periplasmic Adaptor Proteins (PAPs) – relatively understudied. Here we review the current state of knowledge of these versatile proteins which, far from being passive linkers between the OMF and the transporter, emerge as active architects of tripartite assemblies, and play diverse roles in the transport process. Recognition between the PAPs and OMFs is essential for pump assembly and function, and targeting this interaction may provide a novel avenue for combating multidrug resistance. With the recent advances elucidating the drug efflux and energetics of the tripartite assemblies, the understanding of the interaction between the OMFs and PAPs is the last piece remaining in the complete structure of the tripartite pump assembly puzzle. PMID:26074901

  4. Crk adaptor proteins act as key signaling integrators for breast tumorigenesis

    PubMed Central

    2012-01-01

    Introduction CT10 regulator of kinase (Crk) adaptor proteins (CrkI, CrkII and CrkL) play a role in integrating signals for migration and invasion of highly malignant breast cancer cell lines. This has important implications, as elevated CrkI/II protein levels were observed in a small cohort of breast cancer patients, which identified a potential role for Crk proteins in breast cancer progression. Numerous in vitro studies identified a role for Crk proteins in cell motility, but little is known about how Crk proteins contribute to breast cancer progression in vivo. Methods The clinical significance of Crk proteins in human breast cancer was assessed by analyzing published breast cancer datasets using a gene expression signature that was generated following CrkII over-expression and by examining Crk protein expression in tissue microarrays of breast tumors (n = 254). Stable knockdown of Crk (CrkI/CrkII/CrkL) proteins was accomplished using a short hairpin RNA (shRNA)-mediated approach in two basal breast cancer cell lines, MDA-231 1833TR and SUM1315, where the former have a high affinity to form bone metastases. Both in vitro assays (cell migration, invasion, soft agar growth) and in vivo experiments (intra-cardiac, tibial and mammary fat pad injections) were performed to assess the functional significance of Crk proteins in breast cancer. Results A gene signature derived following CrkII over-expression correlated significantly with basal breast cancers and with high grade and poor outcome in general. Moreover, elevated Crk immunostaining on tissue microarrays revealed a significant association with highly proliferative tumors within the basal subtype. RNAi-mediated knockdown of all three Crk proteins in metastatic basal breast cancer cells established a continued requirement for Crk in cell migration and invasion in vitro and metastatic growth in vivo. Furthermore, Crk ablation suppressed anchorage independent growth and in vivo orthotopic tumor growth. This was

  5. Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures.

    PubMed

    Schreiber, Andreas; Huber, Matthias C; Cölfen, Helmut; Schiller, Stefan M

    2015-01-01

    The control over the defined assembly of nano-objects with nm-precision is important to create systems and materials with enhanced properties, for example, metamaterials. In nature, the precise assembly of inorganic nano-objects with unique features, for example, magnetosomes, is accomplished by efficient and reliable recognition schemes involving protein effectors. Here we present a molecular approach using protein-based 'adaptors/connectors' with genetically encoded interaction sites to guide the assembly and functionality of different plasmonically active gold nanoparticle architectures (AuNP). The interaction of the defined geometricaly shaped protein adaptors with the AuNP induces the self-assembly of nanoarchitectures ranging from AuNP encapsulation to one-dimensional chain-like structures, complex networks and stars. Synthetic biology and bionanotechnology are applied to co-translationally encode unnatural amino acids as additional site-specific modification sites to generate functionalized biohybrid nanoarchitectures. This protein adaptor-based nano-object assembly approach might be expanded to other inorganic nano-objects creating biohybrid materials with unique electronic, photonic, plasmonic and magnetic properties. PMID:25813537

  6. Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures

    NASA Astrophysics Data System (ADS)

    Schreiber, Andreas; Huber, Matthias C.; Cölfen, Helmut; Schiller, Stefan M.

    2015-03-01

    The control over the defined assembly of nano-objects with nm-precision is important to create systems and materials with enhanced properties, for example, metamaterials. In nature, the precise assembly of inorganic nano-objects with unique features, for example, magnetosomes, is accomplished by efficient and reliable recognition schemes involving protein effectors. Here we present a molecular approach using protein-based ‘adaptors/connectors’ with genetically encoded interaction sites to guide the assembly and functionality of different plasmonically active gold nanoparticle architectures (AuNP). The interaction of the defined geometricaly shaped protein adaptors with the AuNP induces the self-assembly of nanoarchitectures ranging from AuNP encapsulation to one-dimensional chain-like structures, complex networks and stars. Synthetic biology and bionanotechnology are applied to co-translationally encode unnatural amino acids as additional site-specific modification sites to generate functionalized biohybrid nanoarchitectures. This protein adaptor-based nano-object assembly approach might be expanded to other inorganic nano-objects creating biohybrid materials with unique electronic, photonic, plasmonic and magnetic properties.

  7. The p66Shc Adaptor Protein Controls Oxidative Stress Response in Early Bovine Embryos

    PubMed Central

    Betts, Dean H.; Bain, Nathan T.; Madan, Pavneesh

    2014-01-01

    The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 2–4 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 2–4 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos. PMID:24475205

  8. Tetraspanins and Transmembrane Adaptor Proteins As Plasma Membrane Organizers—Mast Cell Case

    PubMed Central

    Halova, Ivana; Draber, Petr

    2016-01-01

    The plasma membrane contains diverse and specialized membrane domains, which include tetraspanin-enriched domains (TEMs) and transmembrane adaptor protein (TRAP)-enriched domains. Recent biophysical, microscopic, and functional studies indicated that TEMs and TRAP-enriched domains are involved in compartmentalization of physicochemical events of such important processes as immunoreceptor signal transduction and chemotaxis. Moreover, there is evidence of a cross-talk between TEMs and TRAP-enriched domains. In this review we discuss the presence and function of such domains and their crosstalk using mast cells as a model. The combined data based on analysis of selected mast cell-expressed tetraspanins [cluster of differentiation (CD)9, CD53, CD63, CD81, CD151)] or TRAPs [linker for activation of T cells (LAT), non-T cell activation linker (NTAL), and phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG)] using knockout mice or specific antibodies point to a diversity within these two families and bring evidence of the important roles of these molecules in signaling events. An example of this diversity is physical separation of two TRAPs, LAT and NTAL, which are in many aspects similar but show plasma membrane location in different microdomains in both non-activated and activated cells. Although our understanding of TEMs and TRAP-enriched domains is far from complete, pharmaceutical applications of the knowledge about these domains are under way. PMID:27243007

  9. Lymphocyte adaptor protein LNK deficiency exacerbates hypertension and end-organ inflammation

    PubMed Central

    Saleh, Mohamed A.; McMaster, William G.; Wu, Jing; Norlander, Allison E.; Funt, Samuel A.; Thabet, Salim R.; Kirabo, Annet; Xiao, Liang; Chen, Wei; Itani, Hana A.; Michell, Danielle; Huan, Tianxiao; Zhang, Yahua; Takaki, Satoshi; Titze, Jens; Levy, Daniel; Harrison, David G.; Madhur, Meena S.

    2015-01-01

    The lymphocyte adaptor protein LNK (also known as SH2B3) is primarily expressed in hematopoietic and endothelial cells, where it functions as a negative regulator of cytokine signaling and cell proliferation. Single-nucleotide polymorphisms in the gene encoding LNK are associated with autoimmune and cardiovascular disorders; however, it is not known how LNK contributes to hypertension. Here, we determined that loss of LNK exacerbates angiotensin II–induced (Ang II–induced) hypertension and the associated renal and vascular dysfunction. At baseline, kidneys from Lnk–/– mice exhibited greater levels of inflammation, oxidative stress, and glomerular injury compared with WT animals, and these parameters were further exacerbated by Ang II infusion. Aortas from Lnk–/– mice exhibited enhanced inflammation, reduced nitric oxide levels, and impaired endothelial-dependent relaxation. Bone marrow transplantation studies demonstrated that loss of LNK in hematopoietic cells is primarily responsible for the observed renal and vascular inflammation and predisposition to hypertension. Ang II infusion increased IFN-γ–producing CD8+ T cells in the spleen and kidneys of Lnk–/– mice compared with WT mice. Moreover, IFN-γ deficiency resulted in blunted hypertension in response to Ang II infusion. Together, these results suggest that LNK is a potential therapeutic target for hypertension and its associated renal and vascular sequela. PMID:25664851

  10. Src-Like adaptor protein (SLAP) binds to the receptor tyrosine kinase Flt3 and modulates receptor stability and downstream signaling.

    PubMed

    Kazi, Julhash U; Rönnstrand, Lars

    2012-01-01

    Fms-like tyrosine kinase 3 (Flt3) is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML). Src-like adaptor protein (SLAP) is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.

  11. Modulation of Hepatitis C Virus Genome Replication by Glycosphingolipids and Four-Phosphate Adaptor Protein 2

    PubMed Central

    Khan, Irfan; Katikaneni, Divya S.; Han, Qingxia; Sanchez-Felipe, Lorena; Hanada, Kentaro; Ambrose, Rebecca L.; Mackenzie, Jason M.

    2014-01-01

    ABSTRACT Hepatitis C virus (HCV) assembles its replication complex on cytosolic membrane vesicles often clustered in a membranous web (MW). During infection, HCV NS5A protein activates PI4KIIIα enzyme, causing massive production and redistribution of phosphatidylinositol 4-phosphate (PI4P) lipid to the replication complex. However, the role of PI4P in the HCV life cycle is not well understood. We postulated that PI4P recruits host effectors to modulate HCV genome replication or virus particle production. To test this hypothesis, we generated cell lines for doxycycline-inducible expression of short hairpin RNAs (shRNAs) targeting the PI4P effector, four-phosphate adaptor protein 2 (FAPP2). FAPP2 depletion attenuated HCV infectivity and impeded HCV RNA synthesis. Indeed, FAPP2 has two functional lipid-binding domains specific for PI4P and glycosphingolipids. While expression of the PI4P-binding mutant protein was expected to inhibit HCV replication, a marked drop in replication efficiency was observed unexpectedly with the glycosphingolipid-binding mutant protein. These data suggest that both domains are crucial for the role of FAPP2 in HCV genome replication. We also found that HCV significantly increases the level of some glycosphingolipids, whereas adding these lipids to FAPP2-depleted cells partially rescued replication, further arguing for the importance of glycosphingolipids in HCV RNA synthesis. Interestingly, FAPP2 is redistributed to the replication complex (RC) characterized by HCV NS5A, NS4B, or double-stranded RNA (dsRNA) foci. Additionally, FAPP2 depletion disrupts the RC and alters the colocalization of HCV replicase proteins. Altogether, our study implies that HCV coopts FAPP2 for virus genome replication via PI4P binding and glycosphingolipid transport to the HCV RC. IMPORTANCE Like most viruses with a positive-sense RNA genome, HCV replicates its RNA on remodeled host membranes composed of lipids hijacked from various internal membrane compartments

  12. Genetic association study of adaptor protein complex 4 with cerebral palsy in a Han Chinese population.

    PubMed

    Wang, Honglian; Xu, Yiran; Chen, Mingjie; Shang, Qing; Sun, Yanyan; Zhu, Dengna; Wang, Lei; Huang, Zhiheng; Ma, Caiyun; Li, Tongchuan; He, Lin; Xing, Qinghe; Zhu, Changlian

    2013-11-01

    Adaptor protein complex 4 (AP-4) plays a key role in vesicle formation, trafficking, and sorting processes that are critical for brain development and function. AP-4 consists of four subunits encoded by the AP4E1, AP4B1, AP4M1, and AP4S1 genes. A number of studies have pointed to the involvement of AP-4-mediated vesicular trafficking pathways in the etiology of cerebral palsy (CP), the most notable of which are the causative mutations that have recently been identified in each of the AP-4 genes in different CP families. We postulated, therefore, that variations in AP-4 genes might influence an indivual's susceptibility to CP. In the present study, 16 SNPs were genotyped among 517 CP patients and 502 healthy controls from the Han Chinese population. We systematically analyzed the association of the AP4E1, AP4B1, AP4M1, and AP4S1 genes with CP on the basis of clinical characteristics. No significant associations were found between these variants and the overall risk of CP. Subgroup analysis showed that rs1217401 of AP4B1 was significantly associated with CP as a sequela of hypoxic-ischemic encephalopathy (HIE) (CP + HIE) (allele: p = 0.042151; genotype: p = 4.46 × 10(-6)). Our results indicate that the 16 variants studied in the genes of the four subunits of AP-4 have no detectable effects on the overall susceptibility to CP, but AP4B1 appears to be a susceptibility gene for CP + HIE in the Han Chinese population. PMID:24065543

  13. Impaired Fracture Healing Caused by Deficiency of the Immunoreceptor Adaptor Protein DAP12.

    PubMed

    Kamimura, Masayuki; Mori, Yu; Sugahara-Tobinai, Akiko; Takai, Toshiyuki; Itoi, Eiji

    2015-01-01

    Osteoclasts play an important role in bone metabolism, but their exact role in fracture healing remains unclear. DAP12 is an immunoadaptor protein with associated immunoreceptors on myeloid lineage cells, including osteoclasts. Its deficiency causes osteopetrosis due to suppression of osteoclast development and activation. In this report, we assessed the impact of DAP12 on the fracture healing process using C57BL/6 (B6) and DAP12-/- mice. Healing was evaluated using radiography, micro-CT, histology, immunohistochemistry and real-time RT-PCR. Radiography showed lower callus volume and lower callus radiolucency in DAP12-/- mice during later stages. Micro-CT images and quantitative structural analysis indicated that DAP12-/- mice developed calluses of dense trabecular structures and experienced deteriorated cortical shell formation on the surface. Histologically, DAP12-/- mice showed less cartilaginous resorption and woven bone formation. In addition, prominent cortical shell formation was much less in DAP12-/- mice. Immunohistochemistry revealed lower invasion of F4/80 positive monocytes and macrophages into the fracture hematoma in DAP12-/- mice. The expression levels of Col1a1, Col2a1 and Col10a1 in DAP12-/- mice increased and subsequently became higher than those in B6 mice. There was a decrease in the gene expression of Tnf during the early stages in DAP12-/- mice. Our results indicate that DAP12 deficiency impairs fracture healing, suggesting a significant role of DAP12 in the initial inflammatory response, bone remodeling and regeneration. PMID:26030755

  14. The alternate AP-1 adaptor subunit Apm2 interacts with the Mil1 regulatory protein and confers differential cargo sorting

    PubMed Central

    Whitfield, Shawn T.; Burston, Helen E.; Bean, Björn D. M.; Raghuram, Nandini; Maldonado-Báez, Lymarie; Davey, Michael; Wendland, Beverly; Conibear, Elizabeth

    2016-01-01

    Heterotetrameric adaptor protein complexes are important mediators of cargo protein sorting in clathrin-coated vesicles. The cell type–specific expression of alternate μ chains creates distinct forms of AP-1 with altered cargo sorting, but how these subunits confer differential function is unclear. Whereas some studies suggest the μ subunits specify localization to different cellular compartments, others find that the two forms of AP-1 are present in the same vesicle but recognize different cargo. Yeast have two forms of AP-1, which differ only in the μ chain. Here we show that the variant μ chain Apm2 confers distinct cargo-sorting functions. Loss of Apm2, but not of Apm1, increases cell surface levels of the v-SNARE Snc1. However, Apm2 is unable to replace Apm1 in sorting Chs3, which requires a dileucine motif recognized by the γ/σ subunits common to both complexes. Apm2 and Apm1 colocalize at Golgi/early endosomes, suggesting that they do not associate with distinct compartments. We identified a novel, conserved regulatory protein that is required for Apm2-dependent sorting events. Mil1 is a predicted lipase that binds Apm2 but not Apm1 and contributes to its membrane recruitment. Interactions with specific regulatory factors may provide a general mechanism to diversify the functional repertoire of clathrin adaptor complexes. PMID:26658609

  15. SHP2-interacting Transmembrane Adaptor Protein (SIT), A Novel Disulfide-linked Dimer Regulating Human T Cell Activation

    PubMed Central

    Marie-Cardine, Anne; Kirchgessner, Henning; Bruyns, Eddy; Shevchenko, Andrej; Mann, Matthias; Autschbach, Frank; Ratnofsky, Sheldon; Meuer, Stefan; Schraven, Burkhart

    1999-01-01

    T lymphocytes express several low molecular weight transmembrane adaptor proteins that recruit src homology (SH)2 domain–containing intracellular molecules to the cell membrane via tyrosine-based signaling motifs. We describe here a novel molecule of this group termed SIT (SHP2 interacting transmembrane adaptor protein). SIT is a disulfide-linked homodimeric glycoprotein that is expressed in lymphocytes. After tyrosine phosphorylation by src and possibly syk protein tyrosine kinases SIT recruits the SH2 domain–containing tyrosine phosphatase SHP2 via an immunoreceptor tyrosine-based inhibition motif. Overexpression of SIT in Jurkat cells downmodulates T cell receptor– and phytohemagglutinin-mediated activation of the nuclear factor of activated T cells (NF-AT) by interfering with signaling processes that are probably located upstream of activation of phospholipase C. However, binding of SHP2 to SIT is not required for inhibition of NF-AT induction, suggesting that SIT not only regulates NF-AT activity but also controls NF-AT unrelated pathways of T cell activation involving SHP2. PMID:10209036

  16. Impaired Fracture Healing Caused by Deficiency of the Immunoreceptor Adaptor Protein DAP12

    PubMed Central

    Kamimura, Masayuki; Mori, Yu; Sugahara-Tobinai, Akiko; Takai, Toshiyuki; Itoi, Eiji

    2015-01-01

    Osteoclasts play an important role in bone metabolism, but their exact role in fracture healing remains unclear. DAP12 is an immunoadaptor protein with associated immunoreceptors on myeloid lineage cells, including osteoclasts. Its deficiency causes osteopetrosis due to suppression of osteoclast development and activation. In this report, we assessed the impact of DAP12 on the fracture healing process using C57BL/6 (B6) and DAP12–/– mice. Healing was evaluated using radiography, micro-CT, histology, immunohistochemistry and real-time RT-PCR. Radiography showed lower callus volume and lower callus radiolucency in DAP12–/– mice during later stages. Micro-CT images and quantitative structural analysis indicated that DAP12–/– mice developed calluses of dense trabecular structures and experienced deteriorated cortical shell formation on the surface. Histologically, DAP12–/– mice showed less cartilaginous resorption and woven bone formation. In addition, prominent cortical shell formation was much less in DAP12–/– mice. Immunohistochemistry revealed lower invasion of F4/80 positive monocytes and macrophages into the fracture hematoma in DAP12–/– mice. The expression levels of Col1a1, Col2a1 and Col10a1 in DAP12–/– mice increased and subsequently became higher than those in B6 mice. There was a decrease in the gene expression of Tnf during the early stages in DAP12–/– mice. Our results indicate that DAP12 deficiency impairs fracture healing, suggesting a significant role of DAP12 in the initial inflammatory response, bone remodeling and regeneration. PMID:26030755

  17. The Cellulosome System of Acetivibrio cellulolyticus Includes a Novel Type of Adaptor Protein and a Cell Surface Anchoring Protein

    PubMed Central

    Xu, Qi; Gao, Wenchen; Ding, Shi-You; Kenig, Rina; Shoham, Yuval; Bayer, Edward A.; Lamed, Raphael

    2003-01-01

    designated ScaA. In addition, ScaB is thought to assume the role of an adaptor protein, which connects the primary scaffoldin (ScaA) to the cohesin-containing anchoring scaffoldin (ScaC). The cellulosome system of A. cellulolyticus thus appears to exhibit a special type of organization that reflects the function of the ScaB adaptor protein. The intercalation of three multiple cohesin-containing scaffoldins results in marked amplification of the number of enzyme subunits per cellulosome unit. At least 96 enzymes can apparently be incorporated into an individual A. cellulolyticus cellulosome. The role of such amplified enzyme incorporation and the resultant proximity of the enzymes within the cellulosome complex presumably contribute to the enhanced synergistic action and overall efficient digestion of recalcitrant forms of cellulose. Comparison of the emerging organization of the A. cellulolyticus cellulosome with the organizations in other cellulolytic bacteria revealed the diversity of the supramolecular architecture. PMID:12867464

  18. Dissecting nuclear Wingless signalling: recruitment of the transcriptional co-activator Pygopus by a chain of adaptor proteins.

    PubMed

    Städeli, Reto; Basler, Konrad

    2005-11-01

    Members of the Wingless (Wg)/Wnt family of secreted glycoproteins control cell fate during embryonic development and adult homeostasis. Wnt signals regulate the expression of target genes by activating a conserved signal transduction pathway. Upon receptor activation, the signal is transmitted intracellularly by stabilization of Armadillo (Arm)/beta-catenin. Arm/beta-catenin translocates to the nucleus, interacts with DNA-binding factors of the Pangolin (Pan)/TCF/LEF class and activates transcription of target genes in cooperation with the recently identified proteins Legless/BCL9 (Lgs) and Pygopus (Pygo). Here, we analyse the mode of action of Pan, Arm, Lgs, and Pygo in Drosophila cultured cells. We provide evidence that together these four proteins form a 'chain of adaptors' linking the NH2-terminal homology domain (NHD) of Pygo to the DNA-binding domain of Pan. We show that the NHD has potent transcriptional activation capacity, which differs from that of acidic activator domains and depends on a conserved NPF tripeptide. A single point mutation within this NPF motif abolishes the transcriptional activity of the Pygo NHD in vitro and strongly reduces Wg signalling in vivo. Together, our results suggest that the transcriptional output of Wg pathway activity largely relies on a 'chain of adaptors' design to direct the Pygo NHD to Wg target promoters in an Arm-dependent manner. PMID:16169192

  19. Single Amino Acid Substitutions Confer the Antiviral Activity of the TRAF3 Adaptor Protein onto TRAF5

    PubMed Central

    Zhang, Peng; Reichardt, Anna; Liang, Huanhuan; Aliyari, Roghiyh; Cheng, David; Wang, Yaya; Xu, Feng

    2014-01-01

    The TRAF [tumor necrosis factor receptor–associated factor] family of cytoplasmic adaptor proteins link cell-surface receptors to intracellular signaling pathways that regulate innate and adaptive immune responses. In response to activation of RIG-I (retinoic acid–inducible gene I), a component of a pattern recognition receptor that detects viruses, TRAF3 binds to the adaptor protein Cardif [caspase activation and recruitment domain (CARD) adaptor–inducing interferon-b (IFN-b)], leading to induction of type I IFNs. We report the crystal structures of the TRAF domain of TRAF5 and that of TRAF3 bound to a peptide from the TRAF-interacting motif of Cardif. By comparing these structures, we identified two residues located near the Cardif binding pocket in TRAF3 (Tyr440 and Phe473) that potentially contributed to Cardif recognition. In vitro and cellular experiments showed that forms of TRAF5 with mutation of the corresponding residues to those of TRAF3 had TRAF3-like antiviral activity. Our results provide a structural basis for the critical role of TRAF3 in activating RIG-I–mediated IFN production. PMID:23150880

  20. Dissecting nuclear Wingless signalling: recruitment of the transcriptional co-activator Pygopus by a chain of adaptor proteins.

    PubMed

    Städeli, Reto; Basler, Konrad

    2005-11-01

    Members of the Wingless (Wg)/Wnt family of secreted glycoproteins control cell fate during embryonic development and adult homeostasis. Wnt signals regulate the expression of target genes by activating a conserved signal transduction pathway. Upon receptor activation, the signal is transmitted intracellularly by stabilization of Armadillo (Arm)/beta-catenin. Arm/beta-catenin translocates to the nucleus, interacts with DNA-binding factors of the Pangolin (Pan)/TCF/LEF class and activates transcription of target genes in cooperation with the recently identified proteins Legless/BCL9 (Lgs) and Pygopus (Pygo). Here, we analyse the mode of action of Pan, Arm, Lgs, and Pygo in Drosophila cultured cells. We provide evidence that together these four proteins form a 'chain of adaptors' linking the NH2-terminal homology domain (NHD) of Pygo to the DNA-binding domain of Pan. We show that the NHD has potent transcriptional activation capacity, which differs from that of acidic activator domains and depends on a conserved NPF tripeptide. A single point mutation within this NPF motif abolishes the transcriptional activity of the Pygo NHD in vitro and strongly reduces Wg signalling in vivo. Together, our results suggest that the transcriptional output of Wg pathway activity largely relies on a 'chain of adaptors' design to direct the Pygo NHD to Wg target promoters in an Arm-dependent manner.

  1. Molecular basis for the specific recognition of the metazoan cyclic GMP-AMP by the innate immune adaptor protein STING.

    PubMed

    Shi, Heping; Wu, Jiaxi; Chen, Zhijian J; Chen, Chuo

    2015-07-21

    Cyclic GMP-AMP containing a unique combination of mixed phosphodiester linkages (2'3'-cGAMP) is an endogenous second messenger molecule that activates the type-I IFN pathway upon binding to the homodimer of the adaptor protein STING on the surface of endoplasmic reticulum membrane. However, the preferential binding of the asymmetric ligand 2'3'-cGAMP to the symmetric dimer of STING represents a physicochemical enigma. Here we show that 2'3'-cGAMP, but not its linkage isomers, adopts an organized free-ligand conformation that resembles the STING-bound conformation and pays low entropy and enthalpy costs in converting into the active conformation. Our results demonstrate that analyses of free-ligand conformations can be as important as analyses of protein conformations in understanding protein-ligand interactions.

  2. The adaptor proteins p140CAP and p130CAS as molecular hubs in cell migration and invasion of cancer cells

    PubMed Central

    Di Stefano, Paola; Leal, Maria Pilar Camacho; Tornillo, Giusy; Bisaro, Brigitte; Repetto, Daniele; Pincini, Alessandra; Santopietro, Emanuela; Sharma, Nanaocha; Turco, Emilia; Cabodi, Sara; Defilippi, Paola

    2011-01-01

    The assembly of molecular hubs upon integrin and growth factor stimulation represents a preferential way to transduce signals throughout the cell. Among the intracellular kinases that are responsive to integrin and growth factor activation, Src Family Kinases (SFKs) are crucial regulators of cell migration and invasion. Increasing evidence highlight the importance of adaptor proteins in these processes, based on their ability to create signalling platforms that control downstream signals. Among these adaptors we will discuss the molecular features of p130Cas and p140Cap proteins in terms of regulation of cell migration and invasion in normal and transformed cells. PMID:21994904

  3. Functional characterization of the interactions between endosomal adaptor protein APPL1 and the NuRD co-repressor complex

    PubMed Central

    Banach-Orlowska, Magdalena; Pilecka, Iwona; Torun, Anna; Pyrzynska, Beata; Miaczynska, Marta

    2009-01-01

    Multifunctional adaptor protein APPL1 [adaptor protein containing PH (pleckstrin homology) domain, PTB (phosphotyrosine binding) domain and leucine zipper motif] belongs to a growing group of endocytic proteins which actively participate in various stages of signalling pathways. Owing to its interaction with the small GTPase Rab5, APPL1 localizes predominantly to a subpopulation of early endosomes but is also capable of nucleocytoplasmic shuttling. Among its various binding partners, APPL1 was reported to associate with the nuclear co-repressor complex NuRD (nucleosome remodelling and deacetylase), containing both nucleosome remodelling and HDAC (histone deacetylase) activities, but the biochemical basis or functional relevance of this interaction remained unknown. Here we characterized the binding between APPL1 and NuRD in more detail, identifying HDAC2 as the key NuRD subunit responsible for this association. APPL1 interacts with the NuRD complex containing enzymatically active HDAC2 but not HDAC1 as the only deacetylase. However, the cellular levels of HDAC1 can regulate the extent of APPL1–NuRD interactions, which in turn modulates the nucleocytoplasmic distribution of APPL1. Increased binding of APPL1 to NuRD upon silencing of HDAC1 promotes the nuclear localization of APPL1, whereas HDAC1 overexpression exerts an opposite effect. Moreover, we also uncovered a NuRD-independent interaction of APPL1 with HDAC1. APPL1 overexpression affects the composition of the HDAC1-containing NuRD complex and the expression of HDAC1 target p21WAF1/CIP1. Cumulatively, these data reveal a surprising complexity of APPL1 interactions with HDACs, with functional consequences for the modulation of gene expression. In a broader sense, these results contribute to an emerging theme of endocytic proteins playing alternative roles in the cell nucleus. PMID:19686092

  4. Interaction of atypical cadherin Fat1 with SoHo adaptor proteins CAP/ponsin and ArgBP2.

    PubMed

    Braun, Gerald S; Kuszka, Andrzej; Dau, Cécile; Kriz, Wilhelm; Moeller, Marcus J

    2016-03-25

    Mammalian Fat1 is a giant atypical cadherin/tumor suppressor involved in the regulation of cellular orientation, migration, and growth. Fat1 is implicated in the development of the brain, eye, and kidney. Altered expression or mutations of FAT1 are also associated with cancer and facioscapulohumeral muscular dystrophy (FSHD). Yet, the mechanistic functions of this pathway remain incompletely understood. Here, we report the identification of Sorbin-homology (SoHo) proteins as novel interaction partners of Fat1 by virtue of a yeast-two-hybrid screen. SoHo proteins play diverse roles as adaptor proteins in cell signaling, cell adhesion and sarcomere architecture, including altered expression in cancer and FSHD. Specifically, we found SoHo proteins CAP/ponsin-1 and -2 (Sorbs1) and ArgBP2 (Sorbs2) to interact with the cytoplasmic domain of Fat1. We mapped the interaction to a prolin-rich classic type II PXXP motif within Fat1 and to the three Src-homology (SH3) domains within SoHo proteins using mutant expression in yeast, pulldown assays, and cell culture. Functionally, endogenous ponsin-2 expression of NRK-52E cells at cellular leading edges was lost upon knockdown of Fat1. In summary, our data point to an interaction of Fat1 with SoHo proteins that is able to recruit SoHo proteins to sites of Fat1 expression. PMID:26903299

  5. Overexpression of myeloid differentiation protein 88 in mice induces mild cardiac dysfunction, but no deficit in heart morphology

    PubMed Central

    Chen, W.; Huang, Z.; Jiang, X.; Li, C.; Gao, X.

    2015-01-01

    Cardiac remodeling involves changes in heart shape, size, structure, and function after injury to the myocardium. The proinflammatory adaptor protein myeloid differentiation protein 88 (MyD88) contributes to cardiac remodeling. To investigate whether excessive MyD88 levels initiate spontaneous cardiac remodeling at the whole-organism level, we generated a transgenic MyD88 mouse model with a cardiac-specific promoter. MyD88 mice (male, 20-30 g, n=∼80) were born at the expected Mendelian ratio and demonstrated similar morphology of the heart and cardiomyocytes with that of wild-type controls. Although heart weight was unaffected, cardiac contractility of MyD88 hearts was mildly reduced, as shown by echocardiographic examination, compared with wild-type controls. Moreover, the cardiac dysfunction phenotype was associated with elevation of ANF and BNP expression. Collectively, our data provide novel evidence of the critical role of balanced MyD88 signaling in maintaining physiological function in the adult heart. PMID:26628395

  6. Adaptor protein complex 4 deficiency causes severe autosomal-recessive intellectual disability, progressive spastic paraplegia, shy character, and short stature.

    PubMed

    Abou Jamra, Rami; Philippe, Orianne; Raas-Rothschild, Annick; Eck, Sebastian H; Graf, Elisabeth; Buchert, Rebecca; Borck, Guntram; Ekici, Arif; Brockschmidt, Felix F; Nöthen, Markus M; Munnich, Arnold; Strom, Tim M; Reis, Andre; Colleaux, Laurence

    2011-06-10

    Intellectual disability inherited in an autosomal-recessive fashion represents an important fraction of severe cognitive-dysfunction disorders. Yet, the extreme heterogeneity of these conditions markedly hampers gene identification. Here, we report on eight affected individuals who were from three consanguineous families and presented with severe intellectual disability, absent speech, shy character, stereotypic laughter, muscular hypotonia that progressed to spastic paraplegia, microcephaly, foot deformity, decreased muscle mass of the lower limbs, inability to walk, and growth retardation. Using a combination of autozygosity mapping and either Sanger sequencing of candidate genes or next-generation exome sequencing, we identified one mutation in each of three genes encoding adaptor protein complex 4 (AP4) subunits: a nonsense mutation in AP4S1 (NM_007077.3: c.124C>T, p.Arg42(∗)), a frameshift mutation in AP4B1 (NM_006594.2: c.487_488insTAT, p.Glu163_Ser739delinsVal), and a splice mutation in AP4E1 (NM_007347.3: c.542+1_542+4delGTAA, r.421_542del, p.Glu181Glyfs(∗)20). Adaptor protein complexes (AP1-4) are ubiquitously expressed, evolutionarily conserved heterotetrameric complexes that mediate different types of vesicle formation and the selection of cargo molecules for inclusion into these vesicles. Interestingly, two mutations affecting AP4M1 and AP4E1 have recently been found to cause cerebral palsy associated with severe intellectual disability. Combined with previous observations, these results support the hypothesis that AP4-complex-mediated trafficking plays a crucial role in brain development and functioning and demonstrate the existence of a clinically recognizable syndrome due to deficiency of the AP4 complex. PMID:21620353

  7. Linker for activation of T-cell family member2 (LAT2) a lipid raft adaptor protein for AKT signaling, is an early mediator of alkylphospholipid anti-leukemic activity.

    PubMed

    Thomé, Carolina H; dos Santos, Guilherme A; Ferreira, Germano A; Scheucher, Priscila S; Izumi, Clarice; Leopoldino, Andreia M; Simão, Ana Maria; Ciancaglini, Pietro; de Oliveira, Kleber T; Chin, Alice; Hanash, Samir M; Falcão, Roberto P; Rego, Eduardo M; Greene, Lewis J; Faça, Vitor M

    2012-12-01

    Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 μm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 μm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five μm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy.

  8. Linker for Activation of T-cell Family Member2 (LAT2) a Lipid Raft Adaptor Protein for AKT Signaling, Is an Early Mediator of Alkylphospholipid Anti-leukemic Activity*

    PubMed Central

    Thomé, Carolina H.; dos Santos, Guilherme A.; Ferreira, Germano A.; Scheucher, Priscila S.; Izumi, Clarice; Leopoldino, Andreia M.; Simão, Ana Maria; Ciancaglini, Pietro; de Oliveira, Kleber T.; Chin, Alice; Hanash, Samir M.; Falcão, Roberto P.; Rego, Eduardo M.; Greene, Lewis J.; Faça, Vitor M.

    2012-01-01

    Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 μm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 μm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five μm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy. PMID:23001822

  9. Matrilin-2, an extracellular adaptor protein, is needed for the regeneration of muscle, nerve and other tissues

    PubMed Central

    Korpos, Éva; Deák, Ferenc; Kiss, Ibolya

    2015-01-01

    The extracellular matrix (ECM) performs essential functions in the differentiation, maintenance and remodeling of tissues during development and regeneration, and it undergoes dynamic changes during remodeling concomitant to alterations in the cell-ECM interactions. Here we discuss recent data addressing the critical role of the widely expressed ECM protein, matrilin-2 (Matn2) in the timely onset of differentiation and regeneration processes in myogenic, neural and other tissues and in tumorigenesis. As a multiadhesion adaptor protein, it interacts with other ECM proteins and integrins. Matn2 promotes neurite outgrowth, Schwann cell migration, neuromuscular junction formation, skeletal muscle and liver regeneration and skin wound healing. Matn2 deposition by myoblasts is crucial for the timely induction of the global switch toward terminal myogenic differentiation during muscle regeneration by affecting transforming growth factor beta/bone morphogenetic protein 7/Smad and other signal transduction pathways. Depending on the type of tissue and the pathomechanism, Matn2 can also promote or suppress tumor growth. PMID:26199591

  10. A Cyclic di-GMP-binding Adaptor Protein Interacts with Histidine Kinase to Regulate Two-component Signaling.

    PubMed

    Xu, Linghui; Venkataramani, Prabhadevi; Ding, Yichen; Liu, Yang; Deng, Yinyue; Yong, Grace Lisi; Xin, Lingyi; Ye, Ruijuan; Zhang, Lianhui; Yang, Liang; Liang, Zhao-Xun

    2016-07-29

    The bacterial messenger cyclic di-GMP (c-di-GMP) binds to a diverse range of effectors to exert its biological effect. Despite the fact that free-standing PilZ proteins are by far the most prevalent c-di-GMP effectors known to date, their physiological function and mechanism of action remain largely unknown. Here we report that the free-standing PilZ protein PA2799 from the opportunistic pathogen Pseudomonas aeruginosa interacts directly with the hybrid histidine kinase SagS. We show that PA2799 (named as HapZ: histidine kinase associated PilZ) binds directly to the phosphoreceiver (REC) domain of SagS, and that the SagS-HapZ interaction is further enhanced at elevated c-di-GMP concentration. We demonstrate that binding of HapZ to SagS inhibits the phosphotransfer between SagS and the downstream protein HptB in a c-di-GMP-dependent manner. In accordance with the role of SagS as a motile-sessile switch and biofilm growth factor, we show that HapZ impacts surface attachment and biofilm formation most likely by regulating the expression of a large number of genes. The observations suggest a previously unknown mechanism whereby c-di-GMP mediates two-component signaling through a PilZ adaptor protein.

  11. A Cyclic di-GMP-binding Adaptor Protein Interacts with Histidine Kinase to Regulate Two-component Signaling.

    PubMed

    Xu, Linghui; Venkataramani, Prabhadevi; Ding, Yichen; Liu, Yang; Deng, Yinyue; Yong, Grace Lisi; Xin, Lingyi; Ye, Ruijuan; Zhang, Lianhui; Yang, Liang; Liang, Zhao-Xun

    2016-07-29

    The bacterial messenger cyclic di-GMP (c-di-GMP) binds to a diverse range of effectors to exert its biological effect. Despite the fact that free-standing PilZ proteins are by far the most prevalent c-di-GMP effectors known to date, their physiological function and mechanism of action remain largely unknown. Here we report that the free-standing PilZ protein PA2799 from the opportunistic pathogen Pseudomonas aeruginosa interacts directly with the hybrid histidine kinase SagS. We show that PA2799 (named as HapZ: histidine kinase associated PilZ) binds directly to the phosphoreceiver (REC) domain of SagS, and that the SagS-HapZ interaction is further enhanced at elevated c-di-GMP concentration. We demonstrate that binding of HapZ to SagS inhibits the phosphotransfer between SagS and the downstream protein HptB in a c-di-GMP-dependent manner. In accordance with the role of SagS as a motile-sessile switch and biofilm growth factor, we show that HapZ impacts surface attachment and biofilm formation most likely by regulating the expression of a large number of genes. The observations suggest a previously unknown mechanism whereby c-di-GMP mediates two-component signaling through a PilZ adaptor protein. PMID:27231351

  12. High-mobility group box protein 1 promotes the survival of myeloid-derived suppressor cells by inducing autophagy.

    PubMed

    Parker, Katherine H; Horn, Lucas A; Ostrand-Rosenberg, Suzanne

    2016-09-01

    Myeloid-derived suppressor cells are immune-suppressive cells that are elevated in most individuals with cancer, where their accumulation and suppressive activity are driven by inflammation. As myeloid-derived suppressor cells inhibit anti-tumor immunity and promote tumor progression, we are determining how their viability is regulated. Previous studies have established that the damage-associated molecular pattern molecule high-mobility group box protein 1 drives myeloid-derived suppressor cell accumulation and suppressive potency and is ubiquitously present in the tumor microenvironment. As high-mobility group box protein 1 also facilitates tumor cell survival by inducing autophagy, we sought to determine if high-mobility group box protein 1 regulates myeloid-derived suppressor cell survival through induction of autophagy. Inhibition of autophagy increased the quantity of apoptotic myeloid-derived suppressor cells, demonstrating that autophagy extends the survival and increases the viability of myeloid-derived suppressor cells. Inhibition of high-mobility group box protein 1 similarly increased the level of apoptotic myeloid-derived suppressor cells and reduced myeloid-derived suppressor cell autophagy, demonstrating that in addition to inducing the accumulation of myeloid-derived suppressor cells, high-mobility group box protein 1 sustains myeloid-derived suppressor cell viability. Circulating myeloid-derived suppressor cells have a default autophagic phenotype, and tumor-infiltrating myeloid-derived suppressor cells are more autophagic, consistent with the concept that inflammatory and hypoxic conditions within the microenvironment of solid tumors contribute to tumor progression by enhancing immune-suppressive myeloid-derived suppressor cells. Overall, these results demonstrate that in addition to previously recognized protumor effects, high-mobility group box protein 1 contributes to tumor progression by increasing myeloid-derived suppressor cell viability by

  13. The Adaptor Protein CD2AP Is a Coordinator of Neurotrophin Signaling-Mediated Axon Arbor Plasticity

    PubMed Central

    Harrison, Benjamin J.; Venkat, Gayathri; Lamb, James L.; Hutson, Tom H.; Drury, Cassa; Rau, Kristofer K.; Bunge, Mary Barlett; Mendell, Lorne M.; Gage, Fred H.; Johnson, Richard D.; Hill, Caitlin E.; Rouchka, Eric C.; Moon, Lawrence D.F.

    2016-01-01

    Growth of intact axons of noninjured neurons, often termed collateral sprouting, contributes to both adaptive and pathological plasticity in the adult nervous system, but the intracellular factors controlling this growth are largely unknown. An automated functional assay of genes regulated in sensory neurons from the rat in vivo spared dermatome model of collateral sprouting identified the adaptor protein CD2-associated protein (CD2AP; human CMS) as a positive regulator of axon growth. In non-neuronal cells, CD2AP, like other adaptor proteins, functions to selectively control the spatial/temporal assembly of multiprotein complexes that transmit intracellular signals. Although CD2AP polymorphisms are associated with increased risk of late-onset Alzheimer's disease, its role in axon growth is unknown. Assessments of neurite arbor structure in vitro revealed CD2AP overexpression, and siRNA-mediated knockdown, modulated (1) neurite length, (2) neurite complexity, and (3) growth cone filopodia number, in accordance with CD2AP expression levels. We show, for the first time, that CD2AP forms a novel multiprotein complex with the NGF receptor TrkA and the PI3K regulatory subunit p85, with the degree of TrkA:p85 association positively regulated by CD2AP levels. CD2AP also regulates NGF signaling through AKT, but not ERK, and regulates long-range signaling though TrkA+/RAB5+ signaling endosomes. CD2AP mRNA and protein levels were increased in neurons during collateral sprouting but decreased following injury, suggesting that, although typically considered together, these two adult axonal growth processes are fundamentally different. These data position CD2AP as a major intracellular signaling molecule coordinating NGF signaling to regulate collateral sprouting and structural plasticity of intact adult axons. SIGNIFICANCE STATEMENT Growth of noninjured axons in the adult nervous system contributes to adaptive and maladaptive plasticity, and dysfunction of this process may

  14. Characterization of a protein phosphatase 2A holoenzyme that dephosphorylates the clathrin adaptors AP-1 and AP-2.

    PubMed

    Ricotta, Doris; Hansen, Jens; Preiss, Carolin; Teichert, Dominic; Höning, Stefan

    2008-02-29

    The AP-2 complex is a key factor in the formation of endocytic clathrin-coated vesicles (CCVs). AP-2 sorts and packages cargo membrane proteins into CCVs, binds the coat protein clathrin, and recruits numerous other factors to the site of vesicle formation. Structural information on the AP-2 complex and biochemical work have allowed understanding its function on the molecular level, and recent studies showed that cycles of phosphorylation are key steps in the regulation of AP-2 function. The complex is phosphorylated on both large subunits (alpha- and beta2-adaptins) as well as at a single threonine residue (Thr-156) of the medium subunit mu2. Phosphorylation of mu2 is necessary for efficient cargo recruitment, whereas the functional context of the large subunit phosphorylation is unknown. Here, we show that the subunit phosphorylation of AP-2 exhibits striking differences, with calculated half-lives of <1 min for mu2, approximately 25 min for beta2, and approximately 70 min for alpha. We were also able to purify a phosphatase that dephosphorylates the mu2 subunit. The enzyme is a member of the protein phosphatase 2A family and composed of a catalytic Cbeta subunit, a scaffolding Abeta subunit, and a regulatory Balpha subunit. RNA interference knock down of the latter subunit in HeLa cells resulted in increased levels of phosphorylated adaptors and altered endocytosis, showing that a specific PP2A holoenzyme is an important regulatory enzyme in CCV-mediated transport.

  15. The cytoskeleton adaptor protein ankyrin-1 is upregulated by p53 following DNA damage and alters cell migration

    PubMed Central

    Hall, A E; Lu, W-T; Godfrey, J D; Antonov, A V; Paicu, C; Moxon, S; Dalmay, T; Wilczynska, A; Muller, P A J; Bushell, M

    2016-01-01

    The integrity of the genome is maintained by a host of surveillance and repair mechanisms that are pivotal for cellular function. The tumour suppressor protein p53 is a major component of the DNA damage response pathway and plays a vital role in the maintenance of cell-cycle checkpoints. Here we show that a microRNA, miR-486, and its host gene ankyrin-1 (ANK1) are induced by p53 following DNA damage. Strikingly, the cytoskeleton adaptor protein ankyrin-1 was induced over 80-fold following DNA damage. ANK1 is upregulated in response to a variety of DNA damage agents in a range of cell types. We demonstrate that miR-486-5p is involved in controlling G1/S transition following DNA damage, whereas the induction of the ankyrin-1 protein alters the structure of the actin cytoskeleton and sustains limited cell migration during DNA damage. Importantly, we found that higher ANK1 expression correlates with decreased survival in cancer patients. Thus, these observations highlight ANK1 as an important effector downstream of the p53 pathway. PMID:27054339

  16. Role of Adaptor TrfA and ClpPC in Controlling Levels of SsrA-Tagged Proteins and Antitoxins in Staphylococcus aureus

    PubMed Central

    Donegan, Niles P.; Marvin, Jonathan S.

    2014-01-01

    Staphylococcus aureus responds to changing extracellular environments in part by adjusting its proteome through alterations of transcriptional priorities and selective degradation of the preexisting pool of proteins. In Bacillus subtilis, the proteolytic adaptor protein MecA has been shown to play a role in assisting with the proteolytic degradation of proteins involved in competence and the oxidative stress response. However, the targets of TrfA, the MecA homolog in S. aureus, have not been well characterized. In this work, we investigated how TrfA assists chaperones and proteases to regulate the proteolysis of several classes of proteins in S. aureus. By fusing the last 3 amino acids of the SsrA degradation tag to Venus, a rapidly folding yellow fluorescent protein, we obtained both fluorescence-based and Western blot assay-based evidence that TrfA and ClpCP are the adaptor and protease, respectively, responsible for the degradation of the SsrA-tagged protein in S. aureus. Notably, the impact of TrfA on degradation was most prominent during late log phase and early stationary phase, due in part to a combination of transcriptional regulation and proteolytic degradation of TrfA by ClpCP. We also characterized the temporal transcriptional regulation governing TrfA activity, wherein Spx, a redox-sensitive transcriptional regulator degraded by ClpXP, activates trfA transcription while repressing its own promoter. Finally, the scope of TrfA-mediated proteolysis was expanded by identifying TrfA as the adaptor that works with ClpCP to degrade antitoxins in S. aureus. Together, these results indicate that the adaptor TrfA adds temporal nuance to protein degradation by ClpCP in S. aureus. PMID:25225270

  17. Adaptor Protein Complex 2–Mediated Endocytosis Is Crucial for Male Reproductive Organ Development in Arabidopsis[W

    PubMed Central

    Kim, Soo Youn; Xu, Zheng-Yi; Song, Kyungyoung; Kim, Dae Heon; Kang, Hyangju; Reichardt, Ilka; Sohn, Eun Ju; Friml, Jiří; Juergens, Gerd; Hwang, Inhwan

    2013-01-01

    Fertilization in flowering plants requires the temporal and spatial coordination of many developmental processes, including pollen production, anther dehiscence, ovule production, and pollen tube elongation. However, it remains elusive as to how this coordination occurs during reproduction. Here, we present evidence that endocytosis, involving heterotetrameric adaptor protein complex 2 (AP-2), plays a crucial role in fertilization. An Arabidopsis thaliana mutant ap2m displays multiple defects in pollen production and viability, as well as elongation of staminal filaments and pollen tubes, all of which are pivotal processes needed for fertilization. Of these abnormalities, the defects in elongation of staminal filaments and pollen tubes were partially rescued by exogenous auxin. Moreover, DR5rev:GFP (for green fluorescent protein) expression was greatly reduced in filaments and anthers in ap2m mutant plants. At the cellular level, ap2m mutants displayed defects in both endocytosis of N-(3-triethylammonium-propyl)-4-(4-diethylaminophenylhexatrienyl) pyridinium dibromide, a lypophilic dye used as an endocytosis marker, and polar localization of auxin-efflux carrier PIN FORMED2 (PIN2) in the stamen filaments. Moreover, these defects were phenocopied by treatment with Tyrphostin A23, an inhibitor of endocytosis. Based on these results, we propose that AP-2–dependent endocytosis plays a crucial role in coordinating the multiple developmental aspects of male reproductive organs by modulating cellular auxin level through the regulation of the amount and polarity of PINs. PMID:23975898

  18. Positive and negative regulation of FcepsilonRI-mediated signaling by the adaptor protein LAB/NTAL.

    PubMed

    Zhu, Minghua; Liu, Yan; Koonpaew, Surapong; Granillo, Olivia; Zhang, Weiguo

    2004-10-18

    Linker for activation of B cells (LAB, also called NTAL; a product of wbscr5 gene) is a newly identified transmembrane adaptor protein that is expressed in B cells, NK cells, and mast cells. Upon BCR activation, LAB is phosphorylated and interacts with Grb2. LAB is capable of rescuing thymocyte development in LAT-deficient mice. To study the in vivo function of LAB, LAB-deficient mice were generated. Although disruption of the Lab gene did not affect lymphocyte development, it caused mast cells to be hyperresponsive to stimulation via the FcepsilonRI, evidenced by enhanced Erk activation, calcium mobilization, degranulation, and cytokine production. These data suggested that LAB negatively regulates mast cell function. However, mast cells that lacked both linker for activation of T cells (LAT) and LAB proteins had a more severe block in FcepsilonRI-mediated signaling than LAT(-/-) mast cells, demonstrating that LAB also shares a redundant function with LAT to play a positive role in FcepsilonRI-mediated signaling.

  19. Transmembrane Adaptor Protein PAG/CBP Is Involved in both Positive and Negative Regulation of Mast Cell Signaling

    PubMed Central

    Draberova, Lubica; Bugajev, Viktor; Potuckova, Lucie; Halova, Ivana; Bambouskova, Monika; Polakovicova, Iva; Xavier, Ramnik J.; Seed, Brian

    2014-01-01

    The transmembrane adaptor protein PAG/CBP (here, PAG) is expressed in multiple cell types. Tyrosine-phosphorylated PAG serves as an anchor for C-terminal SRC kinase, an inhibitor of SRC-family kinases. The role of PAG as a negative regulator of immunoreceptor signaling has been examined in several model systems, but no functions in vivo have been determined. Here, we examined the activation of bone marrow-derived mast cells (BMMCs) with PAG knockout and PAG knockdown and the corresponding controls. Our data show that PAG-deficient BMMCs exhibit impaired antigen-induced degranulation, extracellular calcium uptake, tyrosine phosphorylation of several key signaling proteins (including the high-affinity IgE receptor subunits, spleen tyrosine kinase, and phospholipase C), production of several cytokines and chemokines, and chemotaxis. The enzymatic activities of the LYN and FYN kinases were increased in nonactivated cells, suggesting the involvement of a LYN- and/or a FYN-dependent negative regulatory loop. When BMMCs from PAG-knockout mice were activated via the KIT receptor, enhanced degranulation and tyrosine phosphorylation of the receptor were observed. In vivo experiments showed that PAG is a positive regulator of passive systemic anaphylaxis. The combined data indicate that PAG can function as both a positive and a negative regulator of mast cell signaling, depending upon the signaling pathway involved. PMID:25246632

  20. Bivalent Motif-Ear Interactions Mediate the Association of the Accessory Protein Tepsin with the AP-4 Adaptor Complex.

    PubMed

    Mattera, Rafael; Guardia, Carlos M; Sidhu, Sachdev S; Bonifacino, Juan S

    2015-12-25

    The heterotetrameric (ϵ-β4-μ4-σ4) complex adaptor protein 4 (AP-4) is a component of a non-clathrin coat involved in protein sorting at the trans-Golgi network (TGN). Considerable interest in this complex has arisen from the recent discovery that mutations in each of its four subunits are the cause of a congenital intellectual disability and movement disorder in humans. Despite its physiological importance, the structure and function of this coat remain poorly understood. To investigate the assembly of the AP-4 coat, we dissected the determinants of interaction of AP-4 with its only known accessory protein, the ENTH/VHS-domain-containing protein tepsin. Using a variety of protein interaction assays, we found that tepsin comprises two phylogenetically conserved peptide motifs, [GS]LFXG[ML]X[LV] and S[AV]F[SA]FLN, within its C-terminal unstructured region, which interact with the C-terminal ear (or appendage) domains of the β4 and ϵ subunits of AP-4, respectively. Structure-based mutational analyses mapped the binding site for the [GS]LFXG[ML]X[LV] motif to a conserved, hydrophobic surface on the β4-ear platform fold. Both peptide-ear interactions are required for efficient association of tepsin with AP-4, and for recruitment of tepsin to the TGN. The bivalency of the interactions increases the avidity of tepsin for AP-4 and may enable cross-linking of multiple AP-4 heterotetramers, thus contributing to the assembly of the AP-4 coat. In addition to revealing critical aspects of this coat, our findings extend the paradigm of peptide-ear interactions, previously established for clathrin-AP-1/AP-2 coats, to a non-clathrin coat. PMID:26542808

  1. Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins*

    PubMed Central

    Chen, Buxin; Siderovski, David P.; Neubig, Richard R.; Lawson, Mark A.; Trejo, JoAnn

    2014-01-01

    The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of “regulator of G protein signaling” (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 420AKKAA424 mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins. PMID:24297163

  2. Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

    PubMed

    Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

    2014-01-17

    The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins. PMID:24297163

  3. The adaptor protein LAD/TSAd mediates laminin-dependent T cell migration via association with the 67 kDa laminin binding protein

    PubMed Central

    Park, Eunkyung; Choi, Youngbong; Ahn, Eunseon; Park, Inyoung

    2009-01-01

    The adaptor protein, LAD/TSAd, plays essential roles in T cell activation. To further understand the functions of this protein, we performed yeast two-hybrid screening using TSAd as bait and identified 67 kDa laminin binding protein (LBP) as the interacting partner. Subsequently, TSAd-LBP interaction was confirmed in D1.1 T cell line. Upon costimulation by T cell receptor (TCR) plus laminin crosslinking or TCR plus integrin α6 crosslinking, LBP was coimmunoprecipitated with TSAd. Moreover, TCR plus laminin costimulation-dependent T cell migration was enhanced in D1.1 T cells overexpressing TSAd but was disrupted in D1.1 cells overexpressing dominant negative form of TSAd or TSAd shRNA. These data show that, upon TCR plus integrin costimulation, TSAd associates with LBP and mediates T lymphocyte migration. PMID:19561400

  4. Early Loss of Telomerase Action in Yeast Creates a Dependence on the DNA Damage Response Adaptor Proteins

    PubMed Central

    Jay, Kyle A.; Smith, Dana L.

    2016-01-01

    Telomeres cap the ends of chromosomes, protecting them from degradation and inappropriate DNA repair processes that can lead to genomic instability. A short telomere elicits increased telomerase action on itself that replenishes telomere length, thereby stabilizing the telomere. In the prolonged absence of telomerase activity in dividing cells, telomeres eventually become critically short, inducing a permanent cell cycle arrest (senescence). We recently showed that even early after telomerase inactivation (ETI), yeast cells have accelerated mother cell aging and mildly perturbed cell cycles. Here, we show that the complete disruption of DNA damage response (DDR) adaptor proteins in ETI cells causes severe growth defects. This synthetic-lethality phenotype was as pronounced as that caused by extensive DNA damage in wild-type cells but showed genetic dependencies distinct from such damage and was completely alleviated by SML1 deletion, which increases deoxynucleoside triphosphate (dNTP) pools. Our results indicated that these deleterious effects in ETI cells cannot be accounted for solely by the slow erosion of telomeres due to incomplete replication that leads to senescence. We propose that normally occurring telomeric DNA replication stress is resolved by telomerase activity and the DDR in two parallel pathways and that deletion of Sml1 prevents this stress. PMID:27161319

  5. The Adaptor Protein-1 μ1B Subunit Expands the Repertoire of Basolateral Sorting Signal Recognition in Epithelial Cells

    PubMed Central

    Guo, Xiaoli; Mattera, Rafael; Ren, Xuefeng; Chen, Yu; Retamal, Claudio; González, Alfonso; Bonifacino, Juan S.

    2014-01-01

    SUMMARY An outstanding question in protein sorting is why polarized epithelial cells express two isoforms of the μ1 subunit of the AP-1 clathrin adaptor complex: the ubiquitous μ1A and the epithelial-specific μ1B. Previous studies led to the notion that μ1A and μ1B mediate basolateral sorting predominantly from the trans-Golgi network (TGN) and recycling endosomes, respectively. Using improved analytical tools, however, we find that μ1A and μ1B largely colocalize with each other. They also colocalize to similar extents with TGN and recycling endosome markers, as well as with basolateral cargoes transiting biosynthetic and endocytic-recycling routes. Instead, the two isoforms differ in their signal-recognition specificity. In particular, μ1B preferentially binds a subset of signals from cargoes that are sorted basolaterally in a μ1B-dependent manner. We conclude that expression of distinct μ1 isoforms in epithelial cells expands the repertoire of signals recognized by AP-1 for sorting of a broader range of cargoes to the basolateral surface. PMID:24229647

  6. Novel signaling collaboration between TGF-β and adaptor protein Crk facilitates EMT in human lung cancer

    PubMed Central

    Elmansuri, Aiman Z.; Tanino, Mishie A.; Mahabir, Roshan; Wang, Lei; Kimura, Taichi; Nishihara, Hiroshi; Kinoshita, Ichiro; Dosaka-Akita, Hirotoshi; Tsuda, Masumi; Tanaka, Shinya

    2016-01-01

    The signaling adaptor protein Crk has been shown to play an important role in various human cancers. However, its regulatory machinery is not clear. Here, we demonstrated that Crk induced EMT in A549 human lung adenocarcinoma cells through differential regulation of Rac1/Snail and RhoA/Slug, leading to decreased expression of E-cadherin and increased N-cadherin, fibronectin, and MMP2 expression. Cancer cells with mesenchymal features produced TGF-β and also increased the levels of TGF-β receptor. TGF-β increased the endogenous levels of Crk and also augmented Crk-dependent expression of Snail and Slug, and conversely TGF-β receptor inhibitor suppressed the levels of Snail and Slug. Overexpression of Crk was observed at the invasive front of human lung cancer tissues and was significantly associated with poor prognosis. Thus, TGF-β and Crk collaborate to form a positive feedback loop to facilitate EMT, which may lead to the malignancy of human cancers possibly being affected by their microenvironment. PMID:27027347

  7. Dual Activation of TRIF and MyD88 Adaptor Proteins by Angiotensin II Evokes Opposing Effects on Pressure, Cardiac Hypertrophy and Inflammatory Gene Expression

    PubMed Central

    Singh, Madhu V.; Cicha, Michael Z.; Meyerholz, David K.; Chapleau, Mark W.; Abboud, François M.

    2015-01-01

    Hypertension is recognized as an immune disorder whereby immune cells play a defining role in the genesis and progression of the disease. The innate immune system and its component toll-like receptors (TLRs) are key determinants of the immunological outcome through their pro-inflammatory response. TLR activated signaling pathways utilize several adaptor proteins of which adaptor proteins MyD88 and TRIF define two major inflammatory pathways. In this study, we compared the contributions of MyD88 and TRIF adaptor proteins to angiotensin II (Ang II)-induced hypertension and cardiac hypertrophy in mice. Deletion of MyD88 did not prevent cardiac hypertrophy and the pressor response to Ang II tended to increase. Moreover, the increase in inflammatory gene expression (Tnfa, Nox4 and Agtr1a) was significantly greater in the heart and kidney of MyD88-deficient mice compared with wild type mice. Thus, pathways involving MyD88 may actually restrain the inflammatory responses. On the other hand, in mice with non-functional TRIF (Trifmut mice), Ang II induced hypertension and cardiac hypertrophy were abrogated, and pro-inflammatory gene expression in heart and kidneys was unchanged or decreased. Our results indicate that Ang II induces activation of a pro-inflammatory innate immune response, causing hypertension, and cardiac hypertrophy. These effects require functional adaptor protein TRIF-mediated pathways. However, the common MyD88 dependent signaling pathway, which is also activated simultaneously by Ang II, paradoxically exerts a negative regulatory influence on these responses. PMID:26195481

  8. Nuclear IKKbeta is an adaptor protein for IkappaBalpha ubiquitination and degradation in UV-induced NF-kappaB activation.

    PubMed

    Tsuchiya, Yoshihiro; Asano, Tomoichiro; Nakayama, Keiko; Kato, Tomohisa; Karin, Michael; Kamata, Hideaki

    2010-08-27

    Proinflammatory cytokines activate NF-kappaB using the IkappaB kinase (IKK) complex that phosphorylates inhibitory proteins (IkappaBs) at N-terminal sites resulting in their ubiquitination and degradation in the cytoplasm. Although ultraviolet (UV) irradiation does not lead to IKK activity, it activates NF-kappaB by an unknown mechanism through IkappaBalpha degradation without N-terminal phosphorylation. Here, we describe an adaptor function of nuclear IKKbeta in UV-induced IkappaBalpha degradation. UV irradiation induces the nuclear translocation of IkappaBalpha and association with IKKbeta, which constitutively interacts with beta-TrCP through heterogeneous ribonucleoprotein-U (hnRNP-U) leading to IkappaBalpha ubiquitination and degradation. Furthermore, casein kinase 2 (CK2) and p38 associate with IKKbeta and promote IkappaBalpha degradation by phosphorylation at C-terminal sites. Thus, nuclear IKKbeta acts as an adaptor protein for IkappaBalpha degradation in UV-induced NF-kappaB activation. NF-kappaB activated by the nuclear IKKbeta adaptor protein suppresses anti-apoptotic gene expression and promotes UV-induced cell death.

  9. Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme.

    PubMed

    Bonnemaison, Mathilde L; Bäck, Nils; Duffy, Megan E; Ralle, Martina; Mains, Richard E; Eipper, Betty A

    2015-08-28

    The adaptor protein-1 complex (AP-1), which transports cargo between the trans-Golgi network and endosomes, plays a role in the trafficking of Atp7a, a copper-transporting P-type ATPase, and peptidylglycine α-amidating monooxygenase (PAM), a copper-dependent membrane enzyme. Lack of any of the four AP-1 subunits impairs function, and patients with MEDNIK syndrome, a rare genetic disorder caused by lack of expression of the σ1A subunit, exhibit clinical and biochemical signs of impaired copper homeostasis. To explore the role of AP-1 in copper homeostasis in neuroendocrine cells, we used corticotrope tumor cells in which AP-1 function was diminished by reducing expression of its μ1A subunit. Copper levels were unchanged when AP-1 function was impaired, but cellular levels of Atp7a declined slightly. The ability of PAM to function was assessed by monitoring 18-kDa fragment-NH2 production from proopiomelanocortin. Reduced AP-1 function made 18-kDa fragment amidation more sensitive to inhibition by bathocuproine disulfonate, a cell-impermeant Cu(I) chelator. The endocytic trafficking of PAM was altered, and PAM-1 accumulated on the cell surface when AP-1 levels were reduced. Reduced AP-1 function increased the Atp7a presence in early/recycling endosomes but did not alter the ability of copper to stimulate its appearance on the plasma membrane. Co-immunoprecipitation of a small fraction of PAM and Atp7a supports the suggestion that copper can be transferred directly from Atp7a to PAM, a process that can occur only when both proteins are present in the same subcellular compartment. Altered luminal cuproenzyme function may contribute to deficits observed when the AP-1 function is compromised.

  10. Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme*

    PubMed Central

    Bonnemaison, Mathilde L.; Bäck, Nils; Duffy, Megan E.; Ralle, Martina; Mains, Richard E.; Eipper, Betty A.

    2015-01-01

    The adaptor protein-1 complex (AP-1), which transports cargo between the trans-Golgi network and endosomes, plays a role in the trafficking of Atp7a, a copper-transporting P-type ATPase, and peptidylglycine α-amidating monooxygenase (PAM), a copper-dependent membrane enzyme. Lack of any of the four AP-1 subunits impairs function, and patients with MEDNIK syndrome, a rare genetic disorder caused by lack of expression of the σ1A subunit, exhibit clinical and biochemical signs of impaired copper homeostasis. To explore the role of AP-1 in copper homeostasis in neuroendocrine cells, we used corticotrope tumor cells in which AP-1 function was diminished by reducing expression of its μ1A subunit. Copper levels were unchanged when AP-1 function was impaired, but cellular levels of Atp7a declined slightly. The ability of PAM to function was assessed by monitoring 18-kDa fragment-NH2 production from proopiomelanocortin. Reduced AP-1 function made 18-kDa fragment amidation more sensitive to inhibition by bathocuproine disulfonate, a cell-impermeant Cu(I) chelator. The endocytic trafficking of PAM was altered, and PAM-1 accumulated on the cell surface when AP-1 levels were reduced. Reduced AP-1 function increased the Atp7a presence in early/recycling endosomes but did not alter the ability of copper to stimulate its appearance on the plasma membrane. Co-immunoprecipitation of a small fraction of PAM and Atp7a supports the suggestion that copper can be transferred directly from Atp7a to PAM, a process that can occur only when both proteins are present in the same subcellular compartment. Altered luminal cuproenzyme function may contribute to deficits observed when the AP-1 function is compromised. PMID:26170456

  11. CD2v Interacts with Adaptor Protein AP-1 during African Swine Fever Infection

    PubMed Central

    Pérez-Núñez, Daniel; García-Urdiales, Eduardo; Martínez-Bonet, Marta; Nogal, María L.; Barroso, Susana; Revilla, Yolanda; Madrid, Ricardo

    2015-01-01

    African swine fever virus (ASFV) CD2v protein is believed to be involved in virulence enhancement, viral hemadsorption, and pathogenesis, although the molecular mechanisms of the function of this viral protein are still not fully understood. Here we describe that CD2v localized around viral factories during ASFV infection, suggesting a role in the generation and/or dynamics of these viral structures and hence in disturbing cellular traffic. We show that CD2v targeted the regulatory trans-Golgi network (TGN) protein complex AP-1, a key element in cellular traffic. This interaction was disrupted by brefeldin A even though the location of CD2v around the viral factory remained unchanged. CD2v-AP-1 binding was independent of CD2v glycosylation and occurred on the carboxy-terminal part of CD2v, where a canonical di-Leu motif previously reported to mediate AP-1 binding in eukaryotic cells, was identified. This motif was shown to be functionally interchangeable with the di-Leu motif present in HIV-Nef protein in an AP-1 binding assay. However, we demonstrated that it was not involved either in CD2v cellular distribution or in CD2v-AP-1 binding. Taken together, these findings shed light on CD2v function during ASFV infection by identifying AP-1 as a cellular factor targeted by CD2v and hence elucidate the cellular pathways used by the virus to enhance infectivity. PMID:25915900

  12. Gedunin Binds to Myeloid Differentiation Protein 2 and Impairs Lipopolysaccharide-Induced Toll-Like Receptor 4 Signaling in Macrophages.

    PubMed

    Borges, Perla Villani; Moret, Katelim Hottz; Maya-Monteiro, Clarissa Menezes; Souza-Silva, Franklin; Alves, Carlos Roberto; Batista, Paulo Ricardo; Caffarena, Ernesto Raúl; Pacheco, Patrícia; Henriques, Maria das Graças; Penido, Carmen

    2015-11-01

    Recognition of bacterial lipopolysaccharide (LPS) by innate immune system is mediated by the cluster of differentiation 14/Toll-like receptor 4/myeloid differentiation protein 2 (MD-2) complex. In this study, we investigated the modulatory effect of gedunin, a limonoid from species of the Meliaceae family described as a heat shock protein Hsp90 inhibitor, on LPS-induced response in immortalized murine macrophages. The pretreatment of wild-type (WT) macrophages with gedunin (0.01-100 µM, noncytotoxic concentrations) inhibited LPS (50 ng/ml)-induced calcium influx, tumor necrosis factor-α, and nitric oxide production in a concentration-dependent manner. The selective effect of gedunin on MyD88-adapter-like/myeloid differentiation primary response 88- and TRIF-related adaptor molecule/TIR domain-containing adapter-inducing interferon-β-dependent signaling pathways was further investigated. The pretreatment of WT, TIR domain-containing adapter-inducing interferon-β knockout, and MyD88 adapter-like knockout macrophages with gedunin (10 µM) significantly inhibited LPS (50 ng/ml)-induced tumor necrosis factor-α and interleukin-6 production, at 6 hours and 24 hours, suggesting that gedunin modulates a common event between both signaling pathways. Furthermore, gedunin (10 µM) inhibited LPS-induced prostaglandin E2 production, cyclooxygenase-2 expression, and nuclear factor κB translocation into the nucleus of WT macrophages, demonstrating a wide-range effect of this chemical compound. In addition to the ability to inhibit LPS-induced proinflammatory mediators, gedunin also triggered anti-inflammatory factors interleukin-10, heme oxygenase-1, and Hsp70 in macrophages stimulated or not with LPS. In silico modeling studies revealed that gedunin efficiently docked into the MD-2 LPS binding site, a phenomenon further confirmed by surface plasmon resonance. Our results reveal that, in addition to Hsp90 modulation, gedunin acts as a competitive inhibitor of LPS, blocking

  13. Association of Genetic Variation in Adaptor Protein APPL1/APPL2 Loci with Non-Alcoholic Fatty Liver Disease

    PubMed Central

    Barbieri, Michelangela; Esposito, Antonietta; Angellotti, Edith; Rizzo, Maria Rosaria; Marfella, Raffaele; Paolisso, Giuseppe

    2013-01-01

    The importance of genetics and epigenetic changes in the pathogenesis of non alcoholic fatty liver disease (NAFLD) has been increasingly recognized. Adiponectin has a central role in regulating glucose and lipid metabolism and controlling inflammation in insulin-sensitive tissues and low adiponectin levels have been linked to NAFLD. APPL1 and APPL2 are adaptor proteins that interact with the intracellular region of adiponectin receptors and mediate adiponectin signaling and its effects on metabolism. The aim of our study was the evaluation of a potential association between variants at APPL1 and APPL2 loci and NAFLD occurrence. The impact on liver damage and hepatic steatosis severity has been also evaluated. To this aim allele frequency and genotype distribution of APPL1- rs3806622 and -rs4640525 and APPL2-rs 11112412 variants were evaluated in 223 subjects with clinical diagnosis of NAFLD and compared with 231 healthy subjects. The impact of APPL1 and APPL2 SNPs on liver damage and hepatic steatosis severity has been also evaluated. The minor-allele combination APPL1-C/APPL2-A was associated with an increased risk of NAFLD (OR = 2.50 95% CI 1.45–4.32; p<0.001) even after adjustment for age, sex, body mass index, insulin resistance (HOMA-IR), triglycerides and adiponectin levels. This allele combination carrier had higher plasma alanine aminotransferase levels (Diff = 15.08 [7.60–22.57] p = 0.001) and an increased frequency of severe steatosis compared to the reference allele combination (OR = 3.88; 95% CI 1.582–9.531; p<0.001). In conclusion, C-APPL1/A-APPL2 allele combination is associated with NAFLD occurrence, with a more severe hepatic steatosis grade and with a reduced adiponectin cytoprotective effect on liver. PMID:23977033

  14. Both TRIF and IPS-1 adaptor proteins contribute to the cerebral innate immune response against herpes simplex virus 1 infection.

    PubMed

    Menasria, Rafik; Boivin, Nicolas; Lebel, Manon; Piret, Jocelyne; Gosselin, Jean; Boivin, Guy

    2013-07-01

    Toll-like receptors (TLRs) and RNA helicases (RLHs) are important cell sensors involved in the immunological control of viral infections through production of type I interferon (IFN). The impact of a deficiency in the TRIF and IPS-1 adaptor proteins, respectively, implicated in TLR3 and RLH signaling pathways, was investigated during herpes simplex virus 1 (HSV-1) encephalitis. TRIF(-/-), IPS-1(-/-), and C57BL/6 wild-type (WT) mice were infected intranasally with 7.5 × 10(5) PFU of HSV-1. Mice were monitored for neurological signs and survival over 20 days. Groups of mice were sacrificed on days 3, 5, 7, 9, and 11 postinfection for determination of brain viral replication by quantitative PCR (qPCR), plaque assay, and immunohistochemistry and for alpha/beta interferon (IFN-α/β) levels and phosphorylation of interferon regulatory factors 3 and 7 (IRF-3 and -7) in brain homogenates by enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. TRIF(-/-) and IPS-1(-/-) mice had higher mortality rates than WT mice (P = 0.02 and P = 0.09, respectively). Viral antigens were more disseminated throughout the brain, correlating with a significant increase in brain viral load for TRIF(-/-) (days 5 to 9) and IPS-1(-/-) (days 7 and 9) mice compared to results for the WT. IFN-β production was reduced in brain homogenates of TRIF(-/-) and IPS-1(-/-) mice on day 5 compared to results for the WT, whereas IFN-α levels were increased on day 7 in TRIF(-/-) mice. Phosphorylation levels of IRF-3 and IRF-7 were decreased in TRIF(-/-) and IPS-1(-/-) mice, respectively. These data suggest that both the TRIF and IPS-1 signaling pathways are important for the control of HSV replication in the brain and survival through IFN-β production. PMID:23596298

  15. Structural and Functional Characterization of Cargo-Binding Sites on the μ4-Subunit of Adaptor Protein Complex 4

    PubMed Central

    Ross, Breyan H.; Lin, Yimo; Corales, Esteban A.; Burgos, Patricia V.; Mardones, Gonzalo A.

    2014-01-01

    Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (μ1-μ4) that recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in μ subunits mediates recognition of YXXØ-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXØ-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the μ4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of μ4 on the recognition of the non-canonical YXXØ-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type μ4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered μ4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of μ4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the non-canonical YXXØ-signal of APP is limited to the non

  16. The deca-GX3 proteins Yae1-Lto1 function as adaptors recruiting the ABC protein Rli1 for iron-sulfur cluster insertion

    PubMed Central

    Paul, Viktoria Désirée; Mühlenhoff, Ulrich; Stümpfig, Martin; Seebacher, Jan; Kugler, Karl G; Renicke, Christian; Taxis, Christof; Gavin, Anne-Claude; Pierik, Antonio J; Lill, Roland

    2015-01-01

    Cytosolic and nuclear iron-sulfur (Fe-S) proteins are involved in many essential pathways including translation and DNA maintenance. Their maturation requires the cytosolic Fe-S protein assembly (CIA) machinery. To identify new CIA proteins we employed systematic protein interaction approaches and discovered the essential proteins Yae1 and Lto1 as binding partners of the CIA targeting complex. Depletion of Yae1 or Lto1 results in defective Fe-S maturation of the ribosome-associated ABC protein Rli1, but surprisingly no other tested targets. Yae1 and Lto1 facilitate Fe-S cluster assembly on Rli1 in a chain of binding events. Lto1 uses its conserved C-terminal tryptophan for binding the CIA targeting complex, the deca-GX3 motifs in both Yae1 and Lto1 facilitate their complex formation, and Yae1 recruits Rli1. Human YAE1D1 and the cancer-related ORAOV1 can replace their yeast counterparts demonstrating evolutionary conservation. Collectively, the Yae1-Lto1 complex functions as a target-specific adaptor that recruits apo-Rli1 to the generic CIA machinery. DOI: http://dx.doi.org/10.7554/eLife.08231.001 PMID:26182403

  17. The deca-GX3 proteins Yae1-Lto1 function as adaptors recruiting the ABC protein Rli1 for iron-sulfur cluster insertion.

    PubMed

    Paul, Viktoria Désirée; Mühlenhoff, Ulrich; Stümpfig, Martin; Seebacher, Jan; Kugler, Karl G; Renicke, Christian; Taxis, Christof; Gavin, Anne-Claude; Pierik, Antonio J; Lill, Roland

    2015-07-16

    Cytosolic and nuclear iron-sulfur (Fe-S) proteins are involved in many essential pathways including translation and DNA maintenance. Their maturation requires the cytosolic Fe-S protein assembly (CIA) machinery. To identify new CIA proteins we employed systematic protein interaction approaches and discovered the essential proteins Yae1 and Lto1 as binding partners of the CIA targeting complex. Depletion of Yae1 or Lto1 results in defective Fe-S maturation of the ribosome-associated ABC protein Rli1, but surprisingly no other tested targets. Yae1 and Lto1 facilitate Fe-S cluster assembly on Rli1 in a chain of binding events. Lto1 uses its conserved C-terminal tryptophan for binding the CIA targeting complex, the deca-GX3 motifs in both Yae1 and Lto1 facilitate their complex formation, and Yae1 recruits Rli1. Human YAE1D1 and the cancer-related ORAOV1 can replace their yeast counterparts demonstrating evolutionary conservation. Collectively, the Yae1-Lto1 complex functions as a target-specific adaptor that recruits apo-Rli1 to the generic CIA machinery.

  18. The AP2 clathrin adaptor protein complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of Caenorhabditis elegans

    PubMed Central

    Garafalo, Steven D.; Luth, Eric S.; Moss, Benjamin J.; Monteiro, Michael I.; Malkin, Emily; Juo, Peter

    2015-01-01

    Regulation of glutamate receptor (GluR) abundance at synapses by clathrin-mediated endocytosis can control synaptic strength and plasticity. We take advantage of viable, null mutations in subunits of the clathrin adaptor protein 2 (AP2) complex in Caenorhabditis elegans to characterize the in vivo role of AP2 in GluR trafficking. In contrast to our predictions for an endocytic adaptor, we found that levels of the GluR GLR-1 are decreased at synapses in the ventral nerve cord (VNC) of animals with mutations in the AP2 subunits APM-2/μ2, APA-2/α, or APS-2/σ2. Rescue experiments indicate that APM-2/μ2 functions in glr-1–expressing interneurons and the mature nervous system to promote GLR-1 levels in the VNC. Genetic analyses suggest that APM-2/μ2 acts upstream of GLR-1 endocytosis in the VNC. Consistent with this, GLR-1 accumulates in cell bodies of apm-2 mutants. However, GLR-1 does not appear to accumulate at the plasma membrane of the cell body as expected, but instead accumulates in intracellular compartments including Syntaxin-13– and RAB-14–labeled endosomes. This study reveals a novel role for the AP2 clathrin adaptor in promoting the abundance of GluRs at synapses in vivo, and implicates AP2 in the regulation of GluR trafficking at an early step in the secretory pathway. PMID:25788288

  19. The adaptor protein Cindr regulates JNK activity to maintain epithelial sheet integrity.

    PubMed

    Yasin, Hannah W R; van Rensburg, Samuel H; Feiler, Christina E; Johnson, Ruth I

    2016-02-15

    Epithelia are essential barrier tissues that must be appropriately maintained for their correct function. To achieve this a plethora of protein interactions regulate epithelial cell number, structure and adhesion, and differentiation. Here we show that Cindr (the Drosophila Cin85 and Cd2ap ortholog) is required to maintain epithelial integrity. Reducing Cindr triggered cell delamination and movement. Most delaminating cells died. These behaviors were consistent with JNK activation previously associated with loss of epithelial integrity in response to ectopic oncogene activity. We confirmed a novel interaction between Cindr and Drosophila JNK (dJNK), which when perturbed caused inappropriate JNK signaling. Genetically reducing JNK signaling activity suppressed the effects of reducing Cindr. Furthermore, ectopic JNK signaling phenocopied loss of Cindr and was partially rescued by concomitant cindr over-expression. Thus, correct Cindr-dJNK stoichiometry is essential to maintain epithelial integrity and disturbing this balance may contribute to the pathogenesis of disease states, including cancer. PMID:26772997

  20. Transmembrane adaptor protein PAG1 is a novel tumor suppressor in neuroblastoma

    PubMed Central

    Agarwal, Saurabh; Ghosh, Rajib; Chen, Zaowen; Lakoma, Anna; Gunaratne, Preethi H.; Kim, Eugene S.; Shohet, Jason M.

    2016-01-01

    (NB) is the most common extracranial pediatric solid tumor with high mortality rates. The tyrosine kinase c-Src has been known to play an important role in differentiation of NB cells, but the mechanism of c-Src regulation has not been defined. Here, we characterize PAG1 (Cbp, Csk binding protein), a central inhibitor of c-Src and other Src family kinases, as a novel tumor suppressor in NB. Clinical cohort analysis demonstrate that low expression of PAG1 is a significant prognostic factor for high stage disease, increased relapse, and worse overall survival for children with NB. PAG1 knockdown in NB cells promotes proliferation and anchorage-independent colony formation with increased activation of AKT and ERK downstream of c-Src, while PAG1 overexpression significantly rescues these effects. In vivo, PAG1 overexpression significantly inhibits NB tumorigenicity in an orthotopic xenograft model. Our results establish PAG1 as a potent tumor suppressor in NB by inhibiting c-Src and downstream effector pathways. Thus, reactivation of PAG1 and inhibition of c-Src kinase activity represents an important novel therapeutic approach for high-risk NB. PMID:26993602

  1. The adaptor protein 3BP2 associates with VAV guanine nucleotide exchange factors to regulate NFAT activation by the B-cell antigen receptor.

    PubMed

    Foucault, Isabelle; Le Bras, Séverine; Charvet, Céline; Moon, Chéol; Altman, Amnon; Deckert, Marcel

    2005-02-01

    Engagement of the B-cell antigen receptor (BCR) activates kinases of the Src and Syk families and signaling complexes assembled by adaptor proteins, which dictate B-cell fate and function. The adaptor 3BP2/SH3BP2, an Abl Src homology domain 3 (SH3)-binding and Syk-kinases interacting protein, exhibits positive regulatory roles in T, natural killer (NK), and basophilic cells. However, its involvement in BCR signaling is completely unknown. Here we show that 3BP2 is tyrosine phosphorylated following BCR aggregation on B lymphoma cells, and that 3BP2 is a substrate for Syk and Fyn, but not Btk. To further explore the function of 3BP2 in B cells, we screened a yeast 2-hybrid B-lymphocyte library and found 3BP2 as a binding partner of Vav proteins. The interaction between 3BP2 and Vav proteins involved both constitutive and inducible mechanisms. 3BP2 also interacted with other components of the BCR signaling pathway, including Syk and phospholipase C gamma (PLC-gamma). Furthermore, overexpression and RNAi blocking experiments showed that 3BP2 regulated BCR-mediated activation of nuclear factor of activated T cells (NFATs). Finally, evidence was provided that 3BP2 functionally cooperates with Vav proteins and Rho GTPases to activate NFATs. Our results show that 3BP2 may regulate BCR-mediated gene activation through Vav proteins.

  2. Role for the adaptor protein Grb10 in the activation of Akt.

    PubMed

    Jahn, Thomas; Seipel, Petra; Urschel, Susanne; Peschel, Christian; Duyster, Justus

    2002-02-01

    Grb10 is a member of the Grb7 family of adapter proteins lacking intrinsic enzymatic function and encodes functional domains including a pleckstrin homology (PH) domain and an SH2 domain. The role of different Grb10 splice variants in signal transduction of growth factors like insulin or insulin-like growth factor has been described as inhibitory or stimulatory depending on the presence of a functional PH and/or SH2 domain. Performing a yeast two-hybrid screen with the c-kit cytoplasmic tail fused to LexA as a bait and a mouse embryo cDNA library as prey, we found that the Grb10 SH2 domain interacted with the c-kit receptor tyrosine kinase. In the course of SCF-mediated activation of c-kit, Grb10 is recruited to the c-kit receptor in an SH2 domain- and phosphotyrosine-dependent but PH domain-independent manner. We found that Akt and Grb10 form a constitutive complex, suggesting a role for Grb10 in the translocation of Akt to the cell membrane. Indeed, coexpression studies revealed that Grb10 and c-kit activate Akt in a synergistic manner. This dose-dependent effect of Grb10 is wortmannin sensitive and was also seen at a lower level in cells in which c-kit was not expressed. Expression of a Grb10 mutant lacking the SH2 domain as well as a mutant lacking the PH domain did not influence Akt activity. Grb10-induced Akt activation was observed without increased phosphatidylinositol 3-kinase (PI3-kinase) activity, suggesting that Grb10 is a positive regulator of Akt downstream of PI3-kinase. Significantly, deficient activation of Akt by a constitutively activated c-kit mutant lacking the binding site for PI3-kinase (c-kitD814V/Y719F) could be fully compensated by overexpression of Grb10. In Ba/F3 cells, the incapacity of c-kitD814V/Y719F to induce interleukin-3 (IL-3)-independent growth could be rescued by overexpression of Grb10. In contrast, expression of the SH2 deletion mutant of Grb10 together with c-kitD814V/Y719F did not render Ba/F3 cells independent of IL-3. In

  3. Palmitoylation of protease-activated receptor-1 regulates adaptor protein complex-2 and -3 interaction with tyrosine-based motifs and endocytic sorting.

    PubMed

    Canto, Isabel; Trejo, JoAnn

    2013-05-31

    Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface. PMID:23580642

  4. Palmitoylation of protease-activated receptor-1 regulates adaptor protein complex-2 and -3 interaction with tyrosine-based motifs and endocytic sorting.

    PubMed

    Canto, Isabel; Trejo, JoAnn

    2013-05-31

    Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface.

  5. Adaptor protein 2–mediated endocytosis of the β-secretase BACE1 is dispensable for amyloid precursor protein processing

    PubMed Central

    Prabhu, Yogikala; Burgos, Patricia V.; Schindler, Christina; Farías, Ginny G.; Magadár, Javier G.; Bonifacino, Juan S.

    2012-01-01

    The β-site amyloid precursor protein (APP)–cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER–Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway. PMID:22553349

  6. The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

    PubMed Central

    Glenewinkel, Florian; Cohen, Michael J.; King, Cason R.; Kaspar, Sophie; Bamberg-Lemper, Simone; Mymryk, Joe S.; Becker, Walter

    2016-01-01

    DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation. PMID:27307198

  7. The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2.

    PubMed

    Glenewinkel, Florian; Cohen, Michael J; King, Cason R; Kaspar, Sophie; Bamberg-Lemper, Simone; Mymryk, Joe S; Becker, Walter

    2016-01-01

    DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation. PMID:27307198

  8. The COPII adaptor protein TMED7 is required to initiate and mediate the anterograde trafficking of Toll-like receptor 4 to the plasma membrane

    PubMed Central

    Liaunardy-Jopeace, Ardiyanto; Bryant, Clare E.; Gay, Nicholas J.

    2015-01-01

    Toll-like receptor 4 (TLR4), the receptor for the bacterial product endotoxin, is subject to multiple points of regulation at the levels of signaling, biogenesis, and trafficking. Dysregulation of TLR4 signaling can cause serious inflammatory diseases, such as sepsis. We found that the p24 family protein TMED7 (transmembrane emp24 protein transport domain containing 7) is required for the trafficking of TLR4 from the endoplasmic reticulum to the cell surface through the Golgi. TMED7 formed a stable complex with the ectodomain of TLR4, an interaction that required the coiled-coil and GOLD domains, but not the cytosolic, COP II sorting motif, of TMED7. Depletion of TMED7 reduced TLR4 signaling mediated by the adaptor protein MyD88, but not that mediated by the adaptor proteins TRAM and TRIF. Truncated forms of TMED7 lacking the COP II sorting motif or the transmembrane domain were mislocalized and resulted in constitutive activation of TLR4 signaling. Together, these results support the hypothesis that p24 proteins perform a quality control step by recognizing correctly folded anterograde cargo, such as TLR4, in early secretory compartments and facilitating the translocation of this cargo to the cell surface. PMID:25074978

  9. Adaptors for radiation detectors

    DOEpatents

    Livesay, Ronald Jason

    2015-07-28

    Described herein are adaptors and other devices for radiation detectors that can be used to make accurate spectral measurements of both small and large bulk sources of radioactivity, such as building structures, soils, vessels, large equipment, and liquid bodies. Some exemplary devices comprise an adaptor for a radiation detector, wherein the adaptor can be configured to collimate radiation passing through the adapter from an external radiation source to the radiation detector and the adaptor can be configured to enclose a radiation source within the adapter to allow the radiation detector to measure radiation emitted from the enclosed radiation source.

  10. Adaptors for radiation detectors

    DOEpatents

    Livesay, Ronald Jason

    2014-04-22

    Described herein are adaptors and other devices for radiation detectors that can be used to make accurate spectral measurements of both small and large bulk sources of radioactivity, such as building structures, soils, vessels, large equipment, and liquid bodies. Some exemplary devices comprise an adaptor for a radiation detector, wherein the adaptor can be configured to collimate radiation passing through the adapter from an external radiation source to the radiation detector and the adaptor can be configured to enclose a radiation source within the adapter to allow the radiation detector to measure radiation emitted from the enclosed radiation source.

  11. Regulation of presynaptic anchoring of the scaffold protein Bassoon by phosphorylation-dependent interaction with 14-3-3 adaptor proteins.

    PubMed

    Schröder, Markus S; Stellmacher, Anne; Romorini, Stefano; Marini, Claudia; Montenegro-Venegas, Carolina; Altrock, Wilko D; Gundelfinger, Eckart D; Fejtova, Anna

    2013-01-01

    The proper organization of the presynaptic cytomatrix at the active zone is essential for reliable neurotransmitter release from neurons. Despite of the virtual stability of this tightly interconnected proteinaceous network it becomes increasingly clear that regulated dynamic changes of its composition play an important role in the processes of synaptic plasticity. Bassoon, a core component of the presynaptic cytomatrix, is a key player in structural organization and functional regulation of presynaptic release sites. It is one of the most highly phosphorylated synaptic proteins. Nevertheless, to date our knowledge about functions mediated by any one of the identified phosphorylation sites of Bassoon is sparse. In this study, we have identified an interaction of Bassoon with the small adaptor protein 14-3-3, which depends on phosphorylation of the 14-3-3 binding motif of Bassoon. In vitro phosphorylation assays indicate that phosphorylation of the critical Ser-2845 residue of Bassoon can be mediated by a member of the 90-kDa ribosomal S6 protein kinase family. Elimination of Ser-2845 from the 14-3-3 binding motif results in a significant decrease of Bassoon's molecular exchange rates at synapses of living rat neurons. We propose that the phosphorylation-induced 14-3-3 binding to Bassoon modulates its anchoring to the presynaptic cytomatrix. This regulation mechanism might participate in molecular and structural presynaptic remodeling during synaptic plasticity.

  12. Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice.

    PubMed

    Kook, Seunghyi; Wang, Ping; Young, Lisa R; Schwake, Michael; Saftig, Paul; Weng, Xialian; Meng, Ying; Neculai, Dante; Marks, Michael S; Gonzales, Linda; Beers, Michael F; Guttentag, Susan

    2016-04-15

    The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients.

  13. Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice.

    PubMed

    Kook, Seunghyi; Wang, Ping; Young, Lisa R; Schwake, Michael; Saftig, Paul; Weng, Xialian; Meng, Ying; Neculai, Dante; Marks, Michael S; Gonzales, Linda; Beers, Michael F; Guttentag, Susan

    2016-04-15

    The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients. PMID:26907692

  14. The Adaptor Proteins p66Shc and Grb2 Regulate the Activation of the GTPases ARF1 and ARF6 in Invasive Breast Cancer Cells*

    PubMed Central

    Haines, Eric; Saucier, Caroline; Claing, Audrey

    2014-01-01

    Signals downstream of growth factor receptors play an important role in mammary carcinogenesis. Recently, we demonstrated that the small GTPases ARF1 and ARF6 were shown to be activated downstream of the epidermal growth factor receptor (EGFR) and act as a key regulator of growth, migration, and invasion of breast cancer cells. However, the mechanism via which the EGFR recruits and activates ARF1 and ARF6 to transmit signals has yet to be fully elucidated. Here, we identify adaptor proteins Grb2 and p66Shc as important regulators mediating ARF activation. We demonstrate that ARF1 can be found in complex with Grb2 and p66Shc upon EGF stimulation of the basal-like breast cancer MDA-MB-231 cell line. However, we report that these two adaptors regulate ARF1 activation differently, with Grb2 promoting ARF1 activation and p66Shc blocking this response. Furthermore, we show that Grb2 is essential for the recruitment of ARF1 to the EGFR, whereas p66Shc hindered ARF1 receptor recruitment. We demonstrate that the negative regulatory role of p66Shc stemmed from its ability to block the recruitment of Grb2/ARF1 to the EGFR. Conversely, p66Shc potentiates ARF6 activation as well as the recruitment of this ARF isoform to the EGFR. Interestingly, we demonstrate that Grb2 is also required for the activation and receptor recruitment of ARF6. Additionally, we show an important role for p66Shc in modulating ARF activation, cell growth, and migration in HER2-positive breast cancer cells. Together, our results highlight a central role for adaptor proteins p66Shc and Grb2 in the regulation of ARF1 and ARF6 activation in invasive breast cancer cells. PMID:24407288

  15. The μ Subunit of Arabidopsis Adaptor Protein-2 Is Involved in Effector-Triggered Immunity Mediated by Membrane-Localized Resistance Proteins.

    PubMed

    Hatsugai, Noriyuki; Hillmer, Rachel; Yamaoka, Shohei; Hara-Nishimura, Ikuko; Katagiri, Fumiaki

    2016-05-01

    Endocytosis has been suggested to be important in the cellular processes of plant immune responses. However, our understanding of its role during effector-triggered immunity (ETI) is still limited. We have previously shown that plant endocytosis, especially clathrin-coated vesicle formation at the plasma membrane, is mediated by the adaptor protein-2 (AP-2) complex and that loss of the μ subunit of AP-2 (AP2M) affects plant growth and floral organ development. Here, we report that AP2M is required for full-strength ETI mediated by the disease resistance (R) genes RPM1 and RPS2 in Arabidopsis. Reduced ETI was observed in an ap2m mutant plant, measured by growth of Pseudomonas syringae pv. tomato DC3000 strains carrying the corresponding effector genes avrRpm1 or avrRpt2 and by hypersensitive cell death response and defense gene expression triggered by these strains. In contrast, RPS4-mediated ETI and its associated immune responses were not affected by the ap2m mutation. While RPM1 and RPS2 are localized to the plasma membrane, RPS4 is localized to the cytoplasm and nucleus. Our results suggest that AP2M is involved in ETI mediated by plasma membrane-localized R proteins, possibly by mediating endocytosis of the immune receptor complex components from the plasma membrane.

  16. Allelic Exclusion of the T Cell Receptor β Locus Requires the Sh2 Domain–Containing Leukocyte Protein (Slp)-76 Adaptor Protein

    PubMed Central

    Aifantis, Iannis; Pivniouk, Vadim I.; Gärtner, Frank; Feinberg, Jacqueline; Swat, Wojciech; Alt, Frederick W.; von Boehmer, Harald; Geha, Raif S.

    1999-01-01

    Signaling via the pre-T cell receptor (TCR) is required for the proliferative expansion and maturation of CD4−CD8− double-negative (DN) thymocytes into CD4+CD8+ double-positive (DP) cells and for TCR-β allelic exclusion. The adaptor protein SH2 domain–containing leukocyte protein (SLP)-76 has been shown to play a crucial role in thymic development, because thymocytes of SLP-76−/− mice are arrested at the CD25+CD44− DN stage. Here we show that SLP-76−/− DN thymocytes express the pre-TCR on their surfaces and that introduction of a TCR-α/β transgene into the SLP-76−/− background fails to cause expansion of DN thymocytes or developmental progression to the DP stage. Moreover, analysis of TCR-β rearrangement in SLP-76−/− TCR-transgenic mice or in single CD25+CD44− DN cells from SLP-76−/− mice indicates an essential role of SLP-76 in TCR-β allelic exclusion. PMID:10523607

  17. High Fat Diet Enhances β-Site Cleavage of Amyloid Precursor Protein (APP) via Promoting β-Site APP Cleaving Enzyme 1/Adaptor Protein 2/Clathrin Complex Formation.

    PubMed

    Maesako, Masato; Uemura, Maiko; Tashiro, Yoshitaka; Sasaki, Kazuki; Watanabe, Kiwamu; Noda, Yasuha; Ueda, Karin; Asada-Utsugi, Megumi; Kubota, Masakazu; Okawa, Katsuya; Ihara, Masafumi; Shimohama, Shun; Uemura, Kengo; Kinoshita, Ayae

    2015-01-01

    Obesity and type 2 diabetes are risk factors of Alzheimer's disease (AD). We reported that a high fat diet (HFD) promotes amyloid precursor protein (APP) cleavage by β-site APP cleaving enzyme 1 (BACE1) without increasing BACE1 levels in APP transgenic mice. However, the detailed mechanism had remained unclear. Here we demonstrate that HFD promotes BACE1/Adaptor protein-2 (AP-2)/clathrin complex formation by increasing AP-2 levels in APP transgenic mice. In Swedish APP overexpressing Chinese hamster ovary (CHO) cells as well as in SH-SY5Y cells, overexpression of AP-2 promoted the formation of BACE1/AP-2/clathrin complex, increasing the level of the soluble form of APP β (sAPPβ). On the other hand, mutant D495R BACE1, which inhibits formation of this trimeric complex, was shown to decrease the level of sAPPβ. Overexpression of AP-2 promoted the internalization of BACE1 from the cell surface, thus reducing the cell surface BACE1 level. As such, we concluded that HFD may induce the formation of the BACE1/AP-2/clathrin complex, which is followed by its transport of BACE1 from the cell surface to the intracellular compartments. These events might be associated with the enhancement of β-site cleavage of APP in APP transgenic mice. Here we present evidence that HFD, by regulation of subcellular trafficking of BACE1, promotes APP cleavage. PMID:26414661

  18. Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration.

    PubMed

    Kuropka, Benno; Witte, Amelie; Sticht, Jana; Waldt, Natalie; Majkut, Paul; Hackenberger, Christian P R; Schraven, Burkhart; Krause, Eberhard; Kliche, Stefanie; Freund, Christian

    2015-11-01

    Stimulation of T cells leads to distinct changes of their adhesive and migratory properties. Signal propagation from activated receptors to integrins depends on scaffolding proteins such as the adhesion and degranulation promoting adaptor protein (ADAP)(1). Here we have comprehensively investigated the phosphotyrosine interactome of ADAP in T cells and define known and novel interaction partners of functional relevance. While most phosphosites reside in unstructured regions of the protein, thereby defining classical SH2 domain interaction sites for master regulators of T cell signaling such as SLP76, Fyn-kinase, and NCK, other binding events depend on structural context. Interaction proteomics using different ADAP constructs comprising most of the known phosphotyrosine motifs as well as the structured domains confirm that a distinct set of proteins is attracted by pY571 of ADAP, including the ζ-chain-associated protein kinase of 70 kDa (ZAP70). The interaction of ADAP and ZAP70 is inducible upon stimulation either of the T cell receptor (TCR) or by chemokine. NMR spectroscopy reveals that the N-terminal SH2 domains within a ZAP70-tandem-SH2 construct is the major site of interaction with phosphorylated ADAP-hSH3(N) and microscale thermophoresis (MST) indicates an intermediate binding affinity (Kd = 2.3 μm). Interestingly, although T cell receptor dependent events such as T cell/antigen presenting cell (APC) conjugate formation and adhesion are not affected by mutation of Y571, migration of T cells along a chemokine gradient is compromised. Thus, although most phospho-sites in ADAP are linked to T cell receptor related functions we have identified a unique phosphotyrosine that is solely required for chemokine induced T cell behavior.

  19. Signaling by myeloid C-type lectin receptors in immunity and homeostasis

    PubMed Central

    Sancho, David; Reis e Sousa, Caetano

    2015-01-01

    Myeloid cells are key drivers of physiological responses to pathogen invasion or tissue damage. Members of the C-type lectin receptor (CLR) family stand out among the specialized receptors utilized by myeloid cells to orchestrate these responses. CLR ligands include carbohydrate, protein and lipid components of both pathogens and self, which variably trigger endocytic, phagocytic, pro-inflammatory or anti-inflammatory reactions. These varied outcomes rely on a versatile system for CLR signaling that includes tyrosine based motifs that recruit kinases, phosphatases or endocytic adaptors, as well as non-tyrosine based signals that modulate the activation of other pathways or couple to the uptake machinery. Here, we review the signaling properties of myeloid CLRs and how they impact the role of myeloid cells in innate and adaptive immunity. PMID:22224766

  20. GrpL, a Grb2-related Adaptor Protein, Interacts with SLP-76 to Regulate Nuclear Factor of Activated T Cell Activation

    PubMed Central

    Law, Che-Leung; Ewings, Maria K.; Chaudhary, Preet M.; Solow, Sasha A.; Yun, Theodore J.; Marshall, Aaron J.; Hood, Leroy; Clark, Edward A.

    1999-01-01

    Propagation of signals from the T cell antigen receptor (TCR) involves a number of adaptor molecules. SH2 domain–containing protein 76 (SLP-76) interacts with the guanine nucleotide exchange factor Vav to activate the nuclear factor of activated cells (NF-AT), and its expression is required for normal T cell development. We report the cloning and characterization of a novel Grb2-like adaptor molecule designated as Grb2-related protein of the lymphoid system (GrpL). Expression of GrpL is restricted to hematopoietic tissues, and it is distinguished from Grb2 by having a proline-rich region. GrpL can be coimmunoprecipitated with SLP-76 but not with Sos1 or Sos2 from Jurkat cell lysates. In contrast, Grb2 can be coimmunoprecipitated with Sos1 and Sos2 but not with SLP-76. Moreover, tyrosine-phosphorylated LAT/pp36/38 in detergent lysates prepared from anti-CD3 stimulated T cells associated with Grb2 but not GrpL. These data reveal the presence of distinct complexes involving GrpL and Grb2 in T cells. A functional role of the GrpL–SLP-76 complex is suggested by the ability of GrpL to act alone or in concert with SLP-76 to augment NF-AT activation in Jurkat T cells. PMID:10209041

  1. Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

    SciTech Connect

    Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon; Nguyen, Hong-Hoa; Yang, Jun-Mo; Kang, Jong-Sun; Hahn, Myong-Joon

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer APPL1 regulates the protein level of EGFR in response to EGF stimulation. Black-Right-Pointing-Pointer Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. Black-Right-Pointing-Pointer Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

  2. Adaptor Autoregulation Promotes Coordinated Binding within Clathrin Coats*

    PubMed Central

    Hung, Chao-Wei; Aoh, Quyen L.; Joglekar, Ajit P.; Payne, Gregory S.; Duncan, Mara C.

    2012-01-01

    Membrane traffic is an essential process that allows protein and lipid exchange between the endocytic, lysosomal, and secretory compartments. Clathrin-mediated traffic between the trans-Golgi network and endosomes mediates responses to the environment through the sorting of biosynthetic and endocytic protein cargo. Traffic through this pathway is initiated by the controlled assembly of a clathrin-adaptor protein coat on the cytosolic surface of the originating organelle. In this process, clathrin is recruited by different adaptor proteins that act as a bridge between clathrin and the transmembrane cargo proteins to be transported. Interactions between adaptors and clathrin and between different types of adaptors lead to the formation of a densely packed protein network within the coat. A key unresolved issue is how the highly complex adaptor-clathrin interaction and adaptor-adaptor interaction landscape lead to the correct spatiotemporal assembly of the clathrin coat. Here we report the discovery of a new autoregulatory motif within the clathrin adaptor Gga2 that drives synergistic binding of Gga2 to clathrin and the adaptor Ent5. This autoregulation influences the temporal and/or spatial location of the Gga2-Ent5 interaction. We propose that this synergistic binding provides built-in regulation to ensure the correct assembly of clathrin coats. PMID:22457357

  3. Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN.

    PubMed

    Takayanagi, Hiroki; Yuzawa, Satoru; Sumimoto, Hideki

    2015-02-01

    The adaptor protein LGN interacts via the N-terminal domain comprising eight tetratricopeptide-repeat (TPR) motifs with its partner proteins mInsc, NuMA, Frmpd1 and Frmpd4 in a mutually exclusive manner. Here, the crystal structure of the LGN TPR domain in complex with human Frmpd4 is described at 1.5 Å resolution. In the complex, the LGN-binding region of Frmpd4 (amino-acid residues 990-1011) adopts an extended structure that runs antiparallel to LGN along the concave surface of the superhelix formed by the TPR motifs. Comparison with the previously determined structures of the LGN-Frmpd1, LGN-mInsc and LGN-NuMA complexes reveals that these partner proteins interact with LGN TPR1-6 via a common core binding region with consensus sequence (E/Q)XEX4-5(E/D/Q)X1-2(K/R)X0-1(V/I). In contrast to Frmpd1, Frmpd4 makes additional contacts with LGN via regions N- and C-terminal to the core sequence. The N-terminal extension is replaced by a specific α-helix in mInsc, which drastically increases the direct contacts with LGN TPR7/8, consistent with the higher affinity of mInsc for LGN. A crystal structure of Frmpd4-bound LGN in an oxidized form is also reported, although oxidation does not appear to strongly affect the interaction with Frmpd4.

  4. RTK SLAP down: the emerging role of Src-like adaptor protein as a key player in receptor tyrosine kinase signaling.

    PubMed

    Wybenga-Groot, Leanne E; McGlade, C Jane

    2015-02-01

    SLAP (Src like adaptor protein) contains adjacent Src homology 3 (SH3) and Src homology 2 (SH2) domains closely related in sequence to that of cytoplasmic Src family tyrosine kinases. Expressed most abundantly in the immune system, SLAP function has been predominantly studied in the context of lymphocyte signaling, where it functions in the Cbl dependent downregulation of antigen receptor signaling. However, accumulating evidence suggests that SLAP plays a role in the regulation of a broad range of membrane receptors including members of the receptor tyrosine kinase (RTK) family. In this review we highlight the role of SLAP in the ubiquitin dependent regulation of type III RTKs PDGFR, CSF-1R, KIT and Flt3, as well as Eph family RTKs. SLAP appears to bind activated type III and Eph RTKs via a conserved autophosphorylated juxtamembrane tyrosine motif in an SH2-dependent manner, suggesting that SLAP is important in regulating RTK signaling.

  5. Endocytic adaptors – social networking at the plasma membrane

    PubMed Central

    Reider, Amanda; Wendland, Beverly

    2011-01-01

    Receptor-mediated endocytosis is a dynamic process that is crucial for maintaining plasma membrane composition and controlling cell-signaling pathways. A variety of entry routes have evolved to ensure that the vast array of molecules on the cell surface can be differentially internalized by endocytosis. This diversity has extended to include a growing list of endocytic adaptor proteins, which are thought to initiate the internalization process. The key function of adaptors is to select the proteins that should be removed from the cell surface. Thus, they have a central role in defining the physiology of a cell. This has made the study of adaptor proteins a very active area of research that is ripe for exciting future discoveries. Here, we review recent work on how adaptors mediate endocytosis and address the following questions: what characteristics define an endocytic adaptor protein? What roles do these proteins fulfill in addition to selecting cargo and how might adaptors function in clathrin-independent endocytic pathways? Through the findings discussed in this Commentary, we hope to stimulate further characterization of known adaptors and expansion of the known repertoire by identification of new adaptors. PMID:21536832

  6. Skb5, an SH3 adaptor protein, regulates Pmk1 MAPK signaling by controlling the intracellular localization of the MAPKKK Mkh1.

    PubMed

    Kanda, Yuki; Satoh, Ryosuke; Matsumoto, Saki; Ikeda, Chisato; Inutsuka, Natsumi; Hagihara, Kanako; Matzno, Sumio; Tsujimoto, Sho; Kita, Ayako; Sugiura, Reiko

    2016-08-15

    The mitogen-activated protein kinase (MAPK) cascade is a highly conserved signaling module composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKK) and MAPKs. The MAPKKK Mkh1 is an initiating kinase in Pmk1 MAPK signaling, which regulates cell integrity in fission yeast (Schizosaccharomyces pombe). Our genetic screen for regulators of Pmk1 signaling identified Shk1 kinase binding protein 5 (Skb5), an SH3-domain-containing adaptor protein. Here, we show that Skb5 serves as an inhibitor of Pmk1 MAPK signaling activation by downregulating Mkh1 localization to cell tips through its interaction with the SH3 domain. Consistent with this, the Mkh1(3PA) mutant protein, with impaired Skb5 binding, remained in the cell tips, even when Skb5 was overproduced. Intriguingly, Skb5 needs Mkh1 to localize to the growing ends as Mkh1 deletion and disruption of Mkh1 binding impairs Skb5 localization. Deletion of Pck2, an upstream activator of Mkh1, impaired the cell tip localization of Mkh1 and Skb5 as well as the Mkh1-Skb5 interaction. Interestingly, both Pck2 and Mkh1 localized to the cell tips at the G1/S phase, which coincided with Pmk1 MAPK activation. Taken together, Mkh1 localization to cell tips is important for transmitting upstream signaling to Pmk1, and Skb5 spatially regulates this process. PMID:27451356

  7. AP-1/σ1A and AP-1/σ1B adaptor-proteins differentially regulate neuronal early endosome maturation via the Rab5/Vps34-pathway

    PubMed Central

    Candiello, Ermes; Kratzke, Manuel; Wenzel, Dirk; Cassel, Dan; Schu, Peter

    2016-01-01

    The σ1 subunit of the AP-1 clathrin-coated-vesicle adaptor-protein complex is expressed as three isoforms. Tissues express σ1A and one of the σ1B and σ1C isoforms. Brain is the tissue with the highest σ1A and σ1B expression. σ1B-deficiency leads to severe mental retardation, accumulation of early endosomes in synapses and fewer synaptic vesicles, whose recycling is slowed down. AP-1/σ1A and AP-1/σ1B regulate maturation of these early endosomes into multivesicular body late endosomes, thereby controlling synaptic vesicle protein transport into a degradative pathway. σ1A binds ArfGAP1, and with higher affinity brain-specific ArfGAP1, which bind Rabex-5. AP-1/σ1A-ArfGAP1-Rabex-5 complex formation leads to more endosomal Rabex-5 and enhanced, Rab5GTP-stimulated Vps34 PI3-kinase activity, which is essential for multivesicular body endosome formation. Formation of AP-1/σ1A-ArfGAP1-Rabex-5 complexes is prevented by σ1B binding of Rabex-5 and the amount of endosomal Rabex-5 is reduced. AP-1 complexes differentially regulate endosome maturation and coordinate protein recycling and degradation, revealing a novel molecular mechanism by which they regulate protein transport besides their established function in clathrin-coated-vesicle formation. PMID:27411398

  8. Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 {mu}1A (AP-1 mu1A)

    SciTech Connect

    Sawasdee, Nunghathai; Junking, Mutita; Ngaojanlar, Piengpaga; Sukomon, Nattakan; Ungsupravate, Duangporn; Limjindaporn, Thawornchai; Akkarapatumwong, Varaporn; Noisakran, Sansanee; Yenchitsomanus, Pa-thai

    2010-10-08

    Research highlights: {yields} Trafficking defect of kAE1 is a cause of dRTA but trafficking pathway of kAE1 has not been clearly described. {yields} Adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) was firstly reported to interact with kAE1. {yields} The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. {yields} AP-1 mu1A knockdown showed a marked reduction of kAE1 on the cell membrane and its accumulation in endoplasmic reticulum. {yields} AP-1 mu1A has a critical role in kAE1 trafficking to the plasma membrane. -- Abstract: Kidney anion exchanger 1 (kAE1) mediates chloride (Cl{sup -}) and bicarbonate (HCO{sub 3}{sup -}) exchange at the basolateral membrane of kidney {alpha}-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl{sup -}/HCO{sub 3}{sup -} exchange at the basolateral membrane and failure of proton (H{sup +}) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1

  9. The Src Homology and Collagen A (ShcA) Adaptor Protein Is Required for the Spatial Organization of the Costamere/Z-disk Network during Heart Development*

    PubMed Central

    Mlih, Mohamed; Host, Lionel; Martin, Sophie; Niederhoffer, Nathalie; Monassier, Laurent; Terrand, Jérôme; Messaddeq, Nadia; Radke, Michael; Gotthardt, Michael; Bruban, Véronique; Kober, Frank; Bernard, Monique; Canet-Soulas, Emmanuelle; Abt-Jijon, Francisco; Boucher, Philippe; Matz, Rachel L.

    2015-01-01

    Src homology and collagen A (ShcA) is an adaptor protein that binds to tyrosine kinase receptors. Its germ line deletion is embryonic lethal with abnormal cardiovascular system formation, and its role in cardiovascular development is unknown. To investigate its functional role in cardiovascular development in mice, ShcA was deleted in cardiomyocytes and vascular smooth muscle cells by crossing ShcA flox mice with SM22a-Cre transgenic mice. Conditional mutant mice developed signs of severe dilated cardiomyopathy, myocardial infarctions, and premature death. No evidence of a vascular contribution to the phenotype was observed. Histological analysis of the heart revealed aberrant sarcomeric Z-disk and M-band structures, and misalignments of T-tubules with Z-disks. We find that not only the ErbB3/Neuregulin signaling pathway but also the baroreceptor reflex response, which have been functionally associated, are altered in the mutant mice. We further demonstrate that ShcA interacts with Caveolin-1 and the costameric protein plasma membrane Ca2+/calmodulin-dependent ATPase (PMCA), and that its deletion leads to abnormal dystrophin signaling. Collectively, these results demonstrate that ShcA interacts with crucial proteins and pathways that link Z-disk and costamere. PMID:25488665

  10. Induction of androgen formation in the male by a TAT-VDAC1 fusion peptide blocking 14-3-3ɛ protein adaptor and mitochondrial VDAC1 interactions.

    PubMed

    Aghazadeh, Yasaman; Martinez-Arguelles, Daniel B; Fan, Jinjiang; Culty, Martine; Papadopoulos, Vassilios

    2014-10-01

    Low testosterone (T), a major cause of male hypogonadism and infertility, is linked to mood changes, fatigue, osteoporosis, reduced bone-mass index, and aging. The treatment of choice, T replacement therapy, has been linked with increased risk for prostate cancer and luteinizing hormone (LH) suppression, and shown to lead to infertility, cardiovascular diseases, and obesity. Alternate methods to induce T with lower side effects are desirable. In search of the mechanisms regulating T synthesis in the testes, we identified the 14-3-3ɛ protein adaptor as a negative regulator of steroidogenesis. Steroidogenesis begins in mitochondria. 14-3-3ɛ interacts with the outer mitochondrial membrane voltage-dependent anion channel (VDAC1) protein, forming a scaffold that limits the availability of cholesterol for steroidogenesis. We report the development of a tool able to induce endogenous T formation. Peptides able to penetrate testes conjugated to 14-3-3ɛ site of interaction with VDAC1 blocked 14-3-3ɛ-VDAC1 interactions while at the same time increased VDAC1-translocator protein (18 kDa) interactions that induced steroid formation in rat testes, leading to increased serum T levels. These peptides rescued intratesticular and serum T formation in adult male rats treated with gonadotropin-releasing hormone antagonist, which dampened LH and T production.

  11. The association between the SH2-containing inositol polyphosphate 5-Phosphatase 2 (SHIP2) and the adaptor protein APS has an impact on biochemical properties of both partners.

    PubMed

    Onnockx, Sheela; De Schutter, Julie; Blockmans, Marianne; Xie, Jingwei; Jacobs, Christine; Vanderwinden, Jean-Marie; Erneux, Christophe; Pirson, Isabelle

    2008-01-01

    SHIP2 (SH2-containing inositol polyphosphate 5-phosphatase 2) is a phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase containing various motifs susceptible to mediate protein-protein interaction. In cell models, SHIP2 negatively regulates insulin signalling through its catalytic PtdIns(3,4,5)P(3) 5-phosphatase activity. We have previously reported that SHIP2 interacts with the c-Cbl associated protein (CAP) and c-Cbl, proteins implicated in the insulin cellular response regulating the small G protein TC10. The first steps of the TC10 pathway are the recruitment and tyrosine phosphorylation by the insulin receptor of the adaptor protein with Pleckstrin Homology and Src Homology 2 domains (APS). Herein, we show that SHIP2 can directly interact with APS in 3T3-L1 adipocytes and in transfected CHO-IR cells (Chinese hamster ovary cells stably transfected with the insulin receptor). Upon insulin stimulation, APS and SHIP2 are recruited to cell membranes as seen by immunofluorescence studies, which is consistent with their interaction. We also observed that SHIP2 negatively regulates APS insulin-induced tyrosine phosphorylation and consequently inhibits APS association with c-Cbl. APS, which specifically interacts with SHIP2, but not PTEN, in turn, increases the PtdIns(3,4,5)P(3) 5-phosphatase activity of SHIP2 in an inositol phosphatase assay. Co-transfection of SHIP2 and APS in CHO-IR cells further increases the inhibitory effect of SHIP2 on Akt insulin-induced phosphorylation. Therefore, the interaction between APS and SHIP2 provides to both proteins potential negative regulatory mechanisms to act on the insulin cascade.

  12. Role of adaptor proteins and clathrin in the trafficking of human kidney anion exchanger 1 (kAE1) to the cell surface.

    PubMed

    Junking, Mutita; Sawasdee, Nunghathai; Duangtum, Natapol; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

    2014-07-01

    Kidney anion exchanger 1 (kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl-/HCO3-) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells. PMID:24698155

  13. Role of adaptor proteins and clathrin in the trafficking of human kidney anion exchanger 1 (kAE1) to the cell surface.

    PubMed

    Junking, Mutita; Sawasdee, Nunghathai; Duangtum, Natapol; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

    2014-07-01

    Kidney anion exchanger 1 (kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl-/HCO3-) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells.

  14. Stromal cells expressing hedgehog-interacting protein regulate the proliferation of myeloid neoplasms

    PubMed Central

    Kobune, M; Iyama, S; Kikuchi, S; Horiguchi, H; Sato, T; Murase, K; Kawano, Y; Takada, K; Ono, K; Kamihara, Y; Hayashi, T; Miyanishi, K; Sato, Y; Takimoto, R; Kato, J

    2012-01-01

    Aberrant reactivation of hedgehog (Hh) signaling has been described in a wide variety of human cancers including cancer stem cells. However, involvement of the Hh-signaling system in the bone marrow (BM) microenvironment during the development of myeloid neoplasms is unknown. In this study, we assessed the expression of Hh-related genes in primary human CD34+ cells, CD34+ blastic cells and BM stromal cells. Both Indian Hh (Ihh) and its signal transducer, smoothened (SMO), were expressed in CD34+ acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)-derived cells. However, Ihh expression was relatively low in BM stromal cells. Remarkably, expression of the intrinsic Hh-signaling inhibitor, human Hh-interacting protein (HHIP) in AML/MDS-derived stromal cells was markedly lower than in healthy donor-derived stromal cells. Moreover, HHIP expression levels in BM stromal cells highly correlated with their supporting activity for SMO+ leukemic cells. Knockdown of HHIP gene in stromal cells increased their supporting activity although control cells marginally supported SMO+ leukemic cell proliferation. The demethylating agent, 5-aza-2′-deoxycytidine rescued HHIP expression via demethylation of HHIP gene and reduced the leukemic cell-supporting activity of AML/MDS-derived stromal cells. This indicates that suppression of stromal HHIP could be associated with the proliferation of AML/MDS cells. PMID:22961059

  15. Involvement of Grb2 adaptor protein in nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-mediated signaling and anaplastic large cell lymphoma growth.

    PubMed

    Riera, Ludovica; Lasorsa, Elena; Ambrogio, Chiara; Surrenti, Nadia; Voena, Claudia; Chiarle, Roberto

    2010-08-20

    Most anaplastic large cell lymphomas (ALCL) express oncogenic fusion proteins derived from chromosomal translocations or inversions of the anaplastic lymphoma kinase (ALK) gene. Frequently ALCL carry the t(2;5) translocation, which fuses the ALK gene to the nucleophosmin (NPM1) gene. The transforming activity mediated by NPM-ALK fusion induces different pathways that control proliferation and survival of lymphoma cells. Grb2 is an adaptor protein thought to play an important role in ALK-mediated transformation, but its interaction with NPM-ALK, as well as its function in regulating ALCL signaling pathways and cell growth, has never been elucidated. Here we show that active NPM-ALK, but not a kinase-dead mutant, bound and induced Grb2 phosphorylation in tyrosine 160. An intact SH3 domain at the C terminus of Grb2 was required for Tyr(160) phosphorylation. Furthermore, Grb2 did not bind to a single region but rather to different regions of NPM-ALK, mainly Tyr(152-156), Tyr(567), and a proline-rich region, Pro(415-417). Finally, shRNA knockdown experiments showed that Grb2 regulates primarily the NPM-ALK-mediated phosphorylation of SHP2 and plays a key role in ALCL cell growth.

  16. Endoproteolytic cleavage of FE65 converts the adaptor protein to a potent suppressor of the sAPPalpha pathway in primates.

    PubMed

    Hu, Qubai; Wang, Lin; Yang, Zheng; Cool, Bethany H; Zitnik, Galynn; Martin, George M

    2005-04-01

    Adaptor protein FE65 (APBB1) specifically binds to the intracellular tail of the type I transmembrane protein, beta-amyloid precursor protein (APP). The formation of this complex may be important for modulation of the processing and function of APP. APP is proteolytically cleaved at multiple sites. The cleavages and their regulation are of central importance in the pathogenesis of dementias of the Alzheimer type. In cell cultures and perhaps in vivo, secretion of the alpha-cleaved APP ectodomain (sAPPalpha) is the major pathway in the most cells. Regulation of the process may require extracellular/intracellular cues. Neither extracellular ligands nor intracellular mediators have been identified, however. Here, we show novel evidence that the major isoform of FE65 (97-kDa FE65, p97FE65) can be converted to a 65-kDa N-terminally truncated C-terminal fragment (p65FE65) via endoproteolysis. The cleavage region locates immediately after an acidic residue cluster but before the three major protein-protein binding domains. The cleavage activity is particularly high in human and non-human primate cells and low in rodent cells; the activity appears to be triggered/enhanced by high cell density, presumably via cell-cell/cell-substrate contact cues. As a result, p65FE65 exhibits extraordinarily high affinity for APP (up to 40-fold higher than p97FE65) and potent suppression (up to 90%) of secretion of sAPPalpha. Strong p65FE65-APP binding is required for the suppression. The results suggest that p65FE65 may be an intracellular mediator in a signaling cascade regulating alpha-secretion of APP, particularly in primates. PMID:15647266

  17. BAG1: The Guardian of Anti-Apoptotic Proteins in Acute Myeloid Leukemia

    PubMed Central

    Aveic, Sanja; Pigazzi, Martina; Basso, Giuseppe

    2011-01-01

    BCL2 associated Athano-Gene 1 (BAG1) is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance. PMID:22016818

  18. BAG1: the guardian of anti-apoptotic proteins in acute myeloid leukemia.

    PubMed

    Aveic, Sanja; Pigazzi, Martina; Basso, Giuseppe

    2011-01-01

    BCL2 associated Athano-Gene 1 (BAG1) is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance.

  19. Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 μ1A (AP-1 mu1A).

    PubMed

    Sawasdee, Nunghathai; Junking, Mutita; Ngaojanlar, Piengpaga; Sukomon, Nattakan; Ungsupravate, Duangporn; Limjindaporn, Thawornchai; Akkarapatumwong, Varaporn; Noisakran, Sansanee; Yenchitsomanus, Pa-Thai

    2010-10-01

    Kidney anion exchanger 1 (kAE1) mediates chloride (Cl⁻) and bicarbonate (HCO₃⁻) exchange at the basolateral membrane of kidney α-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl⁻/HCO₃⁻ exchange at the basolateral membrane and failure of proton (H+) secretion at the apical membrane, causing a kidney disease--distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 μ1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXØ motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1 trafficking of kidney α-intercalated cells. PMID:20833140

  20. The Shc-related adaptor protein, Sck, forms a complex with the vascular-endothelial-growth-factor receptor KDR in transfected cells.

    PubMed Central

    Warner, A J; Lopez-Dee, J; Knight, E L; Feramisco, J R; Prigent, S A

    2000-01-01

    Despite much progress in recent years, the precise signalling events triggered by the vascular-endothelial-growth-factor (VEGF) receptors, fms-like tyrosine kinase (Flt1) and kinase insert domain-containing receptor (KDR), are incompletely defined. Results obtained when Flt1 and KDR are individually expressed in fibroblasts or porcine aortic endothelial cells have not been entirely consistent with those observed in other endothelial cells expressing both receptors endogenously. It has also been difficult to demonstrate VEGF-induced phosphorylation of Flt1, which has led to speculation that KDR may be the more important receptor for the mitogenic action of VEGF on endothelial cells. In an attempt to identify physiologically important effectors which bind to KDR, we have screened a yeast two-hybrid mouse embryo library with the cytoplasmic domain of KDR. Here we describe the identification of the adaptor protein, Shc-like protein (Sck), as a binding partner for KDR. We demonstrate that this interaction requires phosphorylation of KDR, and identify the binding site for the Src-homology 2 (SH2) domain as tyrosine-1175 of KDR. We have also shown that the SH2 domain of Sck, but not that of Src-homology collagen protein (Shc), can precipitate phosphorylated KDR from VEGF-stimulated porcine aortic endothelial cells expressing KDR, and that an N-terminally truncated Sck protein can associate with KDR, in a phosphorylation-dependent fashion, when co-expressed in human embryonic kidney 293 cells. Furthermore, we demonstrate that in the two-hybrid assay, both Shc and Sck SH2 domains can associate with the related receptor Flt1. PMID:10749680

  1. Overexpression of Isoforms of Nitric Oxide Synthase 1 Adaptor Protein, Encoded by a Risk Gene for Schizophrenia, Alters Actin Dynamics and Synaptic Function.

    PubMed

    Hernandez, Kristina; Swiatkowski, Przemyslaw; Patel, Mihir V; Liang, Chen; Dudzinski, Natasha R; Brzustowicz, Linda M; Firestein, Bonnie L

    2016-01-01

    Proper communication between neurons depends upon appropriate patterning of dendrites and correct distribution and structure of spines. Schizophrenia is a neuropsychiatric disorder characterized by alterations in dendrite branching and spine density. Nitric oxide synthase 1 adaptor protein (NOS1AP), a risk gene for schizophrenia, encodes proteins that are upregulated in the dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia. To elucidate the effects of NOS1AP overexpression observed in individuals with schizophrenia, we investigated changes in actin dynamics and spine development when a long (NOS1AP-L) or short (NOS1AP-S) isoform of NOS1AP is overexpressed. Increased NOS1AP-L protein promotes the formation of immature spines when overexpressed in rat cortical neurons from day in vitro (DIV) 14 to DIV 17 and reduces the amplitude of miniature excitatory postsynaptic currents (mEPSCs). In contrast, increased NOS1AP-S protein increases the rate of actin polymerization and the number of immature and mature spines, which may be attributed to a decrease in total Rac1 expression and a reduction in the levels of active cofilin. The increase in the number of mature spines by overexpression of NOS1AP-S is accompanied by an increase in the frequency of mEPSCs. Our findings show that overexpression of NOS1AP-L or NOS1AP-S alters the actin cytoskeleton and synaptic function. However, the mechanisms by which these isoforms induce these changes are distinct. These results are important for understanding how increased expression of NOS1AP isoforms can influence spine development and synaptic function. PMID:26869880

  2. Overexpression of Isoforms of Nitric Oxide Synthase 1 Adaptor Protein, Encoded by a Risk Gene for Schizophrenia, Alters Actin Dynamics and Synaptic Function

    PubMed Central

    Hernandez, Kristina; Swiatkowski, Przemyslaw; Patel, Mihir V.; Liang, Chen; Dudzinski, Natasha R.; Brzustowicz, Linda M.; Firestein, Bonnie L.

    2016-01-01

    Proper communication between neurons depends upon appropriate patterning of dendrites and correct distribution and structure of spines. Schizophrenia is a neuropsychiatric disorder characterized by alterations in dendrite branching and spine density. Nitric oxide synthase 1 adaptor protein (NOS1AP), a risk gene for schizophrenia, encodes proteins that are upregulated in the dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia. To elucidate the effects of NOS1AP overexpression observed in individuals with schizophrenia, we investigated changes in actin dynamics and spine development when a long (NOS1AP-L) or short (NOS1AP-S) isoform of NOS1AP is overexpressed. Increased NOS1AP-L protein promotes the formation of immature spines when overexpressed in rat cortical neurons from day in vitro (DIV) 14 to DIV 17 and reduces the amplitude of miniature excitatory postsynaptic currents (mEPSCs). In contrast, increased NOS1AP-S protein increases the rate of actin polymerization and the number of immature and mature spines, which may be attributed to a decrease in total Rac1 expression and a reduction in the levels of active cofilin. The increase in the number of mature spines by overexpression of NOS1AP-S is accompanied by an increase in the frequency of mEPSCs. Our findings show that overexpression of NOS1AP-L or NOS1AP-S alters the actin cytoskeleton and synaptic function. However, the mechanisms by which these isoforms induce these changes are distinct. These results are important for understanding how increased expression of NOS1AP isoforms can influence spine development and synaptic function. PMID:26869880

  3. The role of Drp1 adaptor proteins MiD49 and MiD51 in mitochondrial fission: implications for human disease.

    PubMed

    Atkins, Kathleen; Dasgupta, Asish; Chen, Kuang-Hueih; Mewburn, Jeff; Archer, Stephen L

    2016-11-01

    Mitochondrial morphology is governed by the balance of mitochondrial fusion, mediated by mitofusins and optic atrophy 1 (OPA1), and fission, mediated by dynamin-related protein 1 (Drp1). Disordered mitochondrial dynamics alters metabolism, proliferation, apoptosis and mitophagy, contributing to human diseases, including neurodegenerative syndromes, pulmonary arterial hypertension (PAH), cancer and ischemia/reperfusion injury. Post-translational regulation of Drp1 (by phosphorylation and SUMOylation) is an established means of modulating Drp1 activation and translocation to the outer mitochondrial membrane (OMM). This review focuses on Drp1 adaptor proteins that also regulate fission. The proteins include fission 1 (Fis1), mitochondrial fission factor (Mff) and mitochondrial dynamics proteins of 49 kDa and 51 kDa (MiD49, MiD51). Heterologous MiD overexpression sequesters inactive Drp1 on the OMM, promoting fusion; conversely, increased endogenous MiD creates focused Drp1 multimers that optimize OMM scission. The triggers that activate MiD-bound Drp1 in disease states are unknown; however, MiD51 has a unique capacity for ADP binding at its nucleotidyltransferase domain. Without ADP, MiD51 inhibits Drp1, whereas ADP promotes MiD51-mediated fission, suggesting a link between metabolism and fission. Confusion over whether MiDs mediate fusion (by sequestering inactive Drp1) or fission (by guiding Drp1 assembly) relates to a failure to consider cell types used and to distinguish endogenous compared with heterologous changes in expression. We speculate that endogenous MiDs serve as Drp1-binding partners that are dysregulated in disease states and may be important targets for inhibiting cell proliferation and ischemia/reperfusion injury. Moreover, it appears that the composition of the fission apparatus varies between disease states and amongst individuals. MiDs may be important targets for inhibiting cell proliferation and attenuating ischemia/reperfusion injury. PMID

  4. Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster.

    PubMed

    Rodriguez-Fernandez, Imilce A; Dell'Angelica, Esteban C

    2015-01-01

    The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in

  5. Sorting for storage in myeloid cells of nonmyeloid proteins and chimeras with the propeptide of myeloperoxidase precursor.

    PubMed

    Bülow, E; Nauseef, W M; Goedken, M; McCormick, S; Calafat, J; Gullberg, U; Olsson, I

    2002-02-01

    During formation of polymorphonuclear neutrophils, proteins are synthesized for storage in granules. Whereas sorting of proteins into distinct subtypes of cytoplasmic granules may reflect the coordinated expression of the proteins contained in them, still the mechanism(s) for the retrieval of proteins from the constitutive secretion is unknown. To investigate the mechanisms of retrieval, nonmyeloid secretory proteins were expressed in myeloid cell lines, and their subcellular fate was assessed. The contribution of the propeptide (MPOpro) of the myeloperoxidase (MPO) precursor was investigated by determining the fate of chimeras containing MPOpro. The nonmyeloid protein alpha(1)-microglobulin (alpha(1)-m) was targeted to storage organelles in 32D cells and colocalized with the lysosomal marker LAMP-1, whereas soluble TNF receptor 1 (sTNFR1) was secreted without granule targeting. Fusion of MPOpro to alpha(1)-m delayed exit from endoplasmic reticulum (ER), but subsequent targeting to dense organelles was indistinguishable from that of alpha(1)-m alone. Fusion proteins between MPOpro and sTNFR1 or green fluorescent protein expressed in myeloid 32D, K562, or PLB-985 cells did not associate stably with calreticulin or calnexin, molecular chaperones that normally interact transiently with the MPO precursor, but were still efficiently retained in the ER followed by degradation. We conclude that normally secreted, nonmyeloid proteins can be targeted efficiently to storage organelles in myeloid cells, that myeloid cells selectively target some proteins for storage but not others, and that MPOpro may contribute to the prolonged ER retention of the MPO precursor independent of the ER-molecular chaperones calreticulin and calnexin.

  6. Nck-2, a Novel Src Homology2/3-containing Adaptor Protein That Interacts with the LIM-only Protein PINCH and Components of Growth Factor Receptor Kinase-signaling Pathways

    PubMed Central

    Tu, Yizeng; Li, Fugang; Wu, Chuanyue

    1998-01-01

    Many of the protein–protein interactions that are essential for eukaryotic intracellular signal transduction are mediated by protein binding modules including SH2, SH3, and LIM domains. Nck is a SH3- and SH2-containing adaptor protein implicated in coordinating various signaling pathways, including those of growth factor receptors and cell adhesion receptors. We report here the identification, cloning, and characterization of a widely expressed, Nck-related adaptor protein termed Nck-2. Nck-2 comprises primarily three N-terminal SH3 domains and one C-terminal SH2 domain. We show that Nck-2 interacts with PINCH, a LIM-only protein implicated in integrin-linked kinase signaling. The PINCH-Nck-2 interaction is mediated by the fourth LIM domain of PINCH and the third SH3 domain of Nck-2. Furthermore, we show that Nck-2 is capable of recognizing several key components of growth factor receptor kinase-signaling pathways including EGF receptors, PDGF receptor-β, and IRS-1. The association of Nck-2 with EGF receptors was regulated by EGF stimulation and involved largely the SH2 domain of Nck-2, although the SH3 domains of Nck-2 also contributed to the complex formation. The association of Nck-2 with PDGF receptor-β was dependent on PDGF activation and was mediated solely by the SH2 domain of Nck-2. Additionally, we have detected a stable association between Nck-2 and IRS-1 that was mediated primarily via the second and third SH3 domain of Nck-2. Thus, Nck-2 associates with PINCH and components of different growth factor receptor-signaling pathways via distinct mechanisms. Finally, we provide evidence indicating that a fraction of the Nck-2 and/or Nck-1 proteins are associated with the cytoskeleton. These results identify a novel Nck-related SH2- and SH3-domain–containing protein and suggest that it may function as an adaptor protein connecting the growth factor receptor-signaling pathways with the integrin-signaling pathways. PMID:9843575

  7. The adaptor protein SAP directly associates with PECAM-1 and regulates PECAM-1-mediated-cell adhesion in T-like cell lines.

    PubMed

    Proust, Richard; Crouin, Catherine; Gandji, Leslie Yewakon; Bertoglio, Jacques; Gesbert, Franck

    2014-04-01

    SAP is a small cytosolic adaptor protein expressed in hematopoietic lineages whose main function is to regulate intracellular signaling pathways induced by the triggering of members of the SLAM receptor family. In this paper, we have identified the adhesion molecule PECAM-1 as a new partner for SAP in a conditional yeast two-hybrid screen. PECAM-1 is an immunoglobulin-like molecule expressed by endothelial cells and leukocytes, which possesses both pro- and anti-inflammatory properties. However, little is known about PECAM-1 functions in T cells. We show that SAP directly and specifically interacts with the cytosolic tyrosine 686 of PECAM-1. We generated different T-like cell lines in which SAP or PECAM-1 are expressed or down modulated and we demonstrate that a diminished SAP expression correlates with a diminished PECAM-1-mediated adhesion. Although SAP has mainly been shown to associate with SLAM receptors, we evidence here that SAP is a new actor downstream of PECAM-1.

  8. Activation of EphA receptors mediates the recruitment of the adaptor protein Slap, contributing to the downregulation of N-methyl-D-aspartate receptors.

    PubMed

    Semerdjieva, Sophia; Abdul-Razak, Hayder H; Salim, Sharifah S; Yáñez-Muñoz, Rafael J; Chen, Philip E; Tarabykin, Victor; Alifragis, Pavlos

    2013-04-01

    Regulation of the activity of N-methyl-d-aspartate receptors (NMDARs) at glutamatergic synapses is essential for certain forms of synaptic plasticity underlying learning and memory and is also associated with neurotoxicity and neurodegenerative diseases. In this report, we investigate the role of Src-like adaptor protein (Slap) in NMDA receptor signaling. We present data showing that in dissociated neuronal cultures, activation of ephrin (Eph) receptors by chimeric preclustered eph-Fc ligands leads to recruitment of Slap and NMDA receptors at the sites of Eph receptor activation. Interestingly, our data suggest that prolonged activation of EphA receptors is as efficient in recruiting Slap and NMDA receptors as prolonged activation of EphB receptors. Using established heterologous systems, we examined whether Slap is an integral part of NMDA receptor signaling. Our results showed that Slap does not alter baseline activity of NMDA receptors and does not affect Src-dependent potentiation of NMDA receptor currents in Xenopus oocytes. We also demonstrate that Slap reduces excitotoxic cell death triggered by activation of NMDARs in HEK293 cells. Finally, we present evidence showing reduced levels of NMDA receptors in the presence of Slap occurring in an activity-dependent manner, suggesting that Slap is part of a mechanism that homeostatically modulates the levels of NMDA receptors.

  9. The adaptor protein SAP directly associates with PECAM-1 and regulates PECAM-1-mediated-cell adhesion in T-like cell lines.

    PubMed

    Proust, Richard; Crouin, Catherine; Gandji, Leslie Yewakon; Bertoglio, Jacques; Gesbert, Franck

    2014-04-01

    SAP is a small cytosolic adaptor protein expressed in hematopoietic lineages whose main function is to regulate intracellular signaling pathways induced by the triggering of members of the SLAM receptor family. In this paper, we have identified the adhesion molecule PECAM-1 as a new partner for SAP in a conditional yeast two-hybrid screen. PECAM-1 is an immunoglobulin-like molecule expressed by endothelial cells and leukocytes, which possesses both pro- and anti-inflammatory properties. However, little is known about PECAM-1 functions in T cells. We show that SAP directly and specifically interacts with the cytosolic tyrosine 686 of PECAM-1. We generated different T-like cell lines in which SAP or PECAM-1 are expressed or down modulated and we demonstrate that a diminished SAP expression correlates with a diminished PECAM-1-mediated adhesion. Although SAP has mainly been shown to associate with SLAM receptors, we evidence here that SAP is a new actor downstream of PECAM-1. PMID:24388971

  10. Recruitment of the adaptor protein Nck to PECAM-1 couples oxidative stress to canonical NF-κB signaling and inflammation.

    PubMed

    Chen, Jie; Leskov, Igor L; Yurdagul, Arif; Thiel, Bonnie; Kevil, Christopher G; Stokes, Karen Y; Orr, A Wayne

    2015-02-24

    Oxidative stress stimulates nuclear factor κB (NF-κB) activation and NF-κB-dependent proinflammatory gene expression in endothelial cells during several pathological conditions, including ischemia/reperfusion injury. We found that the Nck family of adaptor proteins linked tyrosine kinase signaling to oxidative stress-induced activation of NF-κB through the classic IκB kinase-dependent pathway. Depletion of Nck prevented oxidative stress induced by exogenous hydrogen peroxide or hypoxia/reoxygenation injury from activating NF-κB in endothelial cells, increasing the abundance of the proinflammatory molecules ICAM-1 (intracellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) and recruiting leukocytes. Nck depletion also attenuated endothelial cell expression of genes encoding proinflammatory factors but not those encoding antioxidants. Nck promoted oxidative stress-induced activation of NF-κB by coupling the tyrosine phosphorylation of PECAM-1 (platelet endothelial cell adhesion molecule-1) to the activation of p21-activated kinase, which mediates oxidative stress-induced NF-κB signaling. Consistent with this mechanism, treatment of mice subjected to ischemia/reperfusion injury in the cremaster muscle with a Nck inhibitory peptide blocked leukocyte adhesion and emigration and the accompanying vascular leak. Together, these data identify Nck as an important mediator of oxidative stress-induced inflammation and a potential therapeutic target for ischemia/reperfusion injury. PMID:25714462

  11. A novel class of antihyperlipidemic agents with low density lipoprotein receptor up-regulation via the adaptor protein autosomal recessive hypercholesterolemia.

    PubMed

    Asano, Shigehiro; Ban, Hitoshi; Tsuboya, Norie; Uno, Shinsaku; Kino, Kouichi; Ioriya, Katsuhisa; Kitano, Masafumi; Ueno, Yoshihide

    2010-04-22

    We have previously reported compound 2 as a inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase (ACAT) and up-regulator of the low density lipoprotein receptor (LDL-R) expression. In this study we focused on compound 2, a unique LDL-R up-regulator, and describe the discovery of a novel class of up-regulators of LDL-R. Replacement the methylene urea linker in compound 2 with an acylsulfonamide linker kept a potent LDL-R up-regulatory activity, and subsequent optimization work gave compound 39 as a highly potent LDL-R up-regulator (39; EC(25) = 0.047 microM). Compound 39 showed no ACAT inhibitory activity even at 1 microM. The sodium salts of compound 39 reduced plasma total and LDL cholesterol levels in a dose-dependent manner in an experimental animal model of hyperlipidemia. Moreover, we revealed in this study using RNA interference that autosomal recessive hypercholesterolemia (ARH), an adaptor protein of LDL-R, is essential for compound 39 up-regulation of LDL-R expression. PMID:20356098

  12. The Interaction of the Cellular Export Adaptor Protein Aly/REF with ICP27 Contributes to the Efficiency of Herpes Simplex Virus 1 mRNA Export

    PubMed Central

    Tian, Xiaochen; Devi-Rao, Gayathri; Golovanov, Alexander P.

    2013-01-01

    Herpes simplex virus 1 (HSV-1) protein ICP27 enables viral mRNA export by accessing the cellular mRNA export receptor TAP/NXF, which guides mRNA through the nuclear pore complex. ICP27 binds viral mRNAs and interacts with TAP/NXF, providing a link to the cellular mRNA export pathway. ICP27 also interacts with the mRNA export adaptor protein Aly/REF, which binds cellular mRNAs and also interacts with TAP/NXF. Studies using small interfering RNA (siRNA) knockdown indicated that Aly/REF is not required for cellular mRNA export, and similar knockdown studies during HSV-1 infection led us to conclude that Aly/REF may be dispensable for viral RNA export. Recently, the structural basis of the interaction of ICP27 with Aly/REF was elucidated at atomic resolution, and it was shown that three ICP27 residues, W105, R107, and L108, interface with the RNA recognition motif (RRM) domain of Aly/REF. Here, to determine the role the interaction of ICP27 and Aly/REF plays during infection, these residues were mutated to alanine, and a recombinant virus, WRL-A, was constructed. Virus production was reduced about 10-fold during WRL-A infection, and export of ICP27 protein and most viral mRNAs was less efficient. We conclude that interaction of ICP27 with Aly/REF contributes to efficient viral mRNA export. PMID:23637401

  13. The interaction of the cellular export adaptor protein Aly/REF with ICP27 contributes to the efficiency of herpes simplex virus 1 mRNA export.

    PubMed

    Tian, Xiaochen; Devi-Rao, Gayathri; Golovanov, Alexander P; Sandri-Goldin, Rozanne M

    2013-07-01

    Herpes simplex virus 1 (HSV-1) protein ICP27 enables viral mRNA export by accessing the cellular mRNA export receptor TAP/NXF, which guides mRNA through the nuclear pore complex. ICP27 binds viral mRNAs and interacts with TAP/NXF, providing a link to the cellular mRNA export pathway. ICP27 also interacts with the mRNA export adaptor protein Aly/REF, which binds cellular mRNAs and also interacts with TAP/NXF. Studies using small interfering RNA (siRNA) knockdown indicated that Aly/REF is not required for cellular mRNA export, and similar knockdown studies during HSV-1 infection led us to conclude that Aly/REF may be dispensable for viral RNA export. Recently, the structural basis of the interaction of ICP27 with Aly/REF was elucidated at atomic resolution, and it was shown that three ICP27 residues, W105, R107, and L108, interface with the RNA recognition motif (RRM) domain of Aly/REF. Here, to determine the role the interaction of ICP27 and Aly/REF plays during infection, these residues were mutated to alanine, and a recombinant virus, WRL-A, was constructed. Virus production was reduced about 10-fold during WRL-A infection, and export of ICP27 protein and most viral mRNAs was less efficient. We conclude that interaction of ICP27 with Aly/REF contributes to efficient viral mRNA export.

  14. Baculovirus vectors expressing F proteins in combination with virus-induced signaling adaptor (VISA) molecules confer protection against respiratory syncytial virus infection.

    PubMed

    Zhang, Yuan; Qiao, Lei; Hu, Xiao; Zhao, Kang; Zhang, Yanwen; Chai, Feng; Pan, Zishu

    2016-01-01

    Baculovirus has been exploited for use as a novel vaccine vector. To investigate the feasibility and efficacy of recombinant baculoviruses (rBVs) expressing respiratory syncytial virus (RSV) fusion (F) proteins, four constructs (Bac-tF/64, Bac-CF, Bac-CF/tF64 and Bac-CF/tF64-VISA) were generated. Bac-tF64 displays the F ectodomain (tF) on the envelope of rBVs, whereas Bac-CF expresses full-length F protein in transduced mammalian cells. Bac-CF/tF64 not only displays tF on the envelope but also expresses F in cells. Bac-CF/tF64-VISA comprises Bac-CF/tF64 harboring the virus-induced signaling adaptor (VISA) gene. After administration to BALB/c mice, all four vectors elicited RSV neutralizing antibody (Ab), systemic Ab (IgG, IgG1, and IgG2a), and cytokine responses. Compared with Bac-tF64, mice inoculated with Bac-CF and Bac-CF/tF64 exhibited an increased mixed Th1/Th2 cytokine response, increased ratios of IgG2a/IgG1 antibody responses, and reduced immunopathology upon RSV challenge. Intriguingly, co-expression of VISA reduced Th2 cytokine (IL-4, IL-5, and IL-10) production induced by Bac-CF/tF64, thus relieving lung pathology upon a subsequent RSV challenge. Our results indicated that the Bac-CF/tF64 vector incorporated with the VISA molecule may provide an effective vaccine strategy for protection against RSV.

  15. Pseudomonas aeruginosa ExoT Induces Atypical Anoikis Apoptosis in Target Host Cells by Transforming Crk Adaptor Protein into a Cytotoxin

    PubMed Central

    Wood, Stephen; Goldufsky, Josef; Shafikhani, Sasha H.

    2015-01-01

    Previously, we demonstrated that Pseudomonas aeruginosa ExoT induces potent apoptosis in host epithelial cells in a manner that primarily depends on its ADP-ribosyltransferase domain (ADPRT) activity. However, the mechanism underlying ExoT/ADPRT-induced apoptosis remains undetermined. We now report that ExoT/ADPRT disrupts focal adhesion sites, activates p38β and JNK, and interferes with integrin-mediated survival signaling; causing atypical anoikis. We show that ExoT/ADPRT-induced anoikis is mediated by the Crk adaptor protein. We found that Crk-/- knockout cells are significantly more resistant to ExoT-induced apoptosis, while Crk-/- cells complemented with Crk are rendered sensitive to ExoT-induced apoptosis. Moreover, a dominant negative (DN) mutant form of Crk phenocopies ExoT-induced apoptosis both kinetically and mechanistically. Crk is generally believed to be a component of focal adhesion (FA) and its role in cellular survival remains controversial in that it has been found to be either pro-survival or pro-apoptosis. Our data demonstrate that although Crk is recruited to FA sites, its function is likely not required for FA assembly or for survival per se. However, when modified by ExoT or by mutagenesis, it can be transformed into a cytotoxin that induces anoikis by disrupting FA sites and interfering with integrin survival signaling. To our knowledge, this is the first example whereby a bacterial toxin exerts its cytotoxicity by subverting the function of an innocuous host cellular protein and turning it against the host cell. PMID:26020630

  16. Protease-activated Receptor-4 Signaling and Trafficking Is Regulated by the Clathrin Adaptor Protein Complex-2 Independent of β-Arrestins.

    PubMed

    Smith, Thomas H; Coronel, Luisa J; Li, Julia G; Dores, Michael R; Nieman, Marvin T; Trejo, JoAnn

    2016-08-26

    Protease-activated receptor-4 (PAR4) is a G protein-coupled receptor (GPCR) for thrombin and is proteolytically activated, similar to the prototypical PAR1. Due to the irreversible activation of PAR1, receptor trafficking is intimately linked to signal regulation. However, unlike PAR1, the mechanisms that control PAR4 trafficking are not known. Here, we sought to define the mechanisms that control PAR4 trafficking and signaling. In HeLa cells depleted of clathrin by siRNA, activated PAR4 failed to internalize. Consistent with clathrin-mediated endocytosis, expression of a dynamin dominant-negative K44A mutant also blocked activated PAR4 internalization. However, unlike most GPCRs, PAR4 internalization occurred independently of β-arrestins and the receptor's C-tail domain. Rather, we discovered a highly conserved tyrosine-based motif in the third intracellular loop of PAR4 and found that the clathrin adaptor protein complex-2 (AP-2) is important for internalization. Depletion of AP-2 inhibited PAR4 internalization induced by agonist. In addition, mutation of the critical residues of the tyrosine-based motif disrupted agonist-induced PAR4 internalization. Using Dami megakaryocytic cells, we confirmed that AP-2 is required for agonist-induced internalization of endogenous PAR4. Moreover, inhibition of activated PAR4 internalization enhanced ERK1/2 signaling, whereas Akt signaling was markedly diminished. These findings indicate that activated PAR4 internalization requires AP-2 and a tyrosine-based motif and occurs independent of β-arrestins, unlike most classical GPCRs. Moreover, these findings are the first to show that internalization of activated PAR4 is linked to proper ERK1/2 and Akt activation. PMID:27402844

  17. SmShb, the SH2-Containing Adaptor Protein B of Schistosoma mansoni Regulates Venus Kinase Receptor Signaling Pathways

    PubMed Central

    Morel, Marion; Vanderstraete, Mathieu; Cailliau, Katia; Hahnel, Steffen; Grevelding, Christoph G.; Dissous, Colette

    2016-01-01

    Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) formed by an extracellular Venus Fly Trap (VFT) ligand binding domain associated via a transmembrane domain with an intracellular tyrosine kinase (TK) domain. Schistosoma mansoni VKRs, SmVKR1 and SmVKR2, are both implicated in reproductive activities of the parasite. In this work, we show that the SH2 domain-containing protein SmShb is a partner of the phosphorylated form of SmVKR1. Expression of these proteins in Xenopus oocytes allowed us to demonstrate that the SH2 domain of SmShb interacts with the phosphotyrosine residue (pY979) located in the juxtamembrane region of SmVKR1. This interaction leads to phosphorylation of SmShb on tyrosines and promotes SmVKR1 signaling towards the JNK pathway. SmShb transcripts are expressed in all parasite stages and they were found in ovary and testes of adult worms, suggesting a possible colocalization of SmShb and SmVKR1 proteins. Silencing of SmShb in adult S. mansoni resulted in an accumulation of mature sperm in testes, indicating a possible role of SmShb in gametogenesis. PMID:27636711

  18. SmShb, the SH2-Containing Adaptor Protein B of Schistosoma mansoni Regulates Venus Kinase Receptor Signaling Pathways.

    PubMed

    Morel, Marion; Vanderstraete, Mathieu; Cailliau, Katia; Hahnel, Steffen; Grevelding, Christoph G; Dissous, Colette

    2016-01-01

    Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) formed by an extracellular Venus Fly Trap (VFT) ligand binding domain associated via a transmembrane domain with an intracellular tyrosine kinase (TK) domain. Schistosoma mansoni VKRs, SmVKR1 and SmVKR2, are both implicated in reproductive activities of the parasite. In this work, we show that the SH2 domain-containing protein SmShb is a partner of the phosphorylated form of SmVKR1. Expression of these proteins in Xenopus oocytes allowed us to demonstrate that the SH2 domain of SmShb interacts with the phosphotyrosine residue (pY979) located in the juxtamembrane region of SmVKR1. This interaction leads to phosphorylation of SmShb on tyrosines and promotes SmVKR1 signaling towards the JNK pathway. SmShb transcripts are expressed in all parasite stages and they were found in ovary and testes of adult worms, suggesting a possible colocalization of SmShb and SmVKR1 proteins. Silencing of SmShb in adult S. mansoni resulted in an accumulation of mature sperm in testes, indicating a possible role of SmShb in gametogenesis. PMID:27636711

  19. SmShb, the SH2-Containing Adaptor Protein B of Schistosoma mansoni Regulates Venus Kinase Receptor Signaling Pathways.

    PubMed

    Morel, Marion; Vanderstraete, Mathieu; Cailliau, Katia; Hahnel, Steffen; Grevelding, Christoph G; Dissous, Colette

    2016-01-01

    Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) formed by an extracellular Venus Fly Trap (VFT) ligand binding domain associated via a transmembrane domain with an intracellular tyrosine kinase (TK) domain. Schistosoma mansoni VKRs, SmVKR1 and SmVKR2, are both implicated in reproductive activities of the parasite. In this work, we show that the SH2 domain-containing protein SmShb is a partner of the phosphorylated form of SmVKR1. Expression of these proteins in Xenopus oocytes allowed us to demonstrate that the SH2 domain of SmShb interacts with the phosphotyrosine residue (pY979) located in the juxtamembrane region of SmVKR1. This interaction leads to phosphorylation of SmShb on tyrosines and promotes SmVKR1 signaling towards the JNK pathway. SmShb transcripts are expressed in all parasite stages and they were found in ovary and testes of adult worms, suggesting a possible colocalization of SmShb and SmVKR1 proteins. Silencing of SmShb in adult S. mansoni resulted in an accumulation of mature sperm in testes, indicating a possible role of SmShb in gametogenesis.

  20. Semisynthetic homoharringtonine induces apoptosis via inhibition of protein synthesis and triggers rapid myeloid cell leukemia-1 down-regulation in myeloid leukemia cells.

    PubMed

    Tang, Ruoping; Faussat, Anne-Marie; Majdak, Patricia; Marzac, Christophe; Dubrulle, Sabine; Marjanovic, Zora; Legrand, Ollivier; Marie, Jean-Pierre

    2006-03-01

    Semisynthetic homoharringtonine (ssHHT) is now being evaluated in phase II clinical trials for the treatment of chronic myelogenous leukemia and acute myelogenous leukemia patients. Here, we examined the mechanism of the apoptosis induced by ssHHT in myeloid leukemia cells. First, we have shown that ssHHT induces apoptosis in HL60 and HL60/MRP cell lines in a time- and dose-dependent manner, and independently of the expression of Bax. The decrease of mitochondrial membrane potential and the release of cytochrome c were observed in the apoptotic cells induced by ssHHT. To unveil the relationship between ssHHT and the mitochondrial disruption, we have shown that ssHHT decreased myeloid cell leukemia-1 (Mcl-1) expression and induced Bcl-2 cleavage in HL60 and HL60/MRP cell lines. The Bcl-2 cleavage could be inhibited by the Z-VAD.fmk caspase inhibitor. However, Mcl-1 turnover was very rapid and occurred before caspase activation. The Mcl-1 turnover was only induced by ssHHT and cycloheximide, but not by daunorubicin and cytosine arabinoside, and could be restored by proteasome inhibitors. Second, we confirmed that ssHHT rapidly induced massive apoptosis in acute myelogenous leukemia patient cells. We have also confirmed the release of cytochrome c and a rapid turnover of Mcl-1 in these patient cells, taking place only in apoptotic cells induced by ssHHT but not in cells undergoing spontaneous apoptosis. Finally, we have shown that ssHHT inhibits protein synthesis in both cell line and patient cells. We suggest that the inhibition of protein synthesis and resulting Mcl-1 turnover play a key role in the apoptosis induced by ssHHT. Our results encourage further clinical trials for the use of ssHHT in acute myelogenous leukemia.

  1. The Mu subunit of Plasmodium falciparum clathrin-associated adaptor protein 2 modulates in vitro parasite response to artemisinin and quinine.

    PubMed

    Henriques, Gisela; van Schalkwyk, Donelly A; Burrow, Rebekah; Warhurst, David C; Thompson, Eloise; Baker, David A; Fidock, David A; Hallett, Rachel; Flueck, Christian; Sutherland, Colin J

    2015-05-01

    The emergence of drug-resistant parasites is a serious threat faced by malaria control programs. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, ap2-mu (encoding the mu chain of the adaptor protein 2 [AP2] complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi (pcap2-mu). Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum ap2-mu homologue, pfap2-mu, found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here, we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing codon 160 encoding either the wild-type Ser (Ser160) or the Asn mutant (160Asn) form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-h in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow-clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance.

  2. The cytoskeletal adaptor protein band 4.1B is required for the maintenance of paranodal axoglial septate junctions in myelinated axons.

    PubMed

    Buttermore, Elizabeth D; Dupree, Jeffrey L; Cheng, JrGang; An, Xiuli; Tessarollo, Lino; Bhat, Manzoor A

    2011-06-01

    Precise targeting and maintenance of axonal domains in myelinated axons is essential for saltatory conduction. Caspr and Caspr2, which localize at paranodal and juxtaparanodal domains, contain binding sites for the cytoskeletal adaptor protein 4.1B. The exact role of 4.1B in the organization and maintenance of axonal domains is still not clear. Here, we report the generation and characterization of 4.1B-null mice. We show that loss of 4.1B in the PNS results in mislocalization of Caspr at paranodes and destabilization of paranodal axoglial septate junctions (AGSJs) as early as postnatal day 30. In the CNS, Caspr localization is progressively disrupted and ultrastructural analysis showed paranodal regions that were completely devoid of AGSJs, with axolemma separated from the myelin loops, and loops coming off the axolemma. Most importantly, our phenotypic analysis of previously generated 4.1B mutants, used in the study by Horresh et al. (2010), showed that Caspr localization was not affected in the PNS, even after 1 year; and 4.1R was neither expressed, nor enriched at the paranodes. Furthermore, ultrastructural analysis of these 4.1B mutants showed destabilization of CNS AGSJs at ∼ 1 year. We also discovered that the 4.1B locus is differentially expressed in the PNS and CNS, and generates multiple splice isoforms in the PNS, suggesting 4.1B may function differently in the PNS versus CNS. Together, our studies provide direct evidence that 4.1B plays a pivotal role in interactions between the paranodal AGSJs and axonal cytoskeleton, and that 4.1B is critically required for long-term maintenance of axonal domains in myelinated axons. PMID:21632923

  3. The Cytoskeletal Adaptor Protein Band 4.1B is Required for the Maintenance of Paranodal Axo-Glial Septate Junctions in Myelinated Axons

    PubMed Central

    Buttermore, Elizabeth D.; Dupree, Jeffrey L.; Cheng, JrGang; An, Xiuli; Tessarollo, Lino; Bhat, Manzoor A.

    2011-01-01

    Precise targeting and maintenance of axonal domains in myelinated axons is essential for saltatory conduction. Caspr and Caspr2, which localize at paranodal and juxtaparanodal domains, contain binding sites for the cytoskeletal adaptor protein 4.1B. The exact role of 4.1B in the organization and maintenance of axonal domains is still not clear. Here we report the generation and characterization of 4.1B null mice. We show that loss of 4.1B in the PNS results in mislocalization of Caspr at paranodes and destabilization of paranodal axo-glial septate junctions (AGSJs) as early as postnatal day 30. In the CNS, Caspr localization is progressively disrupted and ultrastructural analysis showed paranodal regions that were completely devoid of AGSJs, with axolemma separated from the myelin loops, and loops coming off the axolemma. Most importantly, our phenotypic analysis of previously generated 4.1B mutants, used in Horresh et al. (2010), showed that Caspr localization was not affected in the PNS, even after one year; and 4.1R was neither expressed, nor enriched at the paranodes. Furthermore, ultrastructural analysis of these 4.1B mutants showed destabilization of CNS AGSJs at about one year. We also discovered that the 4.1B locus is differentially expressed in the PNS and CNS, and generates multiple splice isoforms in the PNS, suggesting 4.1B may function differently in the PNS versus CNS. Together, our studies provide direct evidence that 4.1B plays a pivotal role in interactions between the paranodal AGSJs and axonal cytoskeleton, and that 4.1B is critically required for long-term maintenance of axonal domains in myelinated axons. PMID:21632923

  4. Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster

    PubMed Central

    Rodriguez-Fernandez, Imilce A.; Dell’Angelica, Esteban C.

    2015-01-01

    The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions–which together covered most of the autosomal chromosomes–to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated

  5. Structural and functional insights into CARDs of zebrafish (Danio rerio) NOD1 and NOD2, and their interaction with adaptor protein RIP2.

    PubMed

    Maharana, Jitendra; Dehury, Budheswar; Sahoo, Jyoti Ranjan; Jena, Itishree; Bej, Aritra; Panda, Debashis; Sahoo, Bikash Ranjan; Patra, Mahesh Chandra; Pradhan, Sukanta Kumar

    2015-08-01

    Nucleotide-binding and oligomerization domain-containing protein 1 (NOD1) and NOD2 are cytosolic pattern-recognition receptors (PRRs) composed of an N-terminal caspase activation and recruitment domain (CARD), a central NACHT domain and C-terminal leucine-rich repeats (LRRs). They play a vital role in innate immune signaling by activating the NF-κB pathway via recognition of peptidoglycans by LRRs, and ATP-dependent self-oligomerization of NACHT followed by downstream signaling. After oligomerization, CARD/s play a crucial role in activating downstream signaling via the adaptor molecule, RIP2. Due to the inadequacy of experimental 3D structures of CARD/s of NOD2 and RIP2, and results from differential experimental setups, the RIP2-mediated CARD-CARD interaction has remained as a contradictory statement. We employed a combinatorial approach involving protein modeling, docking, molecular dynamics simulation, and binding free energy calculation to illuminate the molecular mechanism that shows the possible involvement of either the acidic or basic patch of zebrafish NOD1/2-CARD/a and RIP2-CARD in CARD-CARD interaction. Herein, we have hypothesized 'type-I' mode of CARD-CARD interaction in NOD1 and NOD2, where NOD1/2-CARD/a involve their acidic surfaces to interact with RIP2. Asp37 and Glu51 (of NOD1) and Arg477, Arg521 and Arg529 (of RIP2) were identified to be crucial for NOD1-RIP2 interaction. However, in NOD2-RIP2, Asp32 (of NOD2) and Arg477 and Arg521 (of RIP2) were anticipated to be significant for downstream signaling. Furthermore, we found that strong electrostatic contacts and salt bridges are crucial for protein-protein interactions. Altogether, our study has provided novel insights into the RIP2-mediated CARD-CARD interaction in zebrafish NOD1 and NOD2, which will be helpful to understand the molecular basis of the NOD1/2 signaling mechanism. PMID:26079944

  6. The adaptor protein SAP regulates type II NKT-cell development, cytokine production, and cytotoxicity against lymphoma.

    PubMed

    Weng, Xiufang; Liao, Chia-Min; Bagchi, Sreya; Cardell, Susanna L; Stein, Paul L; Wang, Chyung-Ru

    2014-12-01

    CD1d-restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT-cell TCR transgenic mouse model, we demonstrated that CD1d-expressing hematopoietic cells, but not thymic epithelial cells, meditate efficient selection of type II NKT cells. Furthermore, we showed that SAP regulates type II NKT-cell development by controlling early growth response 2 protein and promyelocytic leukemia zinc finger expression. SAP-deficient 24αβ transgenic T cells (24αβ T cells) exhibited an immature phenotype with reduced Th2 cytokine-producing capacity and diminished cytotoxicity to CD1d-expressing lymphoma cells. The impaired IL-4 production by SAP-deficient 24αβ T cells was associated with reduced IFN regulatory factor 4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP.

  7. The Arabidopsis adaptor protein AP-3μ interacts with the G-protein β subunit AGB1 and is involved in abscisic acid regulation of germination and post-germination development.

    PubMed

    Kansup, Jeeraporn; Tsugama, Daisuke; Liu, Shenkui; Takano, Tetsuo

    2013-12-01

    Heterotrimeric G-proteins (G-proteins) have been implicated in ubiquitous signalling mechanisms in eukaryotes. In plants, G-proteins modulate hormonal and stress responses and regulate diverse developmental processes. However, the molecular mechanisms of their functions are largely unknown. A yeast two-hybrid screen was performed to identify interacting partners of the Arabidopsis G-protein β subunit AGB1. One of the identified AGB1-interacting proteins is the Arabidopsis adaptor protein AP-3µ. The interaction between AGB1 and AP-3µ was confirmed by an in vitro pull-down assay and bimolecular fluorescence complementation assay. Two ap-3µ T-DNA insertional mutants were found to be hyposensitive to abscisic acid (ABA) during germination and post-germination growth, whereas agb1 mutants were hypersensitive to ABA. During seed germination, agb1/ap-3µ double mutants were more sensitive to ABA than the wild type but less sensitive than agb1 mutants. However, in post-germination growth, the double mutants were as sensitive to ABA as agb1 mutants. These data suggest that AP-3µ positively regulates the ABA responses independently of AGB1 in seed germination, while AP-3µ does require AGB1 to regulate ABA responses during post-germination growth.

  8. Myeloid differentiation primary response protein 88 couples reverse cholesterol transport to inflammation.

    PubMed

    Smoak, Kathleen A; Aloor, Jim J; Madenspacher, Jennifer; Merrick, B Alex; Collins, Jennifer B; Zhu, Xuewei; Cavigiolio, Giorgio; Oda, Michael N; Parks, John S; Fessler, Michael B

    2010-06-01

    Crosstalk exists in mammalian cells between cholesterol trafficking and innate immune signaling. Apolipoprotein A-I (apoA-I), a serum apolipoprotein that induces antiatherogenic efflux of macrophage cholesterol, is widely described as anti-inflammatory because it neutralizes bacterial lipopolysaccharide. Conversely, lipopolysaccharide-induced inflammation is proatherogenic. However, whether innate immunity plays an endogenous, physiological role in host cholesterol homeostasis in the absence of infection is undetermined. We report that apoA-I signals in the macrophage through Toll-like receptor (TLR)2, TLR4, and CD14, utilizing myeloid differentiation primary response protein 88 (MyD88)-dependent and -independent pathways, to activate nuclear factor-kappaB and induce cytokines. MyD88 plays a critical role in reverse cholesterol transport in vitro and in vivo, in part through promoting ATP-binding cassette A1 transporter upregulation. Taken together, this work identifies apoA-I as an endogenous stimulus of innate immunity that couples cholesterol trafficking to inflammation through MyD88 and identifies innate immunity as a physiologic signal in cholesterol homeostasis.

  9. The beta-appendages of the four adaptor-protein (AP) complexes: structure and binding properties, and identification of sorting nexin 9 as an accessory protein to AP-2.

    PubMed Central

    Lundmark, Richard; Carlsson, Sven R

    2002-01-01

    Adaptor protein (AP) complexes are essential components for the formation of coated vesicles and the recognition of cargo proteins for intracellular transport. Each AP complex exposes two appendage domains with that function to bind regulatory accessory proteins in the cytosol. Secondary structure predictions, sequence alignments and CD spectroscopy were used to relate the beta-appendages of all human AP complexes to the previously published crystal structure of AP-2. The results suggested that the beta-appendages of AP-1, AP-2 and AP-3 have similar structures, consisting of two subdomains, whereas that of AP-4 lacks the inner subdomain. Pull-down and overlay assays showed partial overlap in the binding specificities of the beta-appendages of AP-1 and AP-2, whereas the corresponding domain of AP-3 displayed a unique binding pattern. That AP-4 may have a truncated, non-functional domain was indicated by its apparent inability to bind any proteins from cytosol. Of several novel beta-appendage-binding proteins detected, one that had affinity exclusively for AP-2 was identified as sorting nexin 9 (SNX9). SNX9, which contains a phox and an Src homology 3 domain, was found in large complexes and was at least partially associated with AP-2 in the cytosol. SNX9 may function to assist AP-2 in its role at the plasma membrane. PMID:11879186

  10. Structural Basis for Small G Protein Effector Interaction of Ras-related Protein 1 (Rap1) and Adaptor Protein Krev Interaction Trapped 1 (KRIT1)

    SciTech Connect

    Li, Xiaofeng; Zhang, Rong; Draheim, Kyle M.; Liu, Weizhi; Calderwood, David A.; Boggon, Titus J.

    2012-09-17

    Cerebral cavernous malformations (CCMs) affect 0.1-0.5% of the population resulting in leaky vasculature and severe neurological defects. KRIT1 (Krev interaction trapped-1) mutations associate with {approx}40% of familial CCMs. KRIT1 is an effector of Ras-related protein 1 (Rap1) GTPase. Rap1 relocalizes KRIT1 from microtubules to cell membranes to impact integrin activation, potentially important for CCM pathology. We report the 1.95 {angstrom} co-crystal structure of KRIT1 FERM domain in complex with Rap1. Rap1-KRIT1 interaction encompasses an extended surface, including Rap1 Switch I and II and KRIT1 FERM F1 and F2 lobes. Rap1 binds KRIT1-F1 lobe using a GTPase-ubiquitin-like fold interaction but binds KRIT1-F2 lobe by a novel interaction. Point mutagenesis confirms the interaction. High similarity between KRIT1-F2/F3 and talin is revealed. Additionally, the mechanism for FERM domains acting as GTPase effectors is suggested. Finally, structure-based alignment of each lobe suggests classification of FERM domains as ERM-like and TMFK-like (talin-myosin-FAK-KRIT-like) and that FERM lobes resemble domain 'modules.'

  11. Protein Phosphatase 2A as a Therapeutic Target in Acute Myeloid Leukemia

    PubMed Central

    Arriazu, Elena; Pippa, Raffaella; Odero, María D.

    2016-01-01

    Acute myeloid leukemia (AML) is a heterogeneous malignant disorder of hematopoietic progenitor cells in which several genetic and epigenetic aberrations have been described. Despite progressive advances in our understanding of the molecular biology of this disease, the outcome for most patients is poor. It is, therefore, necessary to develop more effective treatment strategies. Genetic aberrations affecting kinases have been widely studied in AML; however, the role of phosphatases remains underexplored. Inactivation of the tumor-suppressor protein phosphatase 2A (PP2A) is frequent in AML patients, making it a promising target for therapy. There are several PP2A inactivating mechanisms reported in this disease. Deregulation or specific post-translational modifications of PP2A subunits have been identified as a cause of PP2A malfunction, which lead to deregulation of proliferation or apoptosis pathways, depending on the subunit affected. Likewise, overexpression of either SET or cancerous inhibitor of protein phosphatase 2A, endogenous inhibitors of PP2A, is a recurrent event in AML that impairs PP2A activity, contributing to leukemogenesis progression. Interestingly, the anticancer activity of several PP2A-activating drugs (PADs) depends on interaction/sequestration of SET. Preclinical studies show that pharmacological restoration of PP2A activity by PADs effectively antagonizes leukemogenesis, and that these drugs have synergistic cytotoxic effects with conventional chemotherapy and kinase inhibitors, opening new possibilities for personalized treatment in AML patients, especially in cases with SET-dependent inactivation of PP2A. Here, we review the role of PP2A as a druggable tumor suppressor in AML. PMID:27092295

  12. Dorsal root ganglion myeloid zinc finger protein 1 contributes to neuropathic pain after peripheral nerve trauma.

    PubMed

    Li, Zhisong; Gu, Xiyao; Sun, Linlin; Wu, Shaogen; Liang, Lingli; Cao, Jing; Lutz, Brianna Marie; Bekker, Alex; Zhang, Wei; Tao, Yuan-Xiang

    2015-04-01

    Peripheral nerve injury-induced changes in gene transcription and translation in primary sensory neurons of the dorsal root ganglion (DRG) are considered to contribute to neuropathic pain genesis. Transcription factors control gene expression. Peripheral nerve injury increases the expression of myeloid zinc finger protein 1 (MZF1), a transcription factor, and promotes its binding to the voltage-gated potassium 1.2 (Kv1.2) antisense (AS) RNA gene in the injured DRG. However, whether DRG MZF1 participates in neuropathic pain is still unknown. Here, we report that blocking the nerve injury-induced increase of DRG MZF1 through microinjection of MZF1 siRNA into the injured DRG attenuated the initiation and maintenance of mechanical, cold, and thermal pain hypersensitivities in rats with chronic constriction injury (CCI) of the sciatic nerve, without affecting locomotor functions and basal responses to acute mechanical, heat, and cold stimuli. Mimicking the nerve injury-induced increase of DRG MZF1 through microinjection of recombinant adeno-associated virus 5 expressing full-length MZF1 into the DRG produced significant mechanical, cold, and thermal pain hypersensitivities in naive rats. Mechanistically, MZF1 participated in CCI-induced reductions in Kv1.2 mRNA and protein and total Kv current and the CCI-induced increase in neuronal excitability through MZF1-triggered Kv1.2 AS RNA expression in the injured DRG neurons. MZF1 is likely an endogenous trigger of neuropathic pain and might serve as a potential target for preventing and treating this disorder. PMID:25630025

  13. A Novel Interaction of the Catalytic Subunit of Protein Phosphatase 2A with the Adaptor Protein CIN85 Suppresses Phosphatase Activity and Facilitates Platelet Outside-in αIIbβ3 Integrin Signaling.

    PubMed

    Khatlani, Tanvir; Pradhan, Subhashree; Da, Qi; Shaw, Tanner; Buchman, Vladimir L; Cruz, Miguel A; Vijayan, K Vinod

    2016-08-12

    The transduction of signals generated by protein kinases and phosphatases are critical for the ability of integrin αIIbβ3 to support stable platelet adhesion and thrombus formation. Unlike kinases, it remains unclear how serine/threonine phosphatases engage the signaling networks that are initiated following integrin ligation. Because protein-protein interactions form the backbone of signal transduction, we searched for proteins that interact with the catalytic subunit of protein phosphatase 2A (PP2Ac). In a yeast two-hybrid study, we identified a novel interaction between PP2Ac and an adaptor protein CIN85 (Cbl-interacting protein of 85 kDa). Truncation and alanine mutagenesis studies revealed that PP2Ac binds to the P3 block ((396)PAIPPKKPRP(405)) of the proline-rich region in CIN85. The interaction of purified PP2Ac with CIN85 suppressed phosphatase activity. Human embryonal kidney 293 αIIbβ3 cells overexpressing a CIN85 P3 mutant, which cannot support PP2Ac binding, displayed decreased adhesion to immobilized fibrinogen. Platelets contain the ∼85 kDa CIN85 protein along with the PP2Ac-CIN85 complex. A myristylated cell-permeable peptide derived from residues 395-407 of CIN85 protein (P3 peptide) disrupted the platelet PP2Ac-CIN85 complex and decreased αIIbβ3 signaling dependent functions such as platelet spreading on fibrinogen and thrombin-mediated fibrin clot retraction. In a phospho-profiling study P3 peptide treated platelets also displayed decreased phosphorylation of several signaling proteins including Src and GSK3β. Taken together, these data support a role for the novel PP2Ac-CIN85 complex in supporting integrin-dependent platelet function by dampening the phosphatase activity. PMID:27334924

  14. Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype-phenotype correlations, codon bias and dominant-negative effects.

    PubMed

    Hannan, Fadil M; Howles, Sarah A; Rogers, Angela; Cranston, Treena; Gorvin, Caroline M; Babinsky, Valerie N; Reed, Anita A; Thakker, Clare E; Bockenhauer, Detlef; Brown, Rosalind S; Connell, John M; Cook, Jacqueline; Darzy, Ken; Ehtisham, Sarah; Graham, Una; Hulse, Tony; Hunter, Steven J; Izatt, Louise; Kumar, Dhavendra; McKenna, Malachi J; McKnight, John A; Morrison, Patrick J; Mughal, M Zulf; O'Halloran, Domhnall; Pearce, Simon H; Porteous, Mary E; Rahman, Mushtaqur; Richardson, Tristan; Robinson, Robert; Scheers, Isabelle; Siddique, Haroon; Van't Hoff, William G; Wang, Timothy; Whyte, Michael P; Nesbit, M Andrew; Thakker, Rajesh V

    2015-09-15

    The adaptor protein-2 sigma subunit (AP2σ2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2σ2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca(2+) o) homeostasis. To elucidate the role of AP2σ2 in Ca(2+) o regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2σ2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2σ2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype-phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2σ2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2σ2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2σ2 mutations to result in a more severe FHH phenotype with genotype-phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue. PMID:26082470

  15. Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype-phenotype correlations, codon bias and dominant-negative effects.

    PubMed

    Hannan, Fadil M; Howles, Sarah A; Rogers, Angela; Cranston, Treena; Gorvin, Caroline M; Babinsky, Valerie N; Reed, Anita A; Thakker, Clare E; Bockenhauer, Detlef; Brown, Rosalind S; Connell, John M; Cook, Jacqueline; Darzy, Ken; Ehtisham, Sarah; Graham, Una; Hulse, Tony; Hunter, Steven J; Izatt, Louise; Kumar, Dhavendra; McKenna, Malachi J; McKnight, John A; Morrison, Patrick J; Mughal, M Zulf; O'Halloran, Domhnall; Pearce, Simon H; Porteous, Mary E; Rahman, Mushtaqur; Richardson, Tristan; Robinson, Robert; Scheers, Isabelle; Siddique, Haroon; Van't Hoff, William G; Wang, Timothy; Whyte, Michael P; Nesbit, M Andrew; Thakker, Rajesh V

    2015-09-15

    The adaptor protein-2 sigma subunit (AP2σ2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2σ2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca(2+) o) homeostasis. To elucidate the role of AP2σ2 in Ca(2+) o regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2σ2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2σ2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype-phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2σ2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2σ2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2σ2 mutations to result in a more severe FHH phenotype with genotype-phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.

  16. Overexpression of SET is a recurrent event associated with poor outcome and contributes to protein phosphatase 2A inhibition in acute myeloid leukemia

    PubMed Central

    Cristóbal, Ion; Garcia-Orti, Laura; Cirauqui, Cristina; Cortes-Lavaud, Xabier; García-Sánchez, María A.; Calasanz, María J.; Odero, María D.

    2012-01-01

    Background Protein phosphatase 2A is a novel potential therapeutic target in several types of chronic and acute leukemia, and its inhibition is a common event in acute myeloid leukemia. Upregulation of SET is essential to inhibit protein phosphatase 2A in chronic myeloid leukemia, but its importance in acute myeloid leukemia has not yet been explored. Design and Methods We quantified SET expression by real time reverse transcriptase polymerase chain reaction in 214 acute myeloid leukemia patients at diagnosis. Western blot was performed in acute myeloid leukemia cell lines and in 16 patients’ samples. We studied the effect of SET using cell viability assays. Bioinformatics analysis of the SET promoter, chromatin immunoprecipitation, and luciferase assays were performed to evaluate the transcriptional regulation of SET. Results SET overexpression was found in 60/214 patients, for a prevalence of 28%. Patients with SET overexpression had worse overall survival (P<0.01) and event-free survival (P<0.01). Deregulation of SET was confirmed by western blot in both cell lines and patients’ samples. Functional analysis showed that SET promotes proliferation, and restores cell viability after protein phosphatase 2A overexpression. We identified EVI1 overexpression as a mechanism involved in SET deregulation in acute myeloid leukemia cells. Conclusions These findings suggest that SET overexpression is a key mechanism in the inhibition of PP2A in acute myeloid leukemia, and that EVI1 overexpression contributes to the deregulation of SET. Furthermore, SET overexpression is associated with a poor outcome in acute myeloid leukemia, and it can be used to identify a subgroup of patients who could benefit from future treatments based on PP2A activators. PMID:22133779

  17. Myeloid differentiation protein-2-dependent and -independent neutrophil accumulation during Escherichia coli pneumonia.

    PubMed

    Cai, Shanshan; Zemans, Rachel L; Young, Scott K; Worthen, G Scott; Jeyaseelan, Samithamby

    2009-06-01

    Bacterial pneumonia remains a serious disease. Pattern recognition receptors play an integral role in neutrophil accumulation during pneumonia. Although myeloid differentiation protein (MD)-2 has been recognized as a key molecule for LPS signaling, the role of MD-2 in neutrophil accumulation in the lung during bacterial infection has not been explored. Here, we investigate the role of MD-2 in Escherichia coli LPS-induced lung inflammation and E. coli-induced pneumonia. LPS-induced CD14-independent neutrophil accumulation was abolished in CD14/MD-2(-/-) mice. MD-2(-/-) mice challenged with LPS displayed attenuated neutrophil influx, NF-kappaB activation, cytokine/chemokine expression, and lung histopathology. MD-2(-/-) mice transplanted with MD-2(+/+) bone marrow demonstrated decreased neutrophil influx and cytokine/chemokine expression in the lungs when challenged by LPS. MD-2(-/-) mice infected with E. coli demonstrated reduced neutrophil influx and cytokine/chemokine expression in the lungs, whereas heat-killed E. coli did not induce either neutrophil accumulation or cytokine/chemokine expression in MD-2(-/-) mice infected with E. coli. Furthermore, MD-2(-/-) mice displayed increased bacterial burden in the lungs and enhanced bacterial dissemination. Toll-like receptor (TLR)-5(-/-) mice infected with E. coli exhibited attenuated neutrophil accumulation, whereas MD-2/TLR5(-/-) mice inoculated with E. coli showed further attenuated neutrophil influx and impaired bacterial clearance. Taken together, these new findings demonstrate: (1) the important role of MD-2 in the CD14-independent LPS-mediated cascade of neutrophil influx; (2) the relative importance of bone marrow- and non-bone marrow cell-derived MD-2 in LPS-induced inflammation; and (3) the essential role of MD-2-dependent and MD-2-independent (TLR5) signaling in E. coli-induced neutrophil accumulation and pulmonary host defense. PMID:18988922

  18. Id2 and E Proteins Orchestrate the Initiation and Maintenance of MLL-Rearranged Acute Myeloid Leukemia.

    PubMed

    Ghisi, Margherita; Kats, Lev; Masson, Frederick; Li, Jason; Kratina, Tobias; Vidacs, Eva; Gilan, Omer; Doyle, Maria A; Newbold, Andrea; Bolden, Jessica E; Fairfax, Kirsten A; de Graaf, Carolyn A; Firth, Matthew; Zuber, Johannes; Dickins, Ross A; Corcoran, Lynn M; Dawson, Mark A; Belz, Gabrielle T; Johnstone, Ricky W

    2016-07-11

    E proteins and their antagonists, the Id proteins, are transcriptional regulators important for normal hematopoiesis. We found that Id2 acts as a key regulator of leukemia stem cell (LSC) potential in MLL-rearranged acute myeloid leukemia (AML). Low endogenous Id2 expression is associated with LSC enrichment while Id2 overexpression impairs MLL-AF9-leukemia initiation and growth. Importantly, MLL-AF9 itself controls the E-protein pathway by suppressing Id2 while directly activating E2-2 expression, and E2-2 depletion phenocopies Id2 overexpression in MLL-AF9-AML cells. Remarkably, Id2 tumor-suppressive function is conserved in t(8;21) AML. Low expression of Id2 and its associated gene signature are associated with poor prognosis in MLL-rearranged and t(8;21) AML patients, identifying the Id2/E-protein axis as a promising new therapeutic target in AML. PMID:27374225

  19. Phosphoprotein Associated with Glycosphingolipid-Enriched Microdomains (Pag), a Novel Ubiquitously Expressed Transmembrane Adaptor Protein, Binds the Protein Tyrosine Kinase Csk and Is Involved in Regulation of T Cell Activation

    PubMed Central

    Brdic̆ka, Tomás̆; Pavlis̆tová, Dagmar; Leo, Albrecht; Bruyns, Eddy; Kor̆ínek, Vladimír; Angelisová, Pavla; Scherer, Jeanette; Shevchenko, Andrej; Shevchenko, Anna; Hilgert, Ivan; C̆erný, Jan; Drbal, Karel; Kuramitsu, Yasuhiro; Kornacker, Birgit; Hor̆ejs̆í, Václav; Schraven, Burkhart

    2000-01-01

    According to a recently proposed hypothesis, initiation of signal transduction via immunoreceptors depends on interactions of the engaged immunoreceptor with glycosphingolipid-enriched membrane microdomains (GEMs). In this study, we describe a novel GEM-associated transmembrane adaptor protein, termed phosphoprotein associated with GEMs (PAG). PAG comprises a short extracellular domain of 16 amino acids and a 397-amino acid cytoplasmic tail containing ten tyrosine residues that are likely phosphorylated by Src family kinases. In lymphoid cell lines and in resting peripheral blood α/β T cells, PAG is expressed as a constitutively tyrosine-phosphorylated protein and binds the major negative regulator of Src kinases, the tyrosine kinase Csk. After activation of peripheral blood α/β T cells, PAG becomes rapidly dephosphorylated and dissociates from Csk. Expression of PAG in COS cells results in recruitment of endogenous Csk, altered Src kinase activity, and impaired phosphorylation of Src-specific substrates. Moreover, overexpression of PAG in Jurkat cells downregulates T cell receptor–mediated activation of the transcription factor nuclear factor of activated T cells. These findings collectively suggest that in the absence of external stimuli, the PAG–Csk complex transmits negative regulatory signals and thus may help to keep resting T cells in a quiescent state. PMID:10790433

  20. Structural analysis of intermolecular interactions in the kinesin adaptor complex fasciculation and elongation protein zeta 1/ short coiled-coil protein (FEZ1/SCOCO).

    PubMed

    Alborghetti, Marcos Rodrigo; Furlan, Ariane da Silva; da Silva, Júlio César; Sforça, Maurício Luís; Honorato, Rodrigo Vargas; Granato, Daniela Campos; dos Santos Migueleti, Deivid Lucas; Neves, Jorge L; de Oliveira, Paulo Sergio Lopes; Paes-Leme, Adriana Franco; Zeri, Ana Carolina de Mattos; de Torriani, Iris Concepcion Linares; Kobarg, Jörg

    2013-01-01

    Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.

  1. Influence of DNA-methylation on zinc homeostasis in myeloid cells: Regulation of zinc transporters and zinc binding proteins.

    PubMed

    Kessels, Jana Elena; Wessels, Inga; Haase, Hajo; Rink, Lothar; Uciechowski, Peter

    2016-09-01

    The distribution of intracellular zinc, predominantly regulated through zinc transporters and zinc binding proteins, is required to support an efficient immune response. Epigenetic mechanisms such as DNA methylation are involved in the expression of these genes. In demethylation experiments using 5-Aza-2'-deoxycytidine (AZA) increased intracellular (after 24 and 48h) and total cellular zinc levels (after 48h) were observed in the myeloid cell line HL-60. To uncover the mechanisms that cause the disturbed zinc homeostasis after DNA demethylation, the expression of human zinc transporters and zinc binding proteins were investigated. Real time PCR analyses of 14 ZIP (solute-linked carrier (SLC) SLC39A; Zrt/IRT-like protein), and 9 ZnT (SLC30A) zinc transporters revealed significantly enhanced mRNA expression of the zinc importer ZIP1 after AZA treatment. Because ZIP1 protein was also enhanced after AZA treatment, ZIP1 up-regulation might be the mediator of enhanced intracellular zinc levels. The mRNA expression of ZIP14 was decreased, whereas zinc exporter ZnT3 mRNA was also significantly increased; which might be a cellular reaction to compensate elevated zinc levels. An enhanced but not significant chromatin accessibility of ZIP1 promoter region I was detected by chromatin accessibility by real-time PCR (CHART) assays after demethylation. Additionally, DNA demethylation resulted in increased mRNA accumulation of zinc binding proteins metallothionein (MT) and S100A8/S100A9 after 48h. MT mRNA was significantly enhanced after 24h of AZA treatment also suggesting a reaction of the cell to restore zinc homeostasis. These data indicate that DNA methylation is an important epigenetic mechanism affecting zinc binding proteins and transporters, and, therefore, regulating zinc homeostasis in myeloid cells.

  2. BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies

    PubMed Central

    Bogenberger, J M; Kornblau, S M; Pierceall, W E; Lena, R; Chow, D; Shi, C-X; Mantei, J; Ahmann, G; Gonzales, I M; Choudhary, A; Valdez, R; Camoriano, J; Fauble, V; Tiedemann, R E; Qiu, Y H; Coombes, K R; Cardone, M; Braggio, E; Yin, H; Azorsa, D O; Mesa, R A; Stewart, A K; Tibes, R

    2014-01-01

    Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response. PMID:24451410

  3. Extracellular vesicle miR-7977 is involved in hematopoietic dysfunction of mesenchymal stromal cells via poly(rC) binding protein 1 reduction in myeloid neoplasms

    PubMed Central

    Horiguchi, Hiroto; Kobune, Masayoshi; Kikuchi, Shohei; Yoshida, Masahiro; Murata, Masaki; Murase, Kazuyuki; Iyama, Satoshi; Takada, Kohichi; Sato, Tsutomu; Ono, Kaoru; Hashimoto, Akari; Tatekoshi, Ayumi; Kamihara, Yusuke; Kawano, Yutaka; Miyanishi, Koji; Sawada, Norimasa; Kato, Junji

    2016-01-01

    The failure of normal hematopoiesis is observed in myeloid neoplasms. However, the precise mechanisms governing the replacement of normal hematopoietic stem cells in their niche by myeloid neoplasm stem cells have not yet been clarified. Primary acute myeloid leukemia and myelodysplastic syndrome cells induced aberrant expression of multiple hematopoietic factors including Jagged-1, stem cell factor and angiopoietin-1 in mesenchymal stem cells even in non-contact conditions, and this abnormality was reverted by extracellular vesicle inhibition. Importantly, the transfer of myeloid neoplasm-derived extracellular vesicles reduced the hematopoietic supportive capacity of mesenchymal stem cells. Analysis of extracellular vesicle microRNA indicated that several species, including miR-7977 from acute myeloid leukemia cells, were higher than those from normal CD34+ cells. Remarkably, the copy number of miR-7977 in bone marrow interstitial fluid was elevated not only in acute myeloid leukemia, but also in myelodysplastic syndrome, as compared with lymphoma without bone marrow localization. The transfection of the miR-7977 mimic reduced the expression of the posttranscriptional regulator, poly(rC) binding protein 1, in mesenchymal stem cells. Moreover, the miR-7977 mimic induced aberrant reduction of hematopoietic growth factors in mesenchymal stem cells, resulting in decreased hematopoietic-supporting capacity of bone marrow CD34+ cells. Furthermore, the reduction of hematopoietic growth factors including Jagged-1, stem cell factor and angiopoietin-1 were reverted by target protection of poly(rC) binding protein 1, suggesting that poly(rC) binding protein 1 could be involved in the stabilization of several growth factors. Thus, miR-7977 in extracellular vesicles may be a critical factor that induces failure of normal hematopoiesis via poly(rC) binding protein 1 suppression. PMID:26802051

  4. Mechanisms that uncouple growth and differentiation in myeloid leukemia cells: restoration of requirement for normal growth-inducing protein without restoring induction of differentiation-inducing protein.

    PubMed Central

    Lotem, J; Sachs, L

    1982-01-01

    There are different macrophage- and granulocyte-inducing (MGI) proteins. Normal myeloid precursors are induced to multiply by one form (MGI-1) and to differentiate by another form (MGI-2). There are clones of myeloid leukemia cells that no longer require MGI-1 for growth but can still be induced to differentiate by MGI-2. After induction of differentiation in these leukemia cells by adding MCI-2 or inducing endogenous production of MGI-2 by lipopolysaccharide, the differentiating leukemia cells, like normal cells, again required MGI-1 for growth. This growth requirement for MGI-1 could not be substituted for by adding other protein growth factors such as epidermal, fibroblast, or nerve growth factor or insulin. Induction of differentiation in these leukemia cells by dexamethasone, arabinonucleoside (cytosine arabinoside), or methotrexate instead of by MGI-2, did not restore the requirement of MGI-1 for growth. Mutant myeloid leukemia cells that could not be induced to differentiate by MGI-2 also did not show this restoration of the requirement of MGI-1 for growth. MGI-1 in normal cells induced cell growth and also induced MGI-2, so that the cells could then differentiate by the endogenously produced MGI-2. However, MGI-1 did not induce production of MGI-2 in the leukemia cells, even though they again required MGI-1 for growth, so that there was no induction of differentiation after adding MGI-1. This lack of induction of differentiation-inducing protein by growth-inducing protein has thus identified an effective mechanism for uncoupling of growth and differentiation in malignant cells. PMID:6981812

  5. A unique feature of Toll/IL-1 receptor domain-containing adaptor protein is partially responsible for lipopolysaccharide insensitivity in zebrafish with a highly conserved function of MyD88.

    PubMed

    Liu, Yanhui; Li, Mengzhen; Fan, Shan; Lin, Yiqun; Lin, Bin; Luo, Fang; Zhang, Chenxu; Chen, Shangwu; Li, Yingqiu; Xu, Anlong

    2010-09-15

    MyD88 and Toll/IL-1R domain-containing adaptor protein (TIRAP) are required for the TLR4 response to LPS stimulation in mammals, but the functions of the two adaptors and their involvement in zebrafish insensitivity to LPS remains unknown. We present a functional analysis of zebrafish Myd88 and Tirap and suggest that Myd88 is more important than Tirap for the activation of Tlr-mediated NF-kappaB, which may be a novel mechanism of Myd88-dependent TLR signaling in teleosts. Zebrafish Tirap lacks the phosphatidylinositol 4,5-bisphosphate binding motif required for human TIRAP location and has leucine at position 233 rather than the conserved proline of human TIRAP, as well as 105 additional aa at the N terminus. Overexpression of zebrafish Tirap in HEK293T cells did not activate NF-kappaB and IFN-beta, but slightly activated NF-kappaB in carp leukocyte cells. Zebrafish Myd88 alone strongly induced the activation of NF-kappaB and IFN-beta both in HEK293T and carp leukocyte cells. The function of Myd88 was dependent on its cellular location and the proline in the Toll/IL-1R domain. Although zebrafish Tirap was distributed throughout the cell rather than localized to the cytoplasmic membrane, its impaired ability to activate downstream Tlr molecules was unlikely to be related to its location because chimera TIRAP with a human TIRAP N terminus and membrane-binding domain also did not activate NF-kappaB. However, the mutation of leucine to proline increased the ability of Tirap to activate NF-kappaB. We suggest that the zebrafish Tirap needs a longer N terminus to perform its function and could be partially responsible for the resistance to LPS in zebrafish.

  6. Oridonin induces NPM mutant protein translocation and apoptosis in NPM1c+ acute myeloid leukemia cells in vitro

    PubMed Central

    Li, Fei-fei; Yi, Sha; Wen, Lu; He, Jing; Yang, Li-jing; Zhao, Jie; Zhang, Ben-ping; Cui, Guo-hui; Chen, Yan

    2014-01-01

    Aim: Skewed cytoplasmic accumulation of NPM mutant protein (NPM1c+) is close related to leukemia pathogenesis. The aim of this study was to investigate whether oridonin, a diterpenoid isolated from the Chinese traditional medicine Rabdosia rubescens, was able to interfere with NPM1c+ protein trafficking and induce apoptosis in NPM1c+ acute myeloid leukemia cells in vitro. Methods: OCI-AML3 cell line harboring a NPM1 gene mutation was examined. Cell growth was detected by MTT assay. Cell apoptosis was evaluated using flow cytometry and Hoechst 33258 staining. The expression and subcellular localization of relevant proteins were detected by Western blot and immunofluorescent staining. The mRNA expression was detected by RT-PCR. Results: Oridonin (2–12 μmol/L) dose-dependently inhibited the viability of OCI-AML3 cells (the IC50 value was 3.27±0.23 μmol/L at 24 h). Moreover, oridonin induced OCI-AML3 cell apoptosis accompanied by activation of caspase-3 and nuclear translocation of NPM1c+ protein. Oridonin did not change the expression of Crm1 (the export receptor for nuclear export signal-containing proteins), but induced nuclear translocation of Crm1. Oridonin markedly increased the expression of nucleoporin98 (Nup98), which had an important role in Crm1-mediated nuclear protein export, and induced nuclear accumulation of Nup98. Furthermore, oridonin markedly increased the expression of p14arf and p53. Conclusion: In NPM1c+ leukemia cells, oridonin induces NPM1c+ protein translocation into the nucleus possibly via nuclear accumulation of Crm1; the compound markedly increases p53 and p14arf expression, which may contribute to cell apoptosis. PMID:24902788

  7. Protein signatures as potential surrogate biomarkers for stratification and prediction of treatment response in chronic myeloid leukemia patients

    PubMed Central

    Alaiya, Ayodele A.; Aljurf, Mahmoud; Shinwari, Zakia; Almohareb, Fahad; Malhan, Hafiz; Alzahrani, Hazzaa; Owaidah, Tarek; Fox, Jonathan; Alsharif, Fahad; Mohamed, Said Y.; Rasheed, Walid; Aldawsari, Ghuzayel; Hanbali, Amr; Ahmed, Syed Osman; Chaudhri, Naeem

    2016-01-01

    There is unmet need for prediction of treatment response for chronic myeloid leukemia (CML) patients. The present study aims to identify disease-specific/disease-associated protein biomarkers detectable in bone marrow and peripheral blood for objective prediction of individual’s best treatment options and prognostic monitoring of CML patients. Bone marrow plasma (BMP) and peripheral blood plasma (PBP) samples from newly-diagnosed chronic-phase CML patients were subjected to expression-proteomics using quantitative two-dimensional gel electrophoresis (2-DE) and label-free liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of 2-DE protein fingerprints preceding therapy commencement accurately predicts 13 individuals that achieved major molecular response (MMR) at 6 months from 12 subjects without MMR (No-MMR). Results were independently validated using LC-MS/MS analysis of BMP and PBP from patients that have more than 24 months followed-up. One hundred and sixty-four and 138 proteins with significant differential expression profiles were identified from PBP and BMP, respectively and only 54 proteins overlap between the two datasets. The protein panels also discriminates accurately patients that stay on imatinib treatment from patients ultimately needing alternative treatment. Among the identified proteins are TYRO3, a member of TAM family of receptor tyrosine kinases (RTKs), the S100A8, and MYC and all of which have been implicated in CML. Our findings indicate analyses of a panel of protein signatures is capable of objective prediction of molecular response and therapy choice for CML patients at diagnosis as ‘personalized-medicine-model’. PMID:27573699

  8. Protein signatures as potential surrogate biomarkers for stratification and prediction of treatment response in chronic myeloid leukemia patients.

    PubMed

    Alaiya, Ayodele A; Aljurf, Mahmoud; Shinwari, Zakia; Almohareb, Fahad; Malhan, Hafiz; Alzahrani, Hazzaa; Owaidah, Tarek; Fox, Jonathan; Alsharif, Fahad; Mohamed, Said Y; Rasheed, Walid; Aldawsari, Ghuzayel; Hanbali, Amr; Ahmed, Syed Osman; Chaudhri, Naeem

    2016-09-01

    There is unmet need for prediction of treatment response for chronic myeloid leukemia (CML) patients. The present study aims to identify disease-specific/disease-associated protein biomarkers detectable in bone marrow and peripheral blood for objective prediction of individual's best treatment options and prognostic monitoring of CML patients. Bone marrow plasma (BMP) and peripheral blood plasma (PBP) samples from newly-diagnosed chronic-phase CML patients were subjected to expression-proteomics using quantitative two-dimensional gel electrophoresis (2-DE) and label-free liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of 2-DE protein fingerprints preceding therapy commencement accurately predicts 13 individuals that achieved major molecular response (MMR) at 6 months from 12 subjects without MMR (No-MMR). Results were independently validated using LC-MS/MS analysis of BMP and PBP from patients that have more than 24 months followed-up. One hundred and sixty-four and 138 proteins with significant differential expression profiles were identified from PBP and BMP, respectively and only 54 proteins overlap between the two datasets. The protein panels also discriminates accurately patients that stay on imatinib treatment from patients ultimately needing alternative treatment. Among the identified proteins are TYRO3, a member of TAM family of receptor tyrosine kinases (RTKs), the S100A8, and MYC and all of which have been implicated in CML. Our findings indicate analyses of a panel of protein signatures is capable of objective prediction of molecular response and therapy choice for CML patients at diagnosis as 'personalized-medicine-model'. PMID:27573699

  9. Connexin expression in human acute myeloid leukemia cells: Identification of patient subsets based on protein and global gene expression profiles

    PubMed Central

    REIKVAM, HÅKON; RYNINGEN, ANITA; SÆTERDAL, LARS RUNE; NEPSTAD, INA; FOSS, BRYNJAR; BRUSERUD, ØYSTEIN

    2015-01-01

    Bone marrow stromal cells support both normal and malignant hematopoiesis. Τhis support is mediated through the local cytokine network and by direct cell-cell interactions mediated via adhesion molecules and the formation of gap junctions by connexins. Previous studies on connexins in human acute myeloid leukemia (AML) have mainly focused on the investigation of leukemia cell lines. In the present study, we therefore investigated the expression of various connexins at the protein (i.e., cell surface expression) and mRNA level in primary human AML cells. The cell surface expression of the connexins, Cx26, Cx32, Cx37, Cx43 and Cx45, varied considerably between patients, and detectable levels were observed only for subsets of patients. On the whole, Cx43 and Cx45 showed the highest cell surface expression. Connexin expression was dependent on AML cell differentiation, but showed no association with cytogenetic abnormalities or mutations of the fms-related tyrosine kinase 3 (FLT3) or nucleophosmin (NPM)‑1 genes. By contrast, only Cx45 showed a significant variation between patients at the mRNA level. A high Cx45 expression was associated with the altered regulation of the mitogen-activated protein kinase (MAPK) pathway and the release of pro-inflammatory cytokines [interleukin (IL)-17, tumor necrosis factor (TNF), interferon-γ], whereas a low Cx45 expression was associated with the altered regulation of protein functions (i.e., ligase activity, protein folding and catabolism). There was no significant correlation observed between the connexin mRNA and protein levels. Thus, differences in connexin expression can be used to subclassify AML patients. Differences in connexin cell surface expression profiles are not reflected at the mRNA level and have to be directly examined, whereas variations in Cx45 mRNA expression are associated with differences in cell signaling and the regulation of protein functions. PMID:25529637

  10. Connexin expression in human acute myeloid leukemia cells: identification of patient subsets based on protein and global gene expression profiles.

    PubMed

    Reikvam, Håkon; Ryningen, Anita; Sæterdal, Lars Rune; Nepstad, Ina; Foss, Brynjar; Bruserud, Øystein

    2015-03-01

    Bone marrow stromal cells support both normal and malignant hematopoiesis. Τhis support is mediated through the local cytokine network and by direct cell‑cell interactions mediated via adhesion molecules and the formation of gap junctions by connexins. Previous studies on connexins in human acute myeloid leukemia (AML) have mainly focused on the investigation of leukemia cell lines. In the present study, we therefore investigated the expression of various connexins at the protein (i.e., cell surface expression) and mRNA level in primary human AML cells. The cell surface expression of the connexins, Cx26, Cx32, Cx37, Cx43 and Cx45, varied considerably between patients, and detectable levels were observed only for subsets of patients. On the whole, Cx43 and Cx45 showed the highest cell surface expression. Connexin expression was dependent on AML cell differentiation, but showed no association with cytogenetic abnormalities or mutations of the fms-related tyrosine kinase 3 (FLT3) or nucleophosmin (NPM)‑1 genes. By contrast, only Cx45 showed a significant variation between patients at the mRNA level. A high Cx45 expression was associated with the altered regulation of the mitogen‑activated protein kinase (MAPK) pathway and the release of pro-inflammatory cytokines [interleukin (IL)‑17, tumor necrosis factor (TNF), interferon‑γ], whereas a low Cx45 expression was associated with the altered regulation of protein functions (i.e., ligase activity, protein folding and catabolism). There was no significant correlation observed between the connexin mRNA and protein levels. Thus, differences in connexin expression can be used to subclassify AML patients. Differences in connexin cell surface expression profiles are not reflected at the mRNA level and have to be directly examined, whereas variations in Cx45 mRNA expression are associated with differences in cell signaling and the regulation of protein functions.

  11. Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3).

    PubMed

    Rouka, Evgenia; Simister, Philip C; Janning, Melanie; Kumbrink, Joerg; Konstantinou, Tassos; Muniz, João R C; Joshi, Dhira; O'Reilly, Nicola; Volkmer, Rudolf; Ritter, Brigitte; Knapp, Stefan; von Delft, Frank; Kirsch, Kathrin H; Feller, Stephan M

    2015-10-16

    CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three Src homology 3 (SH3) domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide array screen based on the recognition motif PxPxPR and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine nucleotide exchange factor. CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry data showed that the preferred binding motif is Px(P/A)xPR. Two high-resolution crystal structures (1.65 and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains for c-CBL, ALIX, and RIN3. PMID:26296892

  12. Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3)*

    PubMed Central

    Rouka, Evgenia; Simister, Philip C.; Janning, Melanie; Kumbrink, Joerg; Konstantinou, Tassos; Muniz, João R. C.; Joshi, Dhira; O'Reilly, Nicola; Volkmer, Rudolf; Ritter, Brigitte; Knapp, Stefan; von Delft, Frank; Kirsch, Kathrin H.; Feller, Stephan M.

    2015-01-01

    CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three Src homology 3 (SH3) domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide array screen based on the recognition motif PxPxPR and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine nucleotide exchange factor. CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry data showed that the preferred binding motif is Px(P/A)xPR. Two high-resolution crystal structures (1.65 and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains for c-CBL, ALIX, and RIN3. PMID:26296892

  13. Early myeloid lineage choice is not initiated by random PU.1 to GATA1 protein ratios.

    PubMed

    Hoppe, Philipp S; Schwarzfischer, Michael; Loeffler, Dirk; Kokkaliaris, Konstantinos D; Hilsenbeck, Oliver; Moritz, Nadine; Endele, Max; Filipczyk, Adam; Gambardella, Adriana; Ahmed, Nouraiz; Etzrodt, Martin; Coutu, Daniel L; Rieger, Michael A; Marr, Carsten; Strasser, Michael K; Schauberger, Bernhard; Burtscher, Ingo; Ermakova, Olga; Bürger, Antje; Lickert, Heiko; Nerlov, Claus; Theis, Fabian J; Schroeder, Timm

    2016-07-14

    The mechanisms underlying haematopoietic lineage decisions remain disputed. Lineage-affiliated transcription factors with the capacity for lineage reprogramming, positive auto-regulation and mutual inhibition have been described as being expressed in uncommitted cell populations. This led to the assumption that lineage choice is cell-intrinsically initiated and determined by stochastic switches of randomly fluctuating cross-antagonistic transcription factors. However, this hypothesis was developed on the basis of RNA expression data from snapshot and/or population-averaged analyses. Alternative models of lineage choice therefore cannot be excluded. Here we use novel reporter mouse lines and live imaging for continuous single-cell long-term quantification of the transcription factors GATA1 and PU.1 (also known as SPI1). We analyse individual haematopoietic stem cells throughout differentiation into megakaryocytic-erythroid and granulocytic-monocytic lineages. The observed expression dynamics are incompatible with the assumption that stochastic switching between PU.1 and GATA1 precedes and initiates megakaryocytic-erythroid versus granulocytic-monocytic lineage decision-making. Rather, our findings suggest that these transcription factors are only executing and reinforcing lineage choice once made. These results challenge the current prevailing model of early myeloid lineage choice. PMID:27411635

  14. The matricellular protein CCN3 regulates NOTCH1 signalling in chronic myeloid leukaemia.

    PubMed

    Suresh, Sukanya; McCallum, Lynn; Crawford, Lisa J; Lu, Wan Hua; Sharpe, Daniel J; Irvine, Alexandra E

    2013-11-01

    Deregulated NOTCH1 has been reported in lymphoid leukaemia, although its role in chronic myeloid leukaemia (CML) is not well established. We previously reported BCR-ABL down-regulation of a novel haematopoietic regulator, CCN3, in CML; CCN3 is a non-canonical NOTCH1 ligand. This study characterizes the NOTCH1–CCN3 signalling axis in CML. In K562 cells, BCR-ABL silencing reduced full-length NOTCH1 (NOTCH1-FL) and inhibited the cleavage of NOTCH1 intracellular domain (NOTCH1-ICD), resulting in decreased expression of the NOTCH1 targets c-MYC and HES1. K562 cells stably overexpressing CCN3 (K562/CCN3) or treated with recombinant CCN3(rCCN3) showed a significant reduction in NOTCH1 signalling (> 50% reduction in NOTCH1-ICD, p < 0.05).Gamma secretase inhibitor (GSI), which blocks NOTCH1 signalling, reduced K562/CCN3 colony formation but increased that of K562/control cells. GSI combined with either rCCN3 or imatinib reduced K562 colony formation with enhanced reduction of NOTCH1 signalling observed with combination treatments. We demonstrate an oncogenic role for NOTCH1 in CML and suggest that BCR-ABL disruption of NOTCH1–CCN3 signalling contributes to the pathogenesis of CML.

  15. The RA domain of Ste50 adaptor protein is required for delivery of Ste11 to the plasma membrane in the filamentous growth signaling pathway of the yeast Saccharomyces cerevisiae.

    PubMed

    Truckses, Dagmar M; Bloomekatz, Joshua E; Thorner, Jeremy

    2006-02-01

    In Saccharomyces cerevisiae, pheromone response requires Ste5 scaffold protein, which ensures efficient G-protein-dependent recruitment of mitogen-activated protein kinase (MAPK) cascade components Ste11 (MAPK kinase kinase), Ste7 (MAPK kinase), and Fus3 (MAPK) to the plasma membrane for activation by Ste20 protein kinase. Ste20, which phosphorylates Ste11 to initiate signaling, is activated by binding to Cdc42 GTPase (membrane anchored via its C-terminal geranylgeranylation). Less clear is how activated and membrane-localized Ste20 contacts Ste11 to trigger invasive growth signaling, which also requires Ste7 and the MAPK Kss1, but not Ste5. Ste50 protein associates constitutively via an N-terminal sterile-alpha motif domain with Ste11, and this interaction is required for optimal invasive growth and hyperosmotic stress (high-osmolarity glycerol [HOG]) signaling but has a lesser role in pheromone response. We show that a conserved C-terminal, so-called "Ras association" (RA) domain in Ste50 is also essential for invasive growth and HOG signaling in vivo. In vitro the Ste50 RA domain is not able to associate with Ras2, but it does associate with Cdc42 and binds to a different face than does Ste20. RA domain function can be replaced by the nine C-terminal, plasma membrane-targeting residues (KKSKKCAIL) of Cdc42, and membrane-targeted Ste50 also suppresses the signaling deficiency of cdc42 alleles specifically defective in invasive growth. Thus, Ste50 serves as an adaptor to tether Ste11 to the plasma membrane and can do so via association with Cdc42, thereby permitting the encounter of Ste11 with activated Ste20. PMID:16428446

  16. Timing of the loss of Pten protein determines disease severity in a mouse model of myeloid malignancy

    PubMed Central

    Yan, Yan; Webster, Cody; Shao, Lijian; Lensing, Shelly Y.; Ni, Hongyu; Feng, Wei; Colorado, Natalia; Pathak, Rupak; Xiang, Zhifu; Hauer-Jensen, Martin; Li, Shaoguang; Zhou, Daohong; Emanuel, Peter D.

    2016-01-01

    Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric mixed myelodysplastic/myeloproliferative neoplasm (MDS/MPN). JMML leukemogenesis is linked to a hyperactivated RAS pathway, with driver mutations in the KRAS, NRAS, NF1, PTPN11, or CBL genes. Previous murine models demonstrated how those genes contributed to the selective hypersensitivity of JMML cells to granulocyte macrophage–colony-stimulating factor (GM-CSF), a unifying characteristic in the disease. However, it is unclear what causes the early death in children with JMML, because transformation to acute leukemia is rare. Here, we demonstrate that loss of Pten (phosphatase and tensin homolog) protein at postnatal day 8 in mice harboring Nf1 haploinsufficiency results in an aggressive MPN with death at a murine prepubertal age of 20 to 35 days (equivalent to an early juvenile age in JMML patients). The death in the mice was due to organ infiltration with monocytes/macrophages. There were elevated activities of protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) in cells at physiological concentrations of GM-CSF. These were more pronounced in mice with Nf1 haploinsufficiency than in littermates with wild-type Nf1, but this model is insufficient to cause cells to be GM-CSF hypersensitive. This new model represents a murine MPN model with features of a pediatric unclassifiable mixed MDS/MPN and mimics many clinical manifestations of JMML in terms of age of onset, aggressiveness, and organ infiltration with monocytes/macrophages. Our data suggest that the timing of the loss of PTEN protein plays a critical role in determining the disease severity in myeloid malignancies. This model may be useful for studying the pathogenesis of pediatric diseases with alterations in the Ras pathway. PMID:26764354

  17. A phosphatase activity present in peripheral blood myeloid cells of chronic myelogenous leukemia patients but not normal individuals alters nuclear protein binding to transcriptional enhancers of interferon-inducible genes.

    PubMed Central

    Seong, D C; Sims, S; Johnson, E; Howard, O M; Reiter, B; Hester, J; Talpaz, M; Kantarjian, H; Deisseroth, A

    1990-01-01

    Cytoplasmic protein from peripheral blood myeloid cells of chronic myelogenous leukemia (CML) patients altered the electrophoretic mobility of complexes formed between nuclear proteins and interferon-inducible transcriptional enhancers. Immature myeloid marrow cells (blasts and promyelocytes) have a higher level of this activity than do mature myeloid marrow cells (bands and polys). This activity, which is not detectable in the peripheral blood cells of normal individuals, is at least 50-fold higher in CML marrow blasts and promyelocytes than that found in marrow blasts and promyelocytes of normal individuals. This activity was inhibited by in vivo incubation of immature myeloid cells with the phosphatase inhibitor, sodium orthovanadate (0.2 mM), and by adding orthovanadate (20 mM) directly to cytoplasmic proteins of myeloid cells. Interferon-alpha (1,000 U/ml) reduced the effects of the CML myeloid cell cytoplasmic protein on the electrophoretic mobility of nuclear protein-DNA complexes. These data suggest that a unique phosphatase may be involved in the abnormalities in CML which are modulated by interferon-alpha. Images PMID:2243138

  18. Inhibition of growth by p205: a nuclear protein and putative tumor suppressor expressed during myeloid cell differentiation.

    PubMed

    Dermott, Jonathan M; Gooya, John M; Asefa, Benyam; Weiler, Sarah R; Smith, Mark; Keller, Jonathan R

    2004-01-01

    p205 belongs to a family of interferon-inducible proteins called the IFI-200 family, which have been implicated in the regulation of cell growth and differentiation. While p205 is induced in hematopoietic stem cells during myeloid cell differentiation, its function is not known. Therefore, the aim of this study was to determine the role of p205 in regulating proliferation in hematopoietic progenitor cells and in nonhematopoietic cell lines. We found that p205 localizes to the nucleus in hematopoietic and nonhematopoietic cell lines. Transient expression of p205 in murine IL-3-dependent BaF3 and 32D-C123 progenitor cell lines inhibited IL-3-induced growth and proliferation. The closely related IFI-200 family members, p204 and p202, similarly inhibited IL-3-dependent progenitor cell proliferation. p205 also inhibited the proliferation and growth of normal hematopoietic progenitor cells. In nonhematopoietic cell lines, p205 and p204 expression inhibited NIH3T3 cell colony formation in vitro, and microinjection of p205 expression vectors into NIH3T3 fibroblasts inhibited serum-induced proliferation. We have determined the functional domains of p205 necessary for activity, which were identified as the N-terminal domain in apoptosis and interferon response (DAPIN)/PYRIN domain, and the C-terminal retinoblastoma protein (Rb)-binding motif. In addition, we have demonstrated that a putative ataxia telangiectasia, mutated (ATM) kinase phosphorylation site specifically regulates the activity of p205. Taken together, these data suggest that p205 is a potent cell growth regulator whose activity is mediated by its protein-binding domains. We propose that during myelomonocytic cell differentiation, induction of p205 expression contributes to cell growth arrest, thus allowing progenitor cells to differentiate. PMID:15342947

  19. Determination of the binding mode for anti-inflammatory natural product xanthohumol with myeloid differentiation protein 2.

    PubMed

    Fu, Weitao; Chen, Lingfeng; Wang, Zhe; Zhao, Chengwei; Chen, Gaozhi; Liu, Xing; Dai, Yuanrong; Cai, Yuepiao; Li, Chenglong; Zhou, Jianmin; Liang, Guang

    2016-01-01

    It is recognized that myeloid differentiation protein 2 (MD-2), a coreceptor of toll-like receptor 4 (TLR4) for innate immunity, plays an essential role in activation of the lipopolysaccharide signaling pathway. MD-2 is known as a neoteric and suitable therapeutical target. Therefore, there is great interest in the development of a potent MD-2 inhibitor for anti-inflammatory therapeutics. Several studies have reported that xanthohumol (XN), an anti-inflammatory natural product from hops and beer, can block the TLR4 signaling by binding to MD-2 directly. However, the interaction between MD-2 and XN remains unknown. Herein, our work aims at characterizing interactions between MD-2 and XN. Using a combination of experimental and theoretical modeling analysis, we found that XN can embed into the hydrophobic pocket of MD-2 and form two stable hydrogen bonds with residues ARG-90 and TYR-102 of MD-2. Moreover, we confirmed that ARG-90 and TYR-102 were two necessary residues during the recognition process of XN binding to MD-2. Results from this study identified the atomic interactions between the MD-2 and XN, which will contribute to future structural design of novel MD-2-targeting molecules for the treatment of inflammatory diseases.

  20. Determination of the binding mode for anti-inflammatory natural product xanthohumol with myeloid differentiation protein 2.

    PubMed

    Fu, Weitao; Chen, Lingfeng; Wang, Zhe; Zhao, Chengwei; Chen, Gaozhi; Liu, Xing; Dai, Yuanrong; Cai, Yuepiao; Li, Chenglong; Zhou, Jianmin; Liang, Guang

    2016-01-01

    It is recognized that myeloid differentiation protein 2 (MD-2), a coreceptor of toll-like receptor 4 (TLR4) for innate immunity, plays an essential role in activation of the lipopolysaccharide signaling pathway. MD-2 is known as a neoteric and suitable therapeutical target. Therefore, there is great interest in the development of a potent MD-2 inhibitor for anti-inflammatory therapeutics. Several studies have reported that xanthohumol (XN), an anti-inflammatory natural product from hops and beer, can block the TLR4 signaling by binding to MD-2 directly. However, the interaction between MD-2 and XN remains unknown. Herein, our work aims at characterizing interactions between MD-2 and XN. Using a combination of experimental and theoretical modeling analysis, we found that XN can embed into the hydrophobic pocket of MD-2 and form two stable hydrogen bonds with residues ARG-90 and TYR-102 of MD-2. Moreover, we confirmed that ARG-90 and TYR-102 were two necessary residues during the recognition process of XN binding to MD-2. Results from this study identified the atomic interactions between the MD-2 and XN, which will contribute to future structural design of novel MD-2-targeting molecules for the treatment of inflammatory diseases. PMID:26869767

  1. Multidrug resistance protein 4/ ATP binding cassette transporter 4: a new potential therapeutic target for acute myeloid leukemia

    PubMed Central

    Copsel, Sabrina; Bruzzone, Ariana; May, Maria; Beyrath, Julien; Wargon, Victoria; Cany, Jeannette; Frans, G.M. Russel; Shayo, Carina; Davio, Carlos

    2014-01-01

    Less than a third of adults patients with acute myeloid leukemia (AML) are cured by current treatments, emphasizing the need for new approaches to therapy. We previously demonstrated that besides playing a role in drug-resistant leukemia cell lines, multidrug resistance protein 4 (MRP4/ABCC4) regulates leukemia cell proliferation and differentiation through the endogenous MRP4/ABCC4 substrate, cAMP. Here, we studied the role of MRP4/ABCC4 in tumor progression in a mouse xenograft model and in leukemic stem cells (LSCs) differentiation. We found a decrease in the mitotic index and an increase in the apoptotic index associated with the inhibition of tumor growth when mice were treated with rolipram (PDE4 inhibitor) and/or probenecid (MRPs inhibitor). Genetic silencing and pharmacologic inhibition of MRP4 reduced tumor growth. Furthermore, MRP4 knockdown induced cell cycle arrest and apoptosis in vivo. Interestingly, when LSC population was isolated, we observed that increased cAMP levels and MRP4/ABCC4 blockade resulted in LSCs differentiation. Taken together, our findings show that MRP4/ABCC4 has a relevant role in tumor growth and apoptosis and in the eradication of LSCs, providing the basis for a novel promising target in AML therapy. PMID:25301721

  2. The microRNA mir-71 inhibits calcium signaling by targeting the TIR-1/Sarm1 adaptor protein to control stochastic L/R neuronal asymmetry in C. elegans.

    PubMed

    Hsieh, Yi-Wen; Chang, Chieh; Chuang, Chiou-Fen

    2012-01-01

    The Caenorhabditis elegans left and right AWC olfactory neurons communicate to establish stochastic asymmetric identities, AWC(ON) and AWC(OFF), by inhibiting a calcium-mediated signaling pathway in the future AWC(ON) cell. NSY-4/claudin-like protein and NSY-5/innexin gap junction protein are the two parallel signals that antagonize the calcium signaling pathway to induce the AWC(ON) fate. However, it is not known how the calcium signaling pathway is downregulated by nsy-4 and nsy-5 in the AWC(ON) cell. Here we identify a microRNA, mir-71, that represses the TIR-1/Sarm1 adaptor protein in the calcium signaling pathway to promote the AWC(ON) identity. Similar to tir-1 loss-of-function mutants, overexpression of mir-71 generates two AWC(ON) neurons. tir-1 expression is downregulated through its 3' UTR in AWC(ON), in which mir-71 is expressed at a higher level than in AWC(OFF). In addition, mir-71 is sufficient to inhibit tir-1 expression in AWC through the mir-71 complementary site in the tir-1 3' UTR. Our genetic studies suggest that mir-71 acts downstream of nsy-4 and nsy-5 to promote the AWC(ON) identity in a cell autonomous manner. Furthermore, the stability of mature mir-71 is dependent on nsy-4 and nsy-5. Together, these results provide insight into the mechanism by which nsy-4 and nsy-5 inhibit calcium signaling to establish stochastic asymmetric AWC differentiation.

  3. Absence of the Adaptor Protein PEA-15 Is Associated with Altered Pattern of Th Cytokines Production by Activated CD4+ T Lymphocytes In Vitro, and Defective Red Blood Cell Alloimmune Response In Vivo

    PubMed Central

    Kerbrat, Stéphane; Vingert, Benoit; Junier, Marie-Pierre; Castellano, Flavia; Renault-Mihara, François; Dos Reis Tavares, Silvina; Surenaud, Mathieu; Noizat-Pirenne, France; Boczkowski, Jorge; Guellaën, Georges; Chneiweiss, Hervé; Le Gouvello, Sabine

    2015-01-01

    TCR-dependent and costimulation signaling, cell division, and cytokine environment are major factors driving cytokines expression induced by CD4+ T cell activation. PEA-15 15 (Protein Enriched in Astrocyte / 15kDa) is an adaptor protein that regulates death receptor-induced apoptosis and proliferation signaling by binding to FADD and relocating ERK1/2 to the cytosol, respectively. By using PEA-15-deficient mice, we examined the role of PEA-15 in TCR-dependent cytokine production in CD4+ T cells. TCR-stimulated PEA-15-deficient CD4+ T cells exhibited defective progression through the cell cycle associated with impaired expression of cyclin E and phosphoRb, two ERK1/2-dependent proteins of the cell cycle. Accordingly, expression of the division cycle-dependent cytokines IL-2 and IFNγ, a Th1 cytokine, was reduced in stimulated PEA-15-deficient CD4+ T cells. This was associated with abnormal subcellular compartmentalization of activated ERK1/2 in PEA-15-deficient T cells. Furthermore, in vitro TCR-dependent differentiation of naive CD4+ CD62L+ PEA-15-deficient T cells was associated with a lower production of the Th2 cytokine, IL-4, whereas expression of the Th17-associated molecule IL4I1 was enhanced. Finally, a defective humoral response was shown in PEA-15-deficient mice in a model of red blood cell alloimmunization performed with Poly IC, a classical adjuvant of Th1 response in vivo. Collectively, our data suggest that PEA-15 contributes to the specification of the cytokine pattern of activated Th cells, thus highlighting a potential new target to interfere with T cell functional polarization and subsequent immune response. PMID:26317969

  4. Absence of the Adaptor Protein PEA-15 Is Associated with Altered Pattern of Th Cytokines Production by Activated CD4+ T Lymphocytes In Vitro, and Defective Red Blood Cell Alloimmune Response In Vivo.

    PubMed

    Kerbrat, Stéphane; Vingert, Benoit; Junier, Marie-Pierre; Castellano, Flavia; Renault-Mihara, François; Dos Reis Tavares, Silvina; Surenaud, Mathieu; Noizat-Pirenne, France; Boczkowski, Jorge; Guellaën, Georges; Chneiweiss, Hervé; Le Gouvello, Sabine

    2015-01-01

    TCR-dependent and costimulation signaling, cell division, and cytokine environment are major factors driving cytokines expression induced by CD4(+) T cell activation. PEA-15 15 (Protein Enriched in Astrocyte / 15 kDa) is an adaptor protein that regulates death receptor-induced apoptosis and proliferation signaling by binding to FADD and relocating ERK1/2 to the cytosol, respectively. By using PEA-15-deficient mice, we examined the role of PEA-15 in TCR-dependent cytokine production in CD4(+) T cells. TCR-stimulated PEA-15-deficient CD4(+) T cells exhibited defective progression through the cell cycle associated with impaired expression of cyclin E and phosphoRb, two ERK1/2-dependent proteins of the cell cycle. Accordingly, expression of the division cycle-dependent cytokines IL-2 and IFNγ, a Th1 cytokine, was reduced in stimulated PEA-15-deficient CD4(+) T cells. This was associated with abnormal subcellular compartmentalization of activated ERK1/2 in PEA-15-deficient T cells. Furthermore, in vitro TCR-dependent differentiation of naive CD4(+) CD62L(+) PEA-15-deficient T cells was associated with a lower production of the Th2 cytokine, IL-4, whereas expression of the Th17-associated molecule IL4I1 was enhanced. Finally, a defective humoral response was shown in PEA-15-deficient mice in a model of red blood cell alloimmunization performed with Poly IC, a classical adjuvant of Th1 response in vivo. Collectively, our data suggest that PEA-15 contributes to the specification of the cytokine pattern of activated Th cells, thus highlighting a potential new target to interfere with T cell functional polarization and subsequent immune response. PMID:26317969

  5. The cellular RNA export receptor TAP/NXF1 is required for ICP27-mediated export of herpes simplex virus 1 RNA, but the TREX complex adaptor protein Aly/REF appears to be dispensable.

    PubMed

    Johnson, Lisa A; Li, Ling; Sandri-Goldin, Rozanne M

    2009-07-01

    Herpes simplex virus 1 (HSV-1) protein ICP27 has been shown to shuttle between the nucleus and cytoplasm and to bind viral RNA during infection. ICP27 was found to interact with the cellular RNA export adaptor protein Aly/REF, which is part of the TREX complex, and to relocalize Aly/REF to viral replication sites. ICP27 is exported to the cytoplasm through the export receptor TAP/NXF1, and ICP27 must be able to interact with TAP/NXF1 for efficient export of HSV-1 early and late transcripts. We examined the dynamics of ICP27 movement and its localization with respect to Aly/REF and TAP/NXF1 in living cells during viral infection. Recombinant viruses with a yellow fluorescent protein (YFP) tag on the N or C terminus of ICP27 were constructed. While the N-terminally tagged ICP27 virus behaved like wild-type HSV-1, the C-terminally tagged virus was defective in viral replication and gene expression, and ICP27 was confined to the nucleus, suggesting that the C-terminal YFP tag interfered with ICP27's C-terminal interactions, including the interaction with TAP/NXF1. To assess the role of Aly/REF and TAP/NXF1 in viral RNA export, these factors were knocked down using small interfering RNA. Knockdown of Aly/REF had little effect on the export of ICP27 or poly(A)(+) RNA during infection. In contrast, a decrease in TAP/NXF1 levels severely impaired export of ICP27 and poly(A)(+) RNA. We conclude that TAP/NXF1 is essential for ICP27-mediated export of RNA during HSV-1 infection, whereas Aly/REF may be dispensable.

  6. NPM-RAR, not the RAR-NPM reciprocal t(5;17)(q35;q21) acute promyelocytic leukemia fusion protein, inhibits myeloid differentiation.

    PubMed

    Pollock, Sheri L; Rush, Elizabeth A; Redner, Robert L

    2014-06-01

    The t(5;17) variant of acute promyelocytic leukemia (APL) fuses the nucleophosmin (NPM) gene at 5q35 with the retinoic acid receptor alpha (RARA) at 17q12-22. We have previously shown that leukemic cells express both NPM-RAR and RAR- NPM reciprocal translocation products. In this study we investigated the potential role of both proteins in modulating myeloid differentiation. Expression of NPM-RAR inhibited vitamin D3/transforming growth factor β (TGFβ)-mediated differentiation of U937 cells by more than 50%. In contrast, RAR-NPM expression did not alter vitamin D3/TGFβ-induced differentiation of U937 clones. These results indicate that NPM-RAR, not RAR-NPM, is the prime mediator of myeloid differentiation arrest in t(5;17) APL.

  7. The adaptor molecule signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) is essential in mechanisms involving the Fyn tyrosine kinase for induction and progression of collagen-induced arthritis.

    PubMed

    Zhong, Ming-Chao; Veillette, André

    2013-11-01

    Signaling lymphocytic activation molecule-associated protein (SAP) is an Src homology 2 domain-only adaptor involved in multiple immune cell functions. It has also been linked to immunodeficiencies and autoimmune diseases, such as systemic lupus erythematosus. Here, we examined the role and mechanism of action of SAP in autoimmunity using a mouse model of autoimmune arthritis, collagen-induced arthritis (CIA). We found that SAP was essential for development of CIA in response to collagen immunization. It was also required for production of collagen-specific antibodies, which play a key role in disease pathogenesis. These effects required SAP expression in T cells, not in B cells. In mice immunized with a high dose of collagen, the activity of SAP was nearly independent of its ability to bind the protein tyrosine kinase Fyn and correlated with the capacity of SAP to promote full differentiation of follicular T helper (TFH) cells. However, with a lower dose of collagen, the role of SAP was more dependent on Fyn binding, suggesting that additional mechanisms other than TFH cell differentiation were involved. Further studies suggested that this might be due to a role of the SAP-Fyn interaction in natural killer T cell development through the ability of SAP-Fyn to promote Vav-1 activation. We also found that removal of SAP expression during progression of CIA attenuated disease severity. However, it had no effect on disease when CIA was clinically established. Together, these results indicate that SAP plays an essential role in CIA because of Fyn-independent and Fyn-dependent effects on TFH cells and, possibly, other T cell types.

  8. Vitamin E (E) supplementation reverses the age associated decline in phosphorylation of the adaptor protein LAT in CD4+ T cells of old mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    T cell proliferation and interleukin (IL-2) production declines with age. Engagement of the T cell receptor (TCR) by antigen (Ag), known as the immune synapse (IS), in coordination with phosphorylation of key signaling proteins, leads to increased IL-2 synthesis and T cell proliferation. Defects in ...

  9. Clathrin binding by the adaptor Ent5 promotes late stages of clathrin coat maturation

    PubMed Central

    Hung, Chao-Wei; Duncan, Mara C.

    2016-01-01

    Clathrin is a ubiquitous protein that mediates membrane traffic at many locations. To function, clathrin requires clathrin adaptors that link it to transmembrane protein cargo. In addition to this cargo selection function, many adaptors also play mechanistic roles in the formation of the transport carrier. However, the full spectrum of these mechanistic roles is poorly understood. Here we report that Ent5, an endosomal clathrin adaptor in Saccharomyces cerevisiae, regulates the behavior of clathrin coats after the recruitment of clathrin. We show that loss of Ent5 disrupts clathrin-dependent traffic and prolongs the lifespan of endosomal structures that contain clathrin and other adaptors, suggesting a defect in coat maturation at a late stage. We find that the direct binding of Ent5 with clathrin is required for its role in coat behavior and cargo traffic. Surprisingly, the interaction of Ent5 with other adaptors is dispensable for coat behavior but not cargo traffic. These findings support a model in which Ent5 clathrin binding performs a mechanistic role in coat maturation, whereas Ent5 adaptor binding promotes cargo incorporation. PMID:26842894

  10. Myeloid Differentiation Protein-2–Dependent and –Independent Neutrophil Accumulation during Escherichia coli Pneumonia

    PubMed Central

    Cai, Shanshan; Zemans, Rachel L.; Young, Scott K.; Worthen, G. Scott; Jeyaseelan, Samithamby

    2009-01-01

    Bacterial pneumonia remains a serious disease. Pattern recognition receptors play an integral role in neutrophil accumulation during pneumonia. Although myeloid differentiation protein (MD)-2 has been recognized as a key molecule for LPS signaling, the role of MD-2 in neutrophil accumulation in the lung during bacterial infection has not been explored. Here, we investigate the role of MD-2 in Escherichia coli LPS–induced lung inflammation and E. coli–induced pneumonia. LPS-induced CD14-independent neutrophil accumulation was abolished in CD14/MD-2−/− mice. MD-2−/− mice challenged with LPS displayed attenuated neutrophil influx, NF-κB activation, cytokine/chemokine expression, and lung histopathology. MD-2−/− mice transplanted with MD-2+/+ bone marrow demonstrated decreased neutrophil influx and cytokine/chemokine expression in the lungs when challenged by LPS. MD-2−/− mice infected with E. coli demonstrated reduced neutrophil influx and cytokine/chemokine expression in the lungs, whereas heat-killed E. coli did not induce either neutrophil accumulation or cytokine/chemokine expression in MD-2−/− mice infected with E. coli. Furthermore, MD-2−/− mice displayed increased bacterial burden in the lungs and enhanced bacterial dissemination. Toll-like receptor (TLR)-5−/− mice infected with E. coli exhibited attenuated neutrophil accumulation, whereas MD-2/TLR5−/− mice inoculated with E. coli showed further attenuated neutrophil influx and impaired bacterial clearance. Taken together, these new findings demonstrate: (1) the important role of MD-2 in the CD14-independent LPS-mediated cascade of neutrophil influx; (2) the relative importance of bone marrow– and non–bone marrow cell–derived MD-2 in LPS-induced inflammation; and (3) the essential role of MD-2–dependent and MD-2–independent (TLR5) signaling in E. coli–induced neutrophil accumulation and pulmonary host defense. PMID:18988922

  11. Isolation and killing of candidate chronic myeloid leukemia stem cells by antibody targeting of IL-1 receptor accessory protein.

    PubMed

    Järås, Marcus; Johnels, Petra; Hansen, Nils; Agerstam, Helena; Tsapogas, Panagiotis; Rissler, Marianne; Lassen, Carin; Olofsson, Tor; Bjerrum, Ole Weis; Richter, Johan; Fioretos, Thoas

    2010-09-14

    Chronic myeloid leukemia (CML) is genetically characterized by the Philadelphia (Ph) chromosome, formed through a reciprocal translocation between chromosomes 9 and 22 and giving rise to the constitutively active tyrosine kinase P210 BCR/ABL1. Therapeutic strategies aiming for a cure of CML will require full eradication of Ph chromosome-positive (Ph(+)) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34(+) cells and also in cord blood CD34(+) cells as a consequence of retroviral BCR/ABL1 expression. To test whether IL1RAP expression distinguishes normal (Ph(-)) and leukemic (Ph(+)) cells within the CML CD34(+)CD38(-) cell compartment, we established a unique protocol for conducting FISH on small numbers of sorted cells. By using this method, we sorted cells directly into drops on slides to investigate their Ph-chromosome status. Interestingly, we found that the CML CD34(+)CD38(-)IL1RAP(+) cells were Ph(+), whereas CML CD34(+)CD38(-)IL1RAP(-) cells were almost exclusively Ph(-). By performing long-term culture-initiating cell assays on the two cell populations, we found that Ph(+) and Ph(-) candidate CML stem cells could be prospectively separated. In addition, by generating an anti-IL1RAP antibody, we provide proof of concept that IL1RAP can be used as a target on CML CD34(+)CD38(-) cells to induce antibody-dependent cell-mediated cytotoxicity. This study thus identifies IL1RAP as a unique cell surface biomarker distinguishing Ph(+) from Ph(-) candidate CML stem cells and opens up a previously unexplored avenue for therapy of CML. PMID:20805474

  12. Phosphatidylinositol 4-phosphate 5-kinase α facilitates Toll-like receptor 4-mediated microglial inflammation through regulation of the Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) location.

    PubMed

    Nguyen, Tu Thi Ngoc; Kim, Yong Min; Kim, T Doohun; Le, Oanh Thi Tu; Kim, Jae Jin; Kang, Ho Chul; Hasegawa, Hiroshi; Kanaho, Yasunori; Jou, Ilo; Lee, Sang Yoon

    2013-02-22

    Phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)), generated by PI 4-phosphate 5-kinase (PIP5K), regulates many critical cellular events. PIP(2) is also known to mediate plasma membrane localization of the Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), required for the MyD88-dependent Toll-like receptor (TLR) 4 signaling pathway. Microglia are the primary immune competent cells in brain tissue, and TLR4 is important for microglial activation. However, a functional role for PIP5K and PIP(2) in TLR4-dependent microglial activation remains unclear. Here, we knocked down PIP5Kα, a PIP5K isoform, in a BV2 microglial cell line using stable expression of lentiviral shRNA constructs or siRNA transfection. PIP5Kα knockdown significantly suppressed induction of inflammatory mediators, including IL-6, IL-1β, and nitric oxide, by lipopolysaccharide. PIP5Kα knockdown also attenuated signaling events downstream of TLR4 activation, including p38 MAPK and JNK phosphorylation, NF-κB p65 nuclear translocation, and IκB-α degradation. Complementation of the PIP5Kα knockdown cells with wild type but not kinase-dead PIP5Kα effectively restored the LPS-mediated inflammatory response. We found that PIP5Kα and TIRAP colocalized at the cell surface and interacted with each other, whereas kinase-dead PIP5Kα rendered TIRAP soluble. Furthermore, in LPS-stimulated control cells, plasma membrane PIP(2) increased and subsequently declined, and TIRAP underwent bi-directional translocation between the membrane and cytosol, which temporally correlated with the changes in PIP(2). In contrast, PIP5Kα knockdown that reduced PIP(2) levels disrupted TIRAP membrane targeting by LPS. Together, our results suggest that PIP5Kα promotes TLR4-associated microglial inflammation by mediating PIP(2)-dependent recruitment of TIRAP to the plasma membrane.

  13. Differential Association of the Na+/H+ Exchanger Regulatory Factor (NHERF) Family of Adaptor Proteins with the Raft-and the Non-Raft Brush Border Membrane Fractions of NHE3

    PubMed Central

    Sultan, Ayesha; Luo, Min; Yu, Qin; Riederer, Brigitte; Xia, Weiliang; Chen, Mingmin; Lissner, Simone; Gessner, Johannes E.; Donowitz, Mark; Yun, C. Chris; deJonge, Hugo; Lamprecht, Georg; Seidler, Ursula

    2014-01-01

    Background/Aims Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Methods Detergent resistant membranes (“lipid rafts”) were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3− mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs. PMID:24297041

  14. Monocyte chemoattractant protein 1 (MCP-1/CCL2) contributes to thymus atrophy in acute myeloid leukemia.

    PubMed

    Driss, Virginie; Quesnel, Bruno; Brinster, Carine

    2015-02-01

    Recent studies on acute myelogenous leukemia (AML) patients have revealed the existence of T-cell immunodeficiencies, characterized by peripheral T lymphocytes that are unable to interact with blasts, reduced thymic emigrants and oligoclonal restricted repertoires. These observations suggest that there is a profound thymic dysregulation, which is difficult to study in AML patients. Using the C1498 AML mouse model, we demonstrated that leukemia development was associated with thymus atrophy, which was defined by abnormal organ weight and reduced cellularity. In addition, we observed a dramatic loss of peripheral CD4(+) and CD8(+) T-cell numbers with increased frequencies of CD4(+) FoxP3(+) regulatory and activated/memory T cells. Investigating the mechanisms leading to this atrophy, we observed a significant accumulation of the monocyte chemoattractant protein 1 (MCP-1/CCL2) in thymi of leukemic mice. Treatment of AML-bearing animals with a blocking anti-CCL2 antibody revealed a lower tumor burden, augmented antileukemic T-cell responses, and improved survival rate compared to nontreated mice. These results were not observed when neutralization of CCL2 was performed in thymectomized mice. Altogether, we show that the CCL2 protein participates in thymic atrophy in AML mice, and this could have important implications for future immunotherapeutic strategies.

  15. Palmitic acid increases pro-oxidant adaptor protein p66Shc expression and affects vascularization factors in angiogenic mononuclear cells: Action of resveratrol.

    PubMed

    Favre, Julie; Yildirim, Cansu; Leyen, Thomas A; Chen, Weena J Y; van Genugten, Renate E; van Golen, Larissa W; Garcia-Vallejo, Juan-Jesus; Musters, Rene; Baggen, Josefien; Fontijn, Ruud; van der Pouw Kraan, Tineke; Serné, Erik; Koolwijk, Pieter; Diamant, Michaela; Horrevoets, Anton J G

    2015-12-01

    A defect in neo-vascularization process involving circulating angiogenic mononuclear cells (CACs) dysfunction is associated with diabetes. We showed that oxidative stress was elevated in CACs cultured from blood of individuals with metabolic syndrome (MetS) and diabetes. We then assessed the action of palmitic acid (PA), a deregulated and increased NEFA in metabolic disorders, focusing on its oxidant potential. We observed that the phyto-polyphenol resveratrol normalized oxidative stress both in CACs isolated from MetS patients or treated with PA. Resveratrol further decreased the deleterious action of PA on gene expression of vascularization factors (TNFα, VEGF-A, SDF1α, PECAM-1, VEGFR2, Tie2 and CXCR4) and improved CAC motility. Particularly, resveratrol abolished the PA-induced over-expression of the pro-oxidant protein p66Shc. Neither KLF2 nor SIRT1, previously shown in resveratrol and p66Shc action, was directly involved. Silencing p66Shc normalized PA action on VEGF-A and TNFα specifically, without abolishing the PA-induced oxidative stress, which suggests a deleterious role of p66Shc independently of any major modulation of the cellular oxidative status in a high NEFA levels context. Besides showing that resveratrol reverses PA-induced harmful effects on human CAC function, certainly through profound cellular modifications, we establish p66Shc as a major therapeutic target in metabolic disorders, independent from glycemic control. PMID:26254104

  16. The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development.

    PubMed

    Rahman, Kazi; Zhao, Peng; Mandalasi, Msano; van der Wel, Hanke; Wells, Lance; Blader, Ira J; West, Christopher M

    2016-02-26

    Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts. PMID:26719340

  17. The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development.

    PubMed

    Rahman, Kazi; Zhao, Peng; Mandalasi, Msano; van der Wel, Hanke; Wells, Lance; Blader, Ira J; West, Christopher M

    2016-02-26

    Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts.

  18. Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein

    PubMed Central

    Cheng, Pingyan; Corzo, Cesar A.; Luetteke, Noreen; Yu, Bin; Nagaraj, Srinivas; Bui, Marylin M.; Ortiz, Myrna; Nacken, Wolfgang; Sorg, Clemens; Vogl, Thomas; Roth, Johannes; Gabrilovich, Dmitry I.

    2008-01-01

    Accumulation of myeloid-derived suppressor cells (MDSCs) associated with inhibition of dendritic cell (DC) differentiation is one of the major immunological abnormalities in cancer and leads to suppression of antitumor immune responses. The molecular mechanism of this phenomenon remains unclear. We report here that STAT3-inducible up-regulation of the myeloid-related protein S100A9 enhances MDSC production in cancer. Mice lacking this protein mounted potent antitumor immune responses and rejected implanted tumors. This effect was reversed by administration of wild-type MDSCs from tumor-bearing mice to S100A9-null mice. Overexpression of S100A9 in cultured embryonic stem cells or transgenic mice inhibited the differentiation of DCs and macrophages and induced accumulation of MDSCs. This study demonstrates that tumor-induced up-regulation of S100A9 protein is critically important for accumulation of MDSCs and reveals a novel molecular mechanism of immunological abnormalities in cancer. PMID:18809714

  19. Subcellular localization of Bcr, Abl, and Bcr-Abl proteins in normal and leukemic cells and correlation of expression with myeloid differentiation.

    PubMed Central

    Wetzler, M; Talpaz, M; Van Etten, R A; Hirsh-Ginsberg, C; Beran, M; Kurzrock, R

    1993-01-01

    We used specific antisera and immunohistochemical methods to investigate the subcellular localization and expression of Bcr, Abl, and Bcr-Abl proteins in leukemic cell lines and in fresh human leukemic and normal samples at various stages of myeloid differentiation. Earlier studies of the subcellular localization of transfected murine type IV c-Abl protein in fibroblasts have shown that this molecule resides largely in the nucleus, whereas transforming deletion variants are localized exclusively in the cytoplasm. Here, we demonstrate that the murine type IV c-Abl protein is also found in the nucleus when overexpressed in a mouse hematopoietic cell line. However, in both normal and leukemic human hematopoietic cells, c-Abl is discerned predominantly in the cytoplasm, with nuclear staining present, albeit at a lower level. In contrast, normal endogenous Bcr protein, as well as the aberrant p210BCR-ABL and p190BCR-ABL proteins consistently localize to the cytoplasm in both cell lines and fresh cells. The results with p210BCR-ABL were confirmed in a unique Ph1-positive chronic myelogenous leukemia (CML) cell line, KBM5, which lacks the normal chromosome 9 and hence the normal c-Abl product. Because the p210BCR-ABL protein appears cytoplasmic in both chronic phase and blast crisis CML cells, as does the p190BCR-ABL in Ph1-positive acute leukemia, a change in subcellular location of Bcr-Abl proteins between cytoplasm and nucleus cannot explain the different spectrum of leukemias associated with p210 and p190, nor the transition from the chronic to the acute leukemia phenotype seen in CML. Further analysis of fresh CML and normal hematopoietic bone marrow cells reveals that p210BCR-ABL, as well as the normal Bcr and Abl proteins, are expressed primarily in the early stages of myeloid maturation, and that levels of expression are reduced significantly as the cells mature to polymorphonuclear leukocytes. Similarly, a decrease in Bcr and Abl levels occurs in HL-60 cells

  20. Adaptor assembly for coupling turbine blades to rotor disks

    DOEpatents

    Garcia-Crespo, Andres Jose; Delvaux, John McConnell

    2014-09-23

    An adaptor assembly for coupling a blade root of a turbine blade to a root slot of a rotor disk is described. The adaptor assembly includes a turbine blade having a blade root and an adaptor body having an adaptor root. The adaptor body defines a slot having an open end configured to receive the blade root of the turbine blade such that the adaptor root of the adaptor body and the blade root of the turbine blade are adjacent to one another when the blade root of the turbine blade is positioned within the slot. Both the adaptor root of the adaptor body and the blade root of the turbine blade are configured to be received within the root slot of the rotor disk.

  1. Role of the clathrin adaptor PICALM in normal hematopoiesis and polycythemia vera pathophysiology

    PubMed Central

    Ishikawa, Yuichi; Maeda, Manami; Pasham, Mithun; Aguet, Francois; Tacheva-Grigorova, Silvia K.; Masuda, Takeshi; Yi, Hai; Lee, Sung-Uk; Xu, Jian; Teruya-Feldstein, Julie; Ericsson, Maria; Mullally, Ann; Heuser, John; Kirchhausen, Tom; Maeda, Takahiro

    2015-01-01

    Clathrin-dependent endocytosis is an essential cellular process shared by all cell types. Despite this, precisely how endocytosis is regulated in a cell-type-specific manner and how this key pathway functions physiologically or pathophysiologically remain largely unknown. PICALM, which encodes the clathrin adaptor protein PICALM, was originally identified as a component of the CALM/AF10 leukemia oncogene. Here we show, by employing a series of conditional Picalm knockout mice, that PICALM critically regulates transferrin uptake in erythroid cells by functioning as a cell-type-specific regulator of transferrin receptor endocytosis. While transferrin receptor is essential for the development of all hematopoietic lineages, Picalm was dispensable for myeloid and B-lymphoid development. Furthermore, global Picalm inactivation in adult mice did not cause gross defects in mouse fitness, except for anemia and a coat color change. Freeze-etch electron microscopy of primary erythroblasts and live-cell imaging of murine embryonic fibroblasts revealed that Picalm function is required for efficient clathrin coat maturation. We showed that the PICALM PIP2 binding domain is necessary for transferrin receptor endocytosis in erythroblasts and absolutely essential for erythroid development from mouse hematopoietic stem/progenitor cells in an erythroid culture system. We further showed that Picalm deletion entirely abrogated the disease phenotype in a Jak2V617F knock-in murine model of polycythemia vera. Our findings provide new insights into the regulation of cell-type-specific transferrin receptor endocytosis in vivo. They also suggest a new strategy to block cellular uptake of transferrin-bound iron, with therapeutic potential for disorders characterized by inappropriate red blood cell production, such as polycythemia vera. PMID:25552701

  2. The exomer cargo adaptor structure reveals a novel GTPase-binding domain

    PubMed Central

    Paczkowski, Jon E; Richardson, Brian C; Strassner, Amanda M; Fromme, J Christopher

    2012-01-01

    Cargo adaptors control intracellular trafficking of transmembrane proteins by sorting them into membrane transport carriers. The COPI, COPII, and clathrin cargo adaptors are structurally well characterized, but other cargo adaptors remain poorly understood. Exomer is a specialized cargo adaptor that sorts specific proteins into trans-Golgi network (TGN)-derived vesicles in response to cellular signals. Exomer is recruited to the TGN by the Arf1 GTPase, a universally conserved trafficking regulator. Here, we report the crystal structure of a tetrameric exomer complex composed of two copies each of the Chs5 and Chs6 subunits. The structure reveals the FN3 and BRCT domains of Chs5, which together we refer to as the FBE domain (FN3–BRCT of exomer), project from the exomer core complex. The overall architecture of the FBE domain is reminiscent of the appendage domains of other cargo adaptors, although it exhibits a distinct topology. In contrast to appendage domains, which bind accessory factors, we show that the primary role of the FBE domain is to bind Arf1 for recruitment of exomer to membranes. PMID:23000721

  3. THREADED ADAPTOR FOR LUGGED PIPE ENDS

    DOEpatents

    Robb, J.E.

    1962-06-01

    An adaptor is designed for enabling a threaded part to be connected to a member at a region having lugs normally receiving bayonet slots of another part for attachment of the latter. It has been found desirable to replace a closure cap connected in a bayonet joint to the end of a coolant tube containing nuclear- reactor fuel elements, with a threaded valve. An adaptor is used which has J- slots receiving lugs on the end of the reactor tube, a thread for connection with the valve, and gear-tooth section enabling a gear-type of tool to rotate the adaptor to seal the valve to the end of the reactor tube. (AEC)

  4. Yeast Golgi-localized, γ-Ear–containing, ADP-Ribosylation Factor-binding Proteins Are but Adaptor Protein-1 Is Not Required for Cell-free Transport of Membrane Proteins from the Trans-Golgi Network to the Prevacuolar Compartment

    PubMed Central

    Abazeed, Mohamed E.

    2008-01-01

    Golgi-localized, γ-Ear–containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein-1 (AP-1) mediate clathrin-dependent trafficking of transmembrane proteins between the trans-Golgi network (TGN) and endosomes. In yeast, the vacuolar sorting receptor Vps10p follows a direct pathway from the TGN to the late endosome/prevacuolar compartment (PVC), whereas, the processing protease Kex2p partitions between the direct pathway and an indirect pathway through the early endosome. To examine the roles of the Ggas and AP-1 in TGN–PVC transport, we used a cell-free assay that measures delivery to the PVC of either Kex2p or a chimeric protein (K-V), in which the Vps10p cytosolic tail replaces the Kex2p tail. Either antibody inhibition or dominant-negative Gga2p completely blocked K-V transport but only partially blocked Kex2p transport. Deletion of APL2, encoding the β subunit of AP-1, did not affect K-V transport but partially blocked Kex2p transport. Residual Kex2p transport seen with apl2Δ membranes was insensitive to dominant-negative Gga2p, suggesting that the apl2Δ mutation causes Kex2p to localize to a compartment that precludes Gga-dependent trafficking. These results suggest that yeast Ggas facilitate the specific and direct delivery of Vps10p and Kex2p from the TGN to the PVC and that AP-1 modulates Kex2p trafficking through a distinct pathway, presumably involving the early endosome. PMID:18784256

  5. Styles of Creativity: Adaptors and Innovators in a Singapore Context

    ERIC Educational Resources Information Center

    Ee, Jessie; Seng, Tan Oon; Kwang, Ng Aik

    2007-01-01

    Kirton (1976) described two creative styles, namely adaptors and innovators. Adaptors prefer to "do things better" whilst, innovators prefer to "do things differently". This study explored the relationship between two creative styles (adaptor and innovator) and the Big Five personality traits (extraversion, agreeableness, conscientiousness,…

  6. 21 CFR 870.3620 - Pacemaker lead adaptor.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Pacemaker lead adaptor. 870.3620 Section 870.3620...) MEDICAL DEVICES CARDIOVASCULAR DEVICES Cardiovascular Prosthetic Devices § 870.3620 Pacemaker lead adaptor. (a) Identification. A pacemaker lead adaptor is a device used to adapt a pacemaker lead so that...

  7. 21 CFR 870.2350 - Electrocardiograph lead switching adaptor.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Electrocardiograph lead switching adaptor. 870... Electrocardiograph lead switching adaptor. (a) Identification. An electrocardiograph lead switching adaptor is a passive switching device to which electrocardiograph limb and chest leads may be attached. This device...

  8. 21 CFR 870.2350 - Electrocardiograph lead switching adaptor.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Electrocardiograph lead switching adaptor. 870... Electrocardiograph lead switching adaptor. (a) Identification. An electrocardiograph lead switching adaptor is a passive switching device to which electrocardiograph limb and chest leads may be attached. This device...

  9. 21 CFR 870.2350 - Electrocardiograph lead switching adaptor.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Electrocardiograph lead switching adaptor. 870... Electrocardiograph lead switching adaptor. (a) Identification. An electrocardiograph lead switching adaptor is a passive switching device to which electrocardiograph limb and chest leads may be attached. This device...

  10. 21 CFR 870.3620 - Pacemaker lead adaptor.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Pacemaker lead adaptor. 870.3620 Section 870.3620...) MEDICAL DEVICES CARDIOVASCULAR DEVICES Cardiovascular Prosthetic Devices § 870.3620 Pacemaker lead adaptor. (a) Identification. A pacemaker lead adaptor is a device used to adapt a pacemaker lead so that...

  11. 21 CFR 870.3620 - Pacemaker lead adaptor.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Pacemaker lead adaptor. 870.3620 Section 870.3620...) MEDICAL DEVICES CARDIOVASCULAR DEVICES Cardiovascular Prosthetic Devices § 870.3620 Pacemaker lead adaptor. (a) Identification. A pacemaker lead adaptor is a device used to adapt a pacemaker lead so that...

  12. 21 CFR 870.3620 - Pacemaker lead adaptor.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Pacemaker lead adaptor. 870.3620 Section 870.3620...) MEDICAL DEVICES CARDIOVASCULAR DEVICES Cardiovascular Prosthetic Devices § 870.3620 Pacemaker lead adaptor. (a) Identification. A pacemaker lead adaptor is a device used to adapt a pacemaker lead so that...

  13. 21 CFR 870.2350 - Electrocardiograph lead switching adaptor.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Electrocardiograph lead switching adaptor. 870... Electrocardiograph lead switching adaptor. (a) Identification. An electrocardiograph lead switching adaptor is a passive switching device to which electrocardiograph limb and chest leads may be attached. This device...

  14. 21 CFR 870.2350 - Electrocardiograph lead switching adaptor.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Electrocardiograph lead switching adaptor. 870... Electrocardiograph lead switching adaptor. (a) Identification. An electrocardiograph lead switching adaptor is a passive switching device to which electrocardiograph limb and chest leads may be attached. This device...

  15. 21 CFR 870.3620 - Pacemaker lead adaptor.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Pacemaker lead adaptor. 870.3620 Section 870.3620...) MEDICAL DEVICES CARDIOVASCULAR DEVICES Cardiovascular Prosthetic Devices § 870.3620 Pacemaker lead adaptor. (a) Identification. A pacemaker lead adaptor is a device used to adapt a pacemaker lead so that...

  16. Inflammation- and tumor-induced anorexia and weight loss require MyD88 in hematopoietic/myeloid cells but not in brain endothelial or neural cells.

    PubMed

    Ruud, Johan; Wilhelms, Daniel Björk; Nilsson, Anna; Eskilsson, Anna; Tang, Yan-Juan; Ströhle, Peter; Caesar, Robert; Schwaninger, Markus; Wunderlich, Thomas; Bäckhed, Fredrik; Engblom, David; Blomqvist, Anders

    2013-05-01

    Loss of appetite is a hallmark of inflammatory diseases. The underlying mechanisms remain undefined, but it is known that myeloid differentiation primary response gene 88 (MyD88), an adaptor protein critical for Toll-like and IL-1 receptor family signaling, is involved. Here we addressed the question of determining in which cells the MyD88 signaling that results in anorexia development occurs by using chimeric mice and animals with cell-specific deletions. We found that MyD88-knockout mice, which are resistant to bacterial lipopolysaccharide (LPS)-induced anorexia, displayed anorexia when transplanted with wild-type bone marrow cells. Furthermore, mice with a targeted deletion of MyD88 in hematopoietic or myeloid cells were largely protected against LPS-induced anorexia and displayed attenuated weight loss, whereas mice with MyD88 deletion in hepatocytes or in neural cells or the cerebrovascular endothelium developed anorexia and weight loss of similar magnitude as wild-type mice. Furthermore, in a model for cancer-induced anorexia-cachexia, deletion of MyD88 in hematopoietic cells attenuated the anorexia and protected against body weight loss. These findings demonstrate that MyD88-dependent signaling within the brain is not required for eliciting inflammation-induced anorexia. Instead, we identify MyD88 signaling in hematopoietic/myeloid cells as a critical component for acute inflammatory-driven anorexia, as well as for chronic anorexia and weight loss associated with malignant disease.

  17. Adaptor-mediated Lon proteolysis restricts Bacillus subtilis hyperflagellation.

    PubMed

    Mukherjee, Sampriti; Bree, Anna C; Liu, Jing; Patrick, Joyce E; Chien, Peter; Kearns, Daniel B

    2015-01-01

    The Lon AAA+ protease is a highly conserved intracellular protease that is considered an anticancer target in eukaryotic cells and a crucial virulence regulator in bacteria. Lon degrades both damaged, misfolded proteins and specific native regulators, but how Lon discriminates among a large pool of candidate targets remains unclear. Here we report that Bacillus subtilis LonA specifically degrades the master regulator of flagellar biosynthesis SwrA governed by the adaptor protein swarming motility inhibitor A (SmiA). SmiA-dependent LonA proteolysis is abrogated upon microbe-substrate contact causing SwrA protein levels to increase and elevate flagellar density above a critical threshold for swarming motility atop solid surfaces. Surface contact-dependent cellular differentiation in bacteria is rapid, and regulated proteolysis may be a general mechanism of transducing surface stimuli.

  18. Syp1 is a conserved endocytic adaptor that contains domains involved in cargo selection and membrane tubulation

    SciTech Connect

    Reider, Amanda; Barker, Sarah L.; Mishra, Sanjay K.; Im, Young Jun; Maldonado-Báez, Lymarie; Hurley, James H.; Traub, Linton M.; Wendland, Beverly

    2010-10-28

    Internalization of diverse transmembrane cargos from the plasma membrane requires a similarly diverse array of specialized adaptors, yet only a few adaptors have been characterized. We report the identification of the muniscin family of endocytic adaptors that is conserved from yeast to human beings. Solving the structures of yeast muniscin domains confirmed the unique combination of an N-terminal domain homologous to the crescent-shaped membrane-tubulating EFC/F-BAR domains and a C-terminal domain homologous to cargo-binding {mu} homology domains ({mu}HDs). In vitro and in vivo assays confirmed membrane-tubulation activity for muniscin EFC/F-BAR domains. The {mu}HD domain has conserved interactions with the endocytic adaptor/scaffold Ede1/eps15, which influences muniscin localization. The transmembrane protein Mid2, earlier implicated in polarized Rho1 signalling, was identified as a cargo of the yeast adaptor protein. These and other data suggest a model in which the muniscins provide a combined adaptor/membrane-tubulation activity that is important for regulating endocytosis.

  19. TIR domain-containing adaptor SARM is a late addition to the ongoing microbe–host dialog

    PubMed Central

    Zhang, Qing; Zmasek, Christian M.; Cai, Xiaohui; Godzik, Adam

    2011-01-01

    Toll/interleukin-1 receptor (TIR) domain-containing proteins play important roles in defense against pathogens in both animals and plants, connecting the immunity signaling pathways via a chain of specific protein–protein interactions. Among them is SARM, the only TIR domain-containing adaptor that can negatively regulate TLR signaling. By extensive phylogenetic analysis, we show here that SARM is closely related to bacterial proteins with TIR domains, suggesting that this family has a different evolutionary history from other animal TIR-containing adaptors, possibly emerging via a lateral gene transfer from bacteria to animals. We also show evidence of several similar, independent transfer events, none of which, however, survived in vertebrates. An evolutionary relationship between the animal SARM adaptor and bacterial proteins with TIR domains illustrates the possible role that bacterial TIR-containing proteins play in regulating eukaryotic immune responses and how this mechanism was possibly adapted by the eukaryotes themselves. PMID:21110998

  20. Regulation of ITAM adaptor molecules and their receptors by inhibition of calcineurin-NFAT signalling during late stage osteoclast differentiation

    SciTech Connect

    Zawawi, M.S.F.; Dharmapatni, A.A.S.S.K.; Cantley, M.D.; McHugh, K.P.; Haynes, D.R.; Crotti, T.N.

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Calcineurin/NFAT inhibitors FK506 and VIVIT treated human PBMC derived osteoclasts in vitro. Black-Right-Pointing-Pointer Differential regulation of ITAM receptors and adaptor molecules by calcineurin/NFAT inhibitors. Black-Right-Pointing-Pointer FK506 and VIVIT suppress ITAM factors during late phase osteoclast differentiation. -- Abstract: Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway in osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcR{gamma}) and DNAX-activating protein 12 kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin ({beta}3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10 days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real

  1. Scaffold functions of 14-3-3 adaptors in B cell immunoglobulin class switch DNA recombination.

    PubMed

    Lam, Tonika; Thomas, Lisa M; White, Clayton A; Li, Guideng; Pone, Egest J; Xu, Zhenming; Casali, Paolo

    2013-01-01

    Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID) expression and AID targeting to switch (S) regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5'-AGCT-3' repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S-S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3β, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit) and PKA-RIα (regulatory inhibitory subunit) and uracil DNA glycosylase (Ung). 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180-198) or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR). 14-3-3 adaptors colocalized with AID and replication protein A (RPA) in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr), an accessory protein of human immunodeficiency virus type-1 (HIV-1), which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR.

  2. Adaptor assembly for coupling turbine blades to rotor disks

    SciTech Connect

    Delvaux, John McConnel; Garcia-Crespo, Andres Jose; Joyce, Kilmer Joseph; Tindell, Allan Randall

    2014-06-03

    An adaptor assembly for coupling a blade root of a turbine blade to a root slot of a rotor disk is disclosed. The adaptor assembly may generally include an adaptor body having a root configured to be received within the root slot. The adaptor body may also define a slot having an open end configured to receive the blade root. The adaptor body may further define a channel. The adaptor assembly may also include a plate having an outwardly extending foot. The foot may be configured to be received within the channel. Additionally, the plate may be configured to cover at least a portion of the open end of the slot when the foot is received within the channel.

  3. The p95-100 kDa ligand of the T cell-specific adaptor (TSAd) protein Src-homology-2 (SH2) domain implicated in TSAd nuclear import is p97 Valosin-containing protein (VCP).

    PubMed

    Marti, Francesc; King, Philip D

    2005-03-15

    T cell-specific adapter protein (TSAd) is required for normal T cell antigen receptor (TCR)-induced transcription of cytokine genes in T cells. How TSAd controls cytokine transcription is unknown. Previously, we have shown that TSAd is actively transported to the nucleus of T cells suggesting that this adapter may in part function within this cellular compartment. Nuclear translocation of TSAd is dependent upon an intact Src-homology-2 (SH2) domain and a p95-100 kDa ligand of the SH2 domain has been implicated in nuclear import. Here, using microchemical techniques, we identify p95-100 as p97 Valosin-containing protein (VCP) whose homolog in yeast is the cell division control protein, CDC48. Physical interaction between TSAd and VCP can be demonstrated between endogenous proteins in T cells. Interaction is direct and is dependent upon phosphorylation of tyrosine residue 805 of VCP that has been previously recognized as a major target of tyrosine kinase(s) involved in TCR signaling. Significantly, with the use of CDC48 mutant yeast, we demonstrate that VCP/CDC48 is required for transport of TSAd into the eukaryotic nucleus. These findings provide important insights into the mechanism of TSAd nuclear import and the role of TSAd in T cell signal transduction.

  4. An organized co-assembly of clathrin adaptors is essential for endocytosis.

    PubMed

    Skruzny, Michal; Desfosses, Ambroise; Prinz, Simone; Dodonova, Svetlana O; Gieras, Anna; Uetrecht, Charlotte; Jakobi, Arjen J; Abella, Marc; Hagen, Wim J H; Schulz, Joachim; Meijers, Rob; Rybin, Vladimir; Briggs, John A G; Sachse, Carsten; Kaksonen, Marko

    2015-04-20

    Clathrin-mediated endocytosis, the main trafficking route from the plasma membrane to the cytoplasm, is critical to many fundamental cellular processes. Clathrin, coupled to the membrane by adaptor proteins, is thought to play a major structural role in endocytosis by self-assembling into a cage-like lattice around the forming vesicle. Although clathrin adaptors are essential for endocytosis, little is known about their structural role in this process. Here we show that the membrane-binding domains of two conserved clathrin adaptors, Sla2 and Ent1, co-assemble in a PI(4,5)P2-dependent manner to form organized lattices on membranes. We determined the structure of the co-assembled lattice by electron cryo-microscopy and designed mutations that specifically impair the lattice formation in vitro. We show that these mutations block endocytosis in vivo. We suggest that clathrin adaptors not only link the polymerized clathrin to the membrane but also form an oligomeric structure, which is essential for membrane remodeling during endocytosis.

  5. Studying the Impact of Presence of Alpha Acid Glycoprotein and Protein Glycoprotein in Chronic Myeloid Leukemia Patients Treated with Imatinib Mesylate in the State of Qatar

    PubMed Central

    Al-Dewik, Nader I.; Jewell, Andrew P.; Yassin, Mohammed A.; Morsi, Hisham M.

    2015-01-01

    Despite the efficacy of imatinib mesylate (IM) in treating chronic myeloid leukemia (CML), there is a high degree of resistance. Alpha- 1-acid glycoprotein may reduce drug efficacy through its ability to interact with IM and blocks it from reaching its target, while protein glycoprotein (PGP) may reduce the intracellular concentration of the drug via an active pump mechanism. We thus investigated the correlation between AGP and PGP levels and the resistance/response to treatment. A total of 26 CML patients were investigated for AGP and PGP levels at diagnosis and during treatment. There was no significant difference or correlation between AGP levels and the different groups of patients. There was also no significant difference in the fluorescence intensities of PGP levels among the different patient groups. The resistance observed in our CML patient population could not be correlated with AGP and PGP levels. There was no significant pattern of AGP and PGP expression, irrespective of the response or resistance to treatment. PMID:26640393

  6. Transcriptome analysis of G protein-coupled receptors in distinct genetic subgroups of acute myeloid leukemia: identification of potential disease-specific targets.

    PubMed

    Maiga, A; Lemieux, S; Pabst, C; Lavallée, V-P; Bouvier, M; Sauvageau, G; Hébert, J

    2016-01-01

    Acute myeloid leukemia (AML) is associated with poor clinical outcome and the development of more effective therapies is urgently needed. G protein-coupled receptors (GPCRs) represent attractive therapeutic targets, accounting for approximately 30% of all targets of marketed drugs. Using next-generation sequencing, we studied the expression of 772 GPCRs in 148 genetically diverse AML specimens, normal blood and bone marrow cell populations as well as cord blood-derived CD34-positive cells. Among these receptors, 30 are overexpressed and 19 are downregulated in AML samples compared with normal CD34-positive cells. Upregulated GPCRs are enriched in chemokine (CCR1, CXCR4, CCR2, CX3CR1, CCR7 and CCRL2), adhesion (CD97, EMR1, EMR2 and GPR114) and purine (including P2RY2 and P2RY13) receptor subfamilies. The downregulated receptors include adhesion GPCRs, such as LPHN1, GPR125, GPR56, CELSR3 and GPR126, protease-activated receptors (F2R and F2RL1) and the Frizzled family receptors SMO and FZD6. Interestingly, specific deregulation was observed in genetically distinct subgroups of AML, thereby identifying different potential therapeutic targets in these frequent AML subgroups. PMID:27258612

  7. The Mitochondrial Fission Adaptors Caf4 and Mdv1 Are Not Functionally Equivalent

    PubMed Central

    Guo, Qian; Koirala, Sajjan; Perkins, Edward M.; McCaffery, J. Michael; Shaw, Janet M.

    2012-01-01

    Mitochondrial fission in eukaryotes is mediated by protein complexes that encircle and divide mitochondrial tubules. In budding yeast, fission requires the membrane-anchored protein Fis1 and the dynamin-related GTPase Dnm1. Dnm1 is recruited to mitochondria via interactions with the adaptor proteins Caf4 and Mdv1, which bind directly to Fis1. Unlike Mdv1, a function for Caf4 in mitochondrial membrane scission has not been established. In this study, we demonstrate that Caf4 is a bona fide fission adaptor that assembles at sites of mitochondrial division. We also show that fission complexes may contain Caf4 alone or both Caf4 and Mdv1 without compromising fission function. Although there is a correspondence between Caf4 and Mdv1 expression levels and their contribution to fission, the two adaptor proteins are not equivalent. Rather, our functional and phylogenetic analyses indicate that Caf4 mitochondrial fission activity has diverged from that of Mdv1. PMID:23300936

  8. The adaptor Grb7 links netrin-1 signaling to regulation of mRNA translation

    PubMed Central

    Tsai, Nien-Pei; Bi, Jing; Wei, Li-Na

    2007-01-01

    We previously reported a novel biological activity of Netrin-1 in translational stimulation of kappa opioid receptor (KOR). We now identify Grb7 as a new RNA-binding protein that serves as the molecular adaptor for transmitting Netrin-1 signals, through focal adhesion kinase (FAK), to the translation machinery. Grb7 binds specifically to the first stem loop of kor mRNA 5′-UTR through a new RNA-binding domain located in its amino terminus. Upon binding to its capped, target mRNA, Grb7 blocks the recruitment of eIF4E, rendering mRNA untranslatable. The RNA-binding and translation-repressive activity is reduced by FAK-mediated hyperphosphorylation on two tyrosine residues of its carboxyl terminus. This study reports an adaptor protein Grb7 that transmits the stimulating signals of Netrin-1 to the translational machinery to rapidly regulate mRNA translation. PMID:17318180

  9. Novel Toll/IL-1 Receptor Homologous Region Adaptors Act as Negative Regulators in Amphioxus TLR Signaling.

    PubMed

    Peng, Jian; Tao, Xin; Li, Rui; Hu, Jingru; Ruan, Jie; Wang, Ruihua; Yang, Manyi; Yang, Rirong; Dong, Xiangru; Chen, Shangwu; Xu, Anlong; Yuan, Shaochun

    2015-10-01

    Studies have shown that the basal chordate amphioxus possesses an extraordinarily complex TLR system, including 39 TLRs and at least 40 Toll/IL-1R homologous region (TIR) adaptors. Besides homologs to MyD88 and TIR domain-containing adaptor molecule (TICAM), most amphioxus TIR adaptors exhibit domain architectures that are not observed in other species. To reveal how these novel TIR adaptors function in amphioxus Branchiostoma belcheri tsingtauense (bbt), four representatives, bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD, were selected for functional analyses. We found bbtTIRA to show a unique inhibitory role in amphioxus TICAM-mediated pathway by interacting with bbtTICAM and bbt receptor interacting protein 1b, whereas bbtTIRC specifically inhibits the amphioxus MyD88-dependent pathway by interacting with bbtMyD88 and depressing the polyubiquitination of bbt TNFR-associated factor 6. Although both bbtTIRB and bbtTIRD are located on endosomes, the TIR domain of bbtTIRB can interact with bbtMyD88 in the cytosol, whereas the TIR domain of bbtTIRD is enclosed in endosome, suggesting that bbtTIRD may be a redundant gene in amphioxus. This study indicated that most expanded TIR adaptors play nonredundant regulatory roles in amphioxus TLR signaling, adding a new layer to understanding the diversity and complexity of innate immunity at basal chordate.

  10. Novel Toll/IL-1 Receptor Homologous Region Adaptors Act as Negative Regulators in Amphioxus TLR Signaling.

    PubMed

    Peng, Jian; Tao, Xin; Li, Rui; Hu, Jingru; Ruan, Jie; Wang, Ruihua; Yang, Manyi; Yang, Rirong; Dong, Xiangru; Chen, Shangwu; Xu, Anlong; Yuan, Shaochun

    2015-10-01

    Studies have shown that the basal chordate amphioxus possesses an extraordinarily complex TLR system, including 39 TLRs and at least 40 Toll/IL-1R homologous region (TIR) adaptors. Besides homologs to MyD88 and TIR domain-containing adaptor molecule (TICAM), most amphioxus TIR adaptors exhibit domain architectures that are not observed in other species. To reveal how these novel TIR adaptors function in amphioxus Branchiostoma belcheri tsingtauense (bbt), four representatives, bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD, were selected for functional analyses. We found bbtTIRA to show a unique inhibitory role in amphioxus TICAM-mediated pathway by interacting with bbtTICAM and bbt receptor interacting protein 1b, whereas bbtTIRC specifically inhibits the amphioxus MyD88-dependent pathway by interacting with bbtMyD88 and depressing the polyubiquitination of bbt TNFR-associated factor 6. Although both bbtTIRB and bbtTIRD are located on endosomes, the TIR domain of bbtTIRB can interact with bbtMyD88 in the cytosol, whereas the TIR domain of bbtTIRD is enclosed in endosome, suggesting that bbtTIRD may be a redundant gene in amphioxus. This study indicated that most expanded TIR adaptors play nonredundant regulatory roles in amphioxus TLR signaling, adding a new layer to understanding the diversity and complexity of innate immunity at basal chordate. PMID:26324776

  11. Characterization and use of an antibody detecting the CBFbeta-SMMHC fusion protein in inv(16)/t(16;16)-associated acute myeloid leukemias.

    PubMed

    Viswanatha, D S; Chen, I; Liu, P P; Slovak, M L; Rankin, C; Head, D R; Willman, C L

    1998-03-15

    The inv(16)(p13q22) and t(16;16)(p13;q22) cytogenetic abnormalities occur commonly in acute myeloid leukemia (AML), typically associated with French-American-British (FAB) AML-M4Eo subtype. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been recently developed to detect the presence of several variants of the resultant CBFB-MYH11 fusion gene that encodes a CBFbeta-smooth muscle myosin heavy chain (SMMHC) fusion protein. We have now determined the clinical use of a polyclonal antibody [anti-inv(16) Ab] directed against a junctional epitope of the most common type of CBFbeta-SMMHC fusion protein (type A), which is present in 90% of inv(16)/t(16;16) AML cases. Using flow cytometry, reproducible methods were developed for detection of CBFbeta-SMMHC proteins in permeabilized cells; flow cytometric results were then correlated with cytogenetics and RT-PCR detection methods. In an analysis of 42 leukemia cases with various cytogenetic abnormalities and several normal controls, the anti-inv(16) Ab specifically detected all 23 cases that were cytogenetically positive for inv(16) or t(16;16), including a single AML case that was RT-PCR-negative. In addition to detecting all type A fusions, the anti-inv(16) Ab also unexpectedly identified the type C and type D CBFbeta-SMMHC fusion proteins. Molecular characterization of one RT-PCR-positive and Ab-positive t(16;16) case with a non-type A product showed a novel previously unreported CBFB-MYH11 fusion (CBFB nt 455-MYH11 nt 1893). Flow cytometric results were analyzed using the Kolmogorov-Smirnov statistic D-value and the median value for positive samples was 0.65 (range, 0.35 to 0.77) versus 0.07 (range, -0.21 to 0.18) in the negative group (P < .0001). The overall concordance between cytogenetics and RT-PCR was 97%, whereas the concordance between flow cytometry and cytogenetics was 100%. Thus, using the anti-inv(16) Ab, all cytogenetically positive and RT-PCR-positive AML cases with inv(16) or t(16

  12. Pathogenic Fungi Regulate Immunity by Inducing Neutrophilic Myeloid-Derived Suppressor Cells

    PubMed Central

    Rieber, Nikolaus; Singh, Anurag; Öz, Hasan; Carevic, Melanie; Bouzani, Maria; Amich, Jorge; Ost, Michael; Ye, Zhiyong; Ballbach, Marlene; Schäfer, Iris; Mezger, Markus; Klimosch, Sascha N.; Weber, Alexander N.R.; Handgretinger, Rupert; Krappmann, Sven; Liese, Johannes; Engeholm, Maik; Schüle, Rebecca; Salih, Helmut Rainer; Marodi, Laszlo; Speckmann, Carsten; Grimbacher, Bodo; Ruland, Jürgen; Brown, Gordon D.; Beilhack, Andreas; Loeffler, Juergen; Hartl, Dominik

    2015-01-01

    Summary Despite continuous contact with fungi, immunocompetent individuals rarely develop pro-inflammatory antifungal immune responses. The underlying tolerogenic mechanisms are incompletely understood. Using both mouse models and human patients, we show that infection with the human pathogenic fungi Aspergillus fumigatus and Candida albicans induces a distinct subset of neutrophilic myeloid-derived suppressor cells (MDSCs), which functionally suppress T and NK cell responses. Mechanistically, pathogenic fungi induce neutrophilic MDSCs through the pattern recognition receptor Dectin-1 and its downstream adaptor protein CARD9. Fungal MDSC induction is further dependent on pathways downstream of Dectin-1 signaling, notably reactive oxygen species (ROS) generation as well as caspase-8 activity and interleukin-1 (IL-1) production. Additionally, exogenous IL-1β induces MDSCs to comparable levels observed during C. albicans infection. Adoptive transfer and survival experiments show that MDSCs are protective during invasive C. albicans infection, but not A. fumigatus infection. These studies define an innate immune mechanism by which pathogenic fungi regulate host defense. PMID:25771792

  13. Optimization of multiplexed RADseq libraries using low-cost adaptors.

    PubMed

    Henri, Hélène; Cariou, Marie; Terraz, Gabriel; Martinez, Sonia; El Filali, Adil; Veyssiere, Marine; Duret, Laurent; Charlat, Sylvain

    2015-04-01

    Reduced representation genomics approaches, of which RADseq is currently the most popular form, offer the possibility to produce genome wide data from potentially any species, without previous genomic information. The application of RADseq to highly multiplexed libraries (including numerous specimens, and potentially numerous different species) is however limited by technical constraints. First, the cost of synthesis of Illumina adaptors including molecular identifiers (MIDs) becomes excessive when numerous specimens are to be multiplexed. Second, the necessity to empirically adjust the ratio of adaptors to genomic DNA concentration impedes the high throughput application of RADseq to heterogeneous samples, of variable DNA concentration and quality. In an attempt to solve these problems, we propose here some adjustments regarding the adaptor synthesis. First, we show that the common and unique (MID) parts of adaptors can be synthesized separately and subsequently ligated, which drastically reduces the synthesis cost, and thus allows multiplexing hundreds of specimens. Second, we show that self-ligation of adaptors, which makes the adaptor concentration so critical, can be simply prevented by using unphosphorylated adaptors, which significantly improves the ligation and sequencing yield.

  14. Prospective Evaluation of Procalcitonin, Soluble Triggering Receptor Expressed on Myeloid Cells-1 and C-Reactive Protein in Febrile Patients with Autoimmune Diseases

    PubMed Central

    Lin, Chou-Han; Hsieh, Song-Chou; Keng, Li-Ta; Lee, Ho-Sheng; Chang, Hou-Tai; Liao, Wei-Yu; Ho, Chao-Chi; Yu, Chong-Jen

    2016-01-01

    Background Both procalcitonin (PCT) and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) have been investigated separately as indicators of infection in patients with autoimmune diseases. Our study simultaneously evaluated both PCT and sTREM-1 along with C-reactive protein (CRP) in febrile patients with autoimmune diseases. Methods Fifty-nine patients were enrolled in the study. The patients were categorized into the infection group (n = 24) or the disease flare group (n = 35). sTREM-1, PCT and CRP concentrations at fever onset were compared between the two groups of patients. Results sTREM-1 and CRP did not differ between the two groups. PCT [median (range), ng/ml] was higher in the infection group than in the disease flare group [0.53 (0.02–12.85) vs. 0.12 (0.02–19.23), p = 0.001]. The area under the receiver-operating characteristic (ROC) for diagnosis of infection was 0.75 for PCT (p = 0.001), 0.63 for CRP (p = 0.09) and 0.52 for sTREM-1 (p = 0.79). Using 0.2 ng/ml as the cutoff value for PCT, sensitivity was 0.75 and specificity was 0.77. Negative predictive values for PCT were 92%, 87% and 82% for a prevalence of infection of 20%, 30%, and 40%, respectively. Neither immunosuppressants nor biomodulators affected the level of the three biomarkers. However, in patients treated with corticosteroids, the levels of sTREM-1 and CRP were significantly decreased compared with the untreated patients. Conclusions Setting PCT at a lower cutoff value could provide useful information on excluding infection in febrile patients with autoimmune diseases. The possible effect of corticosteroids on the level of sTREM-1 as an infection marker deserves further study. PMID:27096761

  15. Cutting edge: tubulin α functions as an adaptor in NFAT-importin β interaction.

    PubMed

    Ishiguro, Kazuhiro; Ando, Takafumi; Maeda, Osamu; Watanabe, Osamu; Goto, Hidemi

    2011-03-01

    Upon T cell stimulation, NFAT is dephosphorylated by calcineurin, leading to nuclear translocation via NFAT-importin β interaction. Whereas the process of NFAT dephosphorylation has been well researched, the molecular mechanism of NFAT-importin β interaction remains unknown. In contrast to NF-κB and STAT, no importin α family members have been reported as adaptor proteins for NFAT. Our study shows that tubulin α, but not tubulin β, binds to the N-terminal region of NFAT containing the regulatory and Rel homology domains. Importin β interacts with the NFAT-tubulin α complex rather than NFAT or tubulin α alone, resulting in cotranslocation of NFAT and tubulin α into the nucleus. Furthermore, the interaction is suppressed by acetate-induced tubulin α acetylation at lysine 40. In conclusion, tubulin α functions as an adaptor in NFAT-importin β interaction, and this function is regulated by acetate-induced acetylation.

  16. Clathrin adaptors. AP2 controls clathrin polymerization with a membrane-activated switch.

    PubMed

    Kelly, Bernard T; Graham, Stephen C; Liska, Nicole; Dannhauser, Philip N; Höning, Stefan; Ungewickell, Ernst J; Owen, David J

    2014-07-25

    Clathrin-mediated endocytosis (CME) is vital for the internalization of most cell-surface proteins. In CME, plasma membrane-binding clathrin adaptors recruit and polymerize clathrin to form clathrin-coated pits into which cargo is sorted. Assembly polypeptide 2 (AP2) is the most abundant adaptor and is pivotal to CME. Here, we determined a structure of AP2 that includes the clathrin-binding β2 hinge and developed an AP2-dependent budding assay. Our findings suggest that an autoinhibitory mechanism prevents clathrin recruitment by cytosolic AP2. A large-scale conformational change driven by the plasma membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate and cargo relieves this autoinhibition, triggering clathrin recruitment and hence clathrin-coated bud formation. This molecular switching mechanism can couple AP2's membrane recruitment to its key functions of cargo and clathrin binding.

  17. Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire.

    PubMed

    Roy, Edwige; Togbe, Dieudonnée; Holdorf, Amy D; Trubetskoy, Dmitry; Nabti, Sabrina; Küblbeck, Günter; Klevenz, Alexandra; Kopp-Schneider, Annette; Leithäuser, Frank; Möller, Peter; Bladt, Friedhelm; Hämmerling, Günter; Arnold, Bernd; Pawson, Tony; Tafuri, Anna

    2010-08-31

    The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, peripheral T cells expressing a TCR with low avidity for self-antigens were strongly reduced, whereas an overall impairment of T-cell activation by weak antigenic stimulation was observed. Mechanistically, Nck deletion resulted in a significant decrease in calcium mobilization and ERK phosphorylation upon TCR engagement. Taken together, our findings unveil a crucial role for the Nck adaptors in shaping the T-cell repertoire to ensure maximal antigenic coverage and optimal T cell excitability.

  18. Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire

    PubMed Central

    Roy, Edwige; Togbe, Dieudonnée; Holdorf, Amy D.; Trubetskoy, Dmitry; Nabti, Sabrina; Küblbeck, Günter; Klevenz, Alexandra; Kopp-Schneider, Annette; Leithäuser, Frank; Möller, Peter; Bladt, Friedhelm; Hämmerling, Günter; Arnold, Bernd; Pawson, Tony; Tafuri, Anna

    2010-01-01

    The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, peripheral T cells expressing a TCR with low avidity for self-antigens were strongly reduced, whereas an overall impairment of T-cell activation by weak antigenic stimulation was observed. Mechanistically, Nck deletion resulted in a significant decrease in calcium mobilization and ERK phosphorylation upon TCR engagement. Taken together, our findings unveil a crucial role for the Nck adaptors in shaping the T-cell repertoire to ensure maximal antigenic coverage and optimal T cell excitability. PMID:20709959

  19. Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire.

    PubMed

    Roy, Edwige; Togbe, Dieudonnée; Holdorf, Amy D; Trubetskoy, Dmitry; Nabti, Sabrina; Küblbeck, Günter; Klevenz, Alexandra; Kopp-Schneider, Annette; Leithäuser, Frank; Möller, Peter; Bladt, Friedhelm; Hämmerling, Günter; Arnold, Bernd; Pawson, Tony; Tafuri, Anna

    2010-08-31

    The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, peripheral T cells expressing a TCR with low avidity for self-antigens were strongly reduced, whereas an overall impairment of T-cell activation by weak antigenic stimulation was observed. Mechanistically, Nck deletion resulted in a significant decrease in calcium mobilization and ERK phosphorylation upon TCR engagement. Taken together, our findings unveil a crucial role for the Nck adaptors in shaping the T-cell repertoire to ensure maximal antigenic coverage and optimal T cell excitability. PMID:20709959

  20. The Transcription Factor Wilms Tumor 1 Confers Resistance in Myeloid Leukemia Cells against the Proapoptotic Therapeutic Agent TRAIL (Tumor Necrosis Factor α-related Apoptosis-inducing Ligand) by Regulating the Antiapoptotic Protein Bcl-xL*

    PubMed Central

    Bansal, Hima; Seifert, Theresea; Bachier, Carlos; Rao, Manjeet; Tomlinson, Gail; Iyer, Swaminathan Padmanabhan; Bansal, Sanjay

    2012-01-01

    Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is considered a promising cancer therapeutic agent due to its ability to induce apoptosis in a variety of cancer cells, while sparing normal cells. However, many human tumors including acute myeloid leukemia (AML) are partially or completely resistant to monotherapy with TRAIL, limiting its therapeutic utility. Therefore, identification of factors that contribute to TRAIL resistance may facilitate future development of more effective TRAIL-based cancer therapies. Here, we report a previously unknown role for WT1 in mediating TRAIL resistance in leukemia. Knockdown of WT1 with shRNA rendered TRAIL-resistant myeloid leukemia cells sensitive to TRAIL-induced cell death, and re-expression of shRNA-resistant WT1 restored TRAIL resistance. Notably, TRAIL-mediated apoptosis in WT1-silenced cells was largely due to down-regulation of the antiapoptotic protein Bcl-xL. Moreover, WT1 expression strongly correlated with overexpression of Bcl-xL in AML cell lines and blasts from AML patients. Furthermore, we found that WT1 transactivates Bcl-xL by directly binding to its promoter. We previously showed that WT1 is a novel client protein of heat shock protein 90 (Hsp90). Consistent with this, pharmacological inhibition of Hsp90 resulted in reduced WT1 and Bcl-xL expression leading to increased sensitivity of leukemia cells to TRAIL-mediated apoptosis. Collectively, our results suggest that WT1-dependent Bcl-xL overexpression contributes to TRAIL resistance in myeloid leukemias. PMID:22898820

  1. The 57Fe hyperfine interactions in iron storage proteins in liver and spleen tissues from normal human and two patients with mantle cell lymphoma and acute myeloid leukemia: a Mössbauer effect study

    NASA Astrophysics Data System (ADS)

    Oshtrakh, M. I.; Alenkina, I. V.; Vinogradov, A. V.; Konstantinova, T. S.; Semionkin, V. A.

    2015-04-01

    Study of human spleen and liver tissues from healthy persons and two patients with mantle cell lymphoma and acute myeloid leukemia was carried out using Mössbauer spectroscopy with a high velocity resolution. Small variations in the 57Fe hyperfine parameters for normal and patient's tissues were detected and related to small variations in the 57Fe local microenvironment in ferrihydrite cores. The differences in the relative parts of more crystalline and more amorphous core regions were also supposed for iron storage proteins in normal and patients' spleen and liver tissues.

  2. Characterization of the DNA-binding properties of the myeloid zinc finger protein MZF1: two independent DNA-binding domains recognize two DNA consensus sequences with a common G-rich core.

    PubMed Central

    Morris, J F; Hromas, R; Rauscher, F J

    1994-01-01

    The myeloid zinc finger gene 1, MZF1, encodes a transcription factor which is expressed in hematopoietic progenitor cells that are committed to myeloid lineage differentiation. MZF1 contains 13 C2H2 zinc fingers arranged in two domains which are separated by a short glycine- and proline-rich sequence. The first domain consists of zinc fingers 1 to 4, and the second domain is formed by zinc fingers 5 to 13. We have determined that both sets of zinc finger domains bind DNA. Purified, recombinant MZF1 proteins containing either the first set of zinc fingers or the second set were prepared and used to affinity select DNA sequences from a library of degenerate oligonucleotides by using successive rounds of gel shift followed by PCR amplification. Surprisingly, both DNA-binding domains of MZF1 selected similar DNA-binding consensus sequences containing a core of four or five guanine residues, reminiscent of an NF-kappa B half-site: 1-4, 5'-AGTGGGGA-3'; 5-13, 5'-CGGGnGAGGGGGAA-3'. The full-length MZF1 protein containing both sets of zinc finger DNA-binding domains recognizes synthetic oligonucleotides containing either the 1-4 or 5-13 consensus binding sites in gel shift assays. Thus, we have identified the core DNA consensus binding sites for each of the two DNA-binding domains of a myeloid-specific zinc finger transcription factor. Identification of these DNA-binding sites will allow us to identify target genes regulated by MZF1 and to assess the role of MZF1 as a transcriptional regulator of hematopoiesis. Images PMID:8114711

  3. What Is Acute Myeloid Leukemia?

    MedlinePlus

    ... about acute myeloid leukemia? What is acute myeloid leukemia? Cancer starts when cells in a part of ... the body from doing their jobs. Types of leukemia Not all leukemias are the same. There are ...

  4. Construction and biological characterization of an interleukin-12 fusion protein (Flexi-12): delivery to acute myeloid leukemic blasts using adeno-associated virus.

    PubMed

    Anderson, R; Macdonald, I; Corbett, T; Hacking, G; Lowdell, M W; Prentice, H G

    1997-06-10

    Interleukin-12 (IL-12) is a cytokine that exhibits pleiotropic effects on lymphocytes and natural killer cells and has been shown to have promise for the immunotherapy of cancer. The combination of the immune costimulatory molecule B7.1 and IL-12 has been shown to be synergistic for T cell activation. By transfecting tumor cells with both IL-12 and B7.1 cDNAs, it may be possible to use these modified targets as vaccines. A major obstacle in designing a vector to deliver these genes results from the structure of IL-12. Functional IL-12 is a heterodimer composed of two distinct subunits that are encoded by separate genes on different chromosomes. Production of functional IL-12 requires the coordinated expression of both genes. This presents several problems in vectors, particularly those in which additional genes, either a co-stimulatory gene or a selectable marker, are inserted. Therefore, we have constructed a single cDNA that encodes a single-chain protein, called Flexi-12, which retains all of the biological characteristics of recombinant IL-12 (rIL-12). The monomeric polypeptide Flexi-12 is able to induce the proliferation of phytohemagglutinin (PHA) blasts, induce PHA blasts to secrete interferon-gamma (IFN-gamma) and additionally, by preincubation, enhance the killing of K562 targets by PBLs. These phenomena are in a dose-dependent manner comparable to that seen with rIL-12. We have also shown that tyrosine phosphorylation of the STAT 4 transcription factor, which has been shown to be unique to the IL-12 signaling pathway, occurs with Flexi-12 at levels similar to those seen with rIL-12. We have packaged Flexi-12 into a recombinant adeno-associated virus (AAV) and used this vector to infect acute myeloid leukemic (AML) blasts. Infected AML blasts produced between 2 and 6 ng of IL-12/10(6) cells per ml per 48 hr. These studies also confirm that AAV is an efficient delivery vehicle for cytokines to leukemic cells. Direct analysis of these modified cells acting

  5. Systematic VCP-UBXD Adaptor Network Proteomics Identifies a Role for UBXN10 in Regulating Ciliogenesis

    PubMed Central

    Raman, Malavika; Sergeev, Mikhail; Garnaas, Maija; Lydeard, John R.; Huttlin, Edward L.; Goessling, Wolfram; Shah, Jagesh V.; Harper, J. Wade

    2015-01-01

    The AAA-ATPase VCP (also known as p97 or CDC48) uses ATP hydrolysis to “segregate” ubiquitinated proteins from their binding partners. VCP acts via UBX-domain containing adaptors that provide target specificity, but targets and functions of UBXD proteins remain poorly understood. Through systematic proteomic analysis of UBXD proteins in human cells, we reveal a network of over 195 interacting proteins, implicating VCP in diverse cellular pathways. We have explored one such complex between an unstudied adaptor UBXN10 and the intraflagellar transport B (IFT-B) complex, which regulates anterograde transport into cilia. UBXN10 localizes to cilia in a VCP-dependent manner and both VCP and UBXN10 are required for ciliogenesis. Pharmacological inhibition of VCP destabilized the IFT-B complex and increased trafficking rates. Depletion of UBXN10 in zebrafish embryos causes defects in left-right asymmetry, which depends on functional cilia. This study provides a resource for exploring the landscape of UBXD proteins in biology and identifies an unexpected requirement for VCP-UBXN10 in ciliogenesis. PMID:26389662

  6. Ubc2, an Ortholog of the Yeast Ste50p Adaptor, Possesses a Basidiomycete-Specific Carboxy terminal Extension Essential for Pathogenicity Independent of Pheromone Response.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins involved in the MAP kinase pathway controlling mating, morphogenesis and pathogenicity have been identified previously in the fungus Ustilago maydis. One of these, the Ubc2 adaptor protein, possesses a basidiomycete-specific structure. In addition to containing SAM and RA domains typical of...

  7. MYC oncogene in myeloid neoplasias.

    PubMed

    Delgado, M Dolores; Albajar, Marta; Gomez-Casares, M Teresa; Batlle, Ana; León, Javier

    2013-02-01

    MYC is a transcription factor that regulates many critical genes for cell proliferation, differentiation, and biomass accumulation. MYC is one of the most prevalent oncogenes found to be altered in human cancer, being deregulated in about 50 % of tumors. Although MYC deregulation has been more frequently associated to lymphoma and lymphoblastic leukemia than to myeloid malignancies, a body of evidence has been gathered showing that MYC plays a relevant role in malignancies derived from the myeloid compartment. The myeloid leukemogenic activity of MYC has been demonstrated in different murine models. Not surprisingly, MYC has been found to be amplified or/and deregulated in the three major types of myeloid neoplasms: acute myeloid leukemia, myelodysplastic syndromes, and myeloproliferative neoplasms, including chronic myeloid leukemia. Here, we review the recent literature describing the involvement of MYC in myeloid tumors.

  8. EVOLUTION OF MYELOID CELLS

    PubMed Central

    Barreda, Daniel R.; Neely, Harold R.; Flajnik, Martin F.

    2015-01-01

    In 1882, Elie Metchnikoff identified myeloid-like cells from starfish larvae responding to the invasion by a foreign body (rose thorn). This marked the origins of the study of innate immunity, and an appreciation that cellular immunity is already well established in these “primitive” organisms. This chapter focuses on these myeloid cells as well as the newest members of this family, the dendritic cells (DC), and explores their evolutionary origins. Our goal is to provide evolutionary context for the development of the multilayered immune system of mammals, where myeloid cells now serve as central effectors of innate immunity and regulators of adaptive immunity. Overall, we find that core contributions of myeloid cells to the regulation of inflammation are based on mechanisms that have been honed over hundreds of millions of years of evolution. Using phagocytosis as a platform, we show how fairly simple beginnings have offered a robust foundation onto which additional control features have been integrated, resulting in central regulatory nodes that now manage multi-factorial aspects of homeostasis and immunity. PMID:27337471

  9. BCOR regulates myeloid cell proliferation and differentiation.

    PubMed

    Cao, Q; Gearhart, M D; Gery, S; Shojaee, S; Yang, H; Sun, H; Lin, D-C; Bai, J-W; Mead, M; Zhao, Z; Chen, Q; Chien, W-W; Alkan, S; Alpermann, T; Haferlach, T; Müschen, M; Bardwell, V J; Koeffler, H P

    2016-05-01

    BCOR is a component of a variant Polycomb group repressive complex 1 (PRC1). Recently, we and others reported recurrent somatic BCOR loss-of-function mutations in myelodysplastic syndrome and acute myelogenous leukemia (AML). However, the role of BCOR in normal hematopoiesis is largely unknown. Here, we explored the function of BCOR in myeloid cells using myeloid murine models with Bcor conditional loss-of-function or overexpression alleles. Bcor mutant bone marrow cells showed significantly higher proliferation and differentiation rates with upregulated expression of Hox genes. Mutation of Bcor reduced protein levels of RING1B, an H2A ubiquitin ligase subunit of PRC1 family complexes and reduced H2AK119ub upstream of upregulated HoxA genes. Global RNA expression profiling in murine cells and AML patient samples with BCOR loss-of-function mutation suggested that loss of BCOR expression is associated with enhanced cell proliferation and myeloid differentiation. Our results strongly suggest that BCOR plays an indispensable role in hematopoiesis by inhibiting myeloid cell proliferation and differentiation and offer a mechanistic explanation for how BCOR regulates gene expression such as Hox genes. PMID:26847029

  10. The role of natural killer cells in chronic myeloid leukemia

    PubMed Central

    Danier, Anna Carolyna Araújo; de Melo, Ricardo Pereira; Napimoga, Marcelo Henrique; Laguna-Abreu, Maria Theresa Cerávolo

    2011-01-01

    Chronic myeloid leukemia is a neoplasia resulting from a translocation between chromosomes 9 and 22 producing the BCR-ABL hybrid known as the Philadelphia chromosome (Ph). In chronic myeloid leukemia a proliferation of malignant myeloid cells occurs in the bone marrow due to excessive tyrosine kinase activity. In order to maintain homeostasis, natural killer cells, by means of receptors, identify the major histocompatibility complex on the surface of tumor cells and subsequently induce apoptosis. The NKG2D receptor in the natural killer cells recognizes the transmembrane proteins related to major histocompatibility complex class I chain-related genes A and B (MICA and MICB), and it is by the interaction between NKG2D and MICA that natural killer cells exert cytotoxic activity against chronic myeloid leukemia tumor cells. However, in the case of chronic exposure of the NKG2D receptor, the MICA ligand releases soluble proteins called sMICA from the tumor cell surface, which negatively modulate NKG2D and enable the tumor cells to avoid lysis mediated by the natural killer cells. Blocking the formation of sMICA may be an important antitumor strategy. Treatment using tyrosine kinase inhibitors induces modulation of NKG2DL expression, which could favor the activity of the natural killer cells. However this mechanism has not been fully described in chronic myeloid leukemia. In the present study, we analyze the role of natural killer cells to reduce proliferation and in the cellular death of tumor cells in chronic myeloid leukemia. PMID:23049299

  11. In vitro and in vivo Analysis of the Binding of the C Terminus of the HDL Receptor Scavenger Receptor Class B type I (SR-BI) to the PDZ1 Domain of its Cytoplasmic Adaptor Protein PDZK1

    SciTech Connect

    O Kocher; G Birrane; K Tsukamoto; S Fenske; A Yesilaltay; R Pal; K Daniels; J Ladias; M Krieger

    2011-12-31

    The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys(14)-Xaa(4)-Asn(19)-Tyr-Gly-Phe-Phe-Leu(24)), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI ((503)VLQEAKL(509)). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (K(d)) of the K14A and F22A mutants were 3.2 and 4.0 ?M, respectively, similar to 2.6 ?M measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI ((505)QEAKL(509)) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10-20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1.

  12. Testicular myeloid sarcoma: case report

    PubMed Central

    Zago, Luzia Beatriz Ribeiro; Ladeia, Antônio Alexandre Lisbôa; Etchebehere, Renata Margarida; de Oliveira, Leonardo Rodrigues

    2013-01-01

    Myeloid sarcomas are extramedullary solid tumors composed of immature granulocytic precursor cells. In association with acute myeloid leukemia and other myeloproliferative disorders, they may arise concurrently with compromised bone marrow related to acute myeloid leukemia, as a relapsed presentation, or occur as the first manifestation. The testicles are considered to be an uncommon site for myeloid sarcomas. No therapeutic strategy has been defined as best but may include chemotherapy, radiotherapy and/or hematopoietic stem cell transplantation. This study reports the evolution of a patient with testicular myeloid sarcoma as the first manifestation of acute myeloid leukemia. The patient initially refused medical treatment and died five months after the clinical condition started. PMID:23580888

  13. Gene structure, cDNA characterization and RNAi-based functional analysis of a myeloid differentiation factor 88 homolog in Tenebrio molitor larvae exposed to Staphylococcus aureus infection.

    PubMed

    Patnaik, Bharat Bhusan; Patnaik, Hongray Howrelia; Seo, Gi Won; Jo, Yong Hun; Lee, Yong Seok; Lee, Bok Luel; Han, Yeon Soo

    2014-10-01

    Myeloid differentiation factor 88 (MyD88), an intracellular adaptor protein involved in Toll/Toll-like receptor (TLR) signal processing, triggers activation of nuclear factor-kappaB (NF-κB) transcription factors. In the present study, we analyzed the gene structure and biological function of MyD88 in a coleopteran insect, Tenebrio molitor (TmMyD88). The TmMyD88 gene was 1380 bp in length and consisted of five exons and four introns. The 5'-flanking sequence revealed several putative transcription factor binding sites, such as STAT-4, AP-1, cJun, cfos, NF-1 and many heat shock factor binding elements. The cDNA contained a typical death domain, a conservative Toll-like interleukin-1 receptor (TIR) domain, and a C-terminal extension (CTE). The TmMyD88 TIR domain showed three significantly conserved motifs for interacting with the TIR domain of TLRs. TmMyD88 was grouped within the invertebrate cluster of the phylogenetic tree and shared 75% sequence identity with the TIR domain of Tribolium castaneum MyD88. Homology modeling of the TmMyD88 TIR domain revealed five parallel β-strands surrounded by five α-helices that adopted loop conformations to function as an adaptor. TmMyD88 expression was upregulated 7.3- and 4.79-fold after 12 and 6h, respectively, of challenge with Staphylococcus aureus and fungal β-1,3 glucan. Silencing of the TmMyD88 transcript by RNA interference led to reduced resistance of the host to infection by S. aureus. These results indicate that TmMyD88 is required for survival against Staphylococcus infection. PMID:24755285

  14. Gene structure, cDNA characterization and RNAi-based functional analysis of a myeloid differentiation factor 88 homolog in Tenebrio molitor larvae exposed to Staphylococcus aureus infection.

    PubMed

    Patnaik, Bharat Bhusan; Patnaik, Hongray Howrelia; Seo, Gi Won; Jo, Yong Hun; Lee, Yong Seok; Lee, Bok Luel; Han, Yeon Soo

    2014-10-01

    Myeloid differentiation factor 88 (MyD88), an intracellular adaptor protein involved in Toll/Toll-like receptor (TLR) signal processing, triggers activation of nuclear factor-kappaB (NF-κB) transcription factors. In the present study, we analyzed the gene structure and biological function of MyD88 in a coleopteran insect, Tenebrio molitor (TmMyD88). The TmMyD88 gene was 1380 bp in length and consisted of five exons and four introns. The 5'-flanking sequence revealed several putative transcription factor binding sites, such as STAT-4, AP-1, cJun, cfos, NF-1 and many heat shock factor binding elements. The cDNA contained a typical death domain, a conservative Toll-like interleukin-1 receptor (TIR) domain, and a C-terminal extension (CTE). The TmMyD88 TIR domain showed three significantly conserved motifs for interacting with the TIR domain of TLRs. TmMyD88 was grouped within the invertebrate cluster of the phylogenetic tree and shared 75% sequence identity with the TIR domain of Tribolium castaneum MyD88. Homology modeling of the TmMyD88 TIR domain revealed five parallel β-strands surrounded by five α-helices that adopted loop conformations to function as an adaptor. TmMyD88 expression was upregulated 7.3- and 4.79-fold after 12 and 6h, respectively, of challenge with Staphylococcus aureus and fungal β-1,3 glucan. Silencing of the TmMyD88 transcript by RNA interference led to reduced resistance of the host to infection by S. aureus. These results indicate that TmMyD88 is required for survival against Staphylococcus infection.

  15. 21 CFR 870.4290 - Cardiopulmonary bypass adaptor, stopcock, manifold, or fitting.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cardiopulmonary bypass adaptor, stopcock, manifold... Devices § 870.4290 Cardiopulmonary bypass adaptor, stopcock, manifold, or fitting. (a) Identification. A cardiopulmonary bypass adaptor, stopcock, manifold, or fitting is a device used in cardiovascular...

  16. A liquid crystal of ascorbyl palmitate, used as vaccine platform, provides sustained release of antigen and has intrinsic pro-inflammatory and adjuvant activities which are dependent on MyD88 adaptor protein.

    PubMed

    Sánchez Vallecillo, María F; Minguito de la Escalera, María M; Aguirre, María V; Ullio Gamboa, Gabriela V; Palma, Santiago D; González-Cintado, Leticia; Chiodetti, Ana L; Soldano, Germán; Morón, Gabriel; Allemandi, Daniel A; Ardavín, Carlos; Pistoresi-Palencia, María C; Maletto, Belkys A

    2015-09-28

    Modern subunit vaccines require the development of new adjuvant strategies. Recently, we showed that CpG-ODN formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate (Coa-ASC16) is an attractive system for promoting an antigen-specific immune response to weak antigens. Here, we showed that after subcutaneous injection of mice with near-infrared fluorescent dye-labeled OVA antigen formulated with Coa-ASC16, the dye-OVA was retained at the injection site for a longer period than when soluble dye-OVA was administered. Coa-ASC16 alone elicited a local inflammation, but how this material triggers this response has not been described yet. Although it is known that some materials used as a platform are not immunologically inert, very few studies have directly focused on this topic. In this study, we explored the underlying mechanisms concerning the interaction between Coa-ASC16 and the immune system and we found that the whole inflammatory response elicited by Coa-ASC16 (leukocyte recruitment and IL-1β, IL-6 and IL-12 production) was dependent on the MyD88 protein. TLR2, TLR4, TLR7 and NLRP3-inflammasome signaling were not required for induction of this inflammatory response. Coa-ASC16 induced local release of self-DNA, and in TLR9-deficient mice IL-6 production was absent. In addition, Coa-ASC16 revealed an intrinsic adjuvant activity which was affected by MyD88 and IL-6 absence. Taken together these results indicate that Coa-ASC16 used as a vaccine platform is effective due to the combination of the controlled release of antigen and its intrinsic pro-inflammatory activity. Understanding how Coa-ASC16 works might have significant implications for rational vaccine design. PMID:26188153

  17. Anti-adaptors use distinct modes of binding to inhibit the RssB-dependent turnover of RpoS (σS) by ClpXP

    PubMed Central

    Micevski, Dimce; Zammit, Jessica E.; Truscott, Kaye N.; Dougan, David A.

    2015-01-01

    In Escherichia coli, σS is the master regulator of the general stress response. The level of σS changes in response to multiple stress conditions and it is regulated at many levels including protein turnover. In the absence of stress, σS is rapidly degraded by the AAA+ protease, ClpXP in a regulated manner that depends on the adaptor protein RssB. This two-component response regulator mediates the recognition of σS and its delivery to ClpXP. The turnover of σS however, can be inhibited in a stress specific manner, by one of three anti-adaptor proteins. Each anti-adaptor binds to RssB and inhibits its activity, but how this is achieved is not fully understood at a molecular level. Here, we describe details of the interaction between each anti-adaptor and RssB that leads to the stabilization of σS. By defining the domains of RssB using partial proteolysis we demonstrate that each anti-adaptor uses a distinct mode of binding to inhibit RssB activity. IraD docks specifically to the N-terminal domain of RssB, IraP interacts primarily with the C-terminal domain, while IraM interacts with both domains. Despite these differences in binding, we propose that docking of each anti-adaptor induces a conformational change in RssB, which resembles the inactive dimer of RssB. This dimer-like state of RssB not only prevents substrate binding but also triggers substrate release from a pre-bound complex. PMID:25988182

  18. UIF, a New mRNA export adaptor that works together with REF/ALY, requires FACT for recruitment to mRNA.

    PubMed

    Hautbergue, Guillaume M; Hung, Ming-Lung; Walsh, Matthew J; Snijders, Ambrosius P L; Chang, Chung-Te; Jones, Rachel; Ponting, Chris P; Dickman, Mark J; Wilson, Stuart A

    2009-12-01

    Messenger RNA (mRNA) export adaptors play an important role in the transport of mRNA from the nucleus to the cytoplasm. They couple early mRNA processing events such as 5' capping and 3' end formation with loading of the TAP/NXF1 export receptor onto mRNA. The canonical adaptor REF/ALY/Yra1 is recruited to mRNA via UAP56 and subsequently delivers the mRNA to NXF1 [1]. Knockdown of UAP56 [2, 3] and NXF1 [4-7] in higher eukaryotes efficiently blocks mRNA export, whereas knockdown of REF only causes a modest reduction, suggesting the existence of additional adaptors [8-10]. Here we identify a new UAP56-interacting factor, UIF, which functions as an export adaptor, binding NXF1 and delivering mRNA to the nuclear pore. REF and UIF are simultaneously found on the same mRNA molecules, and both proteins are required for efficient export of mRNA. We show that the histone chaperone FACT specifically binds UIF, but not REF, via the SSRP1 subunit, and this interaction is required for recruitment of UIF to mRNA. Together the results indicate that REF and UIF represent key human adaptors for the export of cellular mRNAs via the UAP56-NXF1 pathway.

  19. Phosphatidylcholine-specific phospholipase C and phospholipase D are respectively implicated in mitogen-activated protein kinase and nuclear factor kappaB activation in tumour-necrosis-factor-alpha-treated immature acute-myeloid-leukaemia cells.

    PubMed Central

    Plo, I; Lautier, D; Levade, T; Sekouri, H; Jaffrézou, J P; Laurent, G; Bettaïeb, A

    2000-01-01

    Tumour necrosis factor-alpha (TNFalpha) has been reported to induce potent growth inhibition of committed myeloid progenitor cells, whereas it is a potential growth stimulator of human CD34(+)CD38(-) multipotent haematopoietic cells. The present study was aimed at evaluating the respective role of two phospholipases, phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD) in the response of the CD34(+) CD38(-) KG1a cells to TNFalpha. In these cells TNFalpha triggered phosphoinositide 3-kinase (PI3K)-dependent PC hydrolysis within 4-8 min with concomitant production of both diacylglycerol (DAG) and phosphocholine (P-chol). DAG and P-chol production was accompanied by extracellular-signal-related protein kinase-1 ('ERK-1') activation and DNA-synthesis stimulation. PC-PLC stimulation was followed by PI3K-independent PLD activation with concomitant phosphatidic acid (PA) production followed by PA-derived DAG accumulation and sustained nuclear factor kappaB (NF-kappaB) activation. PLD/NF-kappaB signalling activation played no role in the TNFalpha proliferative effect and conferred no consistent protection of KG1a cells towards antileukaemic agents. Altogether these results suggest that, in KG1a cells, TNFalpha can stimulate in parallel PC-PLC and PLD, whose lipid products activate in turn mitogen-activated protein kinase (MAP kinase) and NF-kappaB signalling respectively. Finally, our study suggests that PC-PLC, but not PLD, plays a role in the TNFalpha proliferative effect in immature myeloid cells. PMID:11023832

  20. Detection of a rare BCR-ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS).

    PubMed

    Breitkopf, Susanne B; Yuan, Min; Pihan, German A; Asara, John M

    2012-10-01

    Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.

  1. Cell cycle-dependent adaptor complex for ClpXP-mediated proteolysis directly integrates phosphorylation and second messenger signals.

    PubMed

    Smith, Stephen C; Joshi, Kamal K; Zik, Justin J; Trinh, Katherine; Kamajaya, Aron; Chien, Peter; Ryan, Kathleen R

    2014-09-30

    The cell-division cycle of Caulobacter crescentus depends on periodic activation and deactivation of the essential response regulator CtrA. Although CtrA is critical for transcription during some parts of the cell cycle, its activity must be eliminated before chromosome replication because CtrA also blocks the initiation of DNA replication. CtrA activity is down-regulated both by dephosphorylation and by proteolysis, mediated by the ubiquitous ATP-dependent protease ClpXP. Here we demonstrate that proteins needed for rapid CtrA proteolysis in vivo form a phosphorylation-dependent and cyclic diguanylate (cdG)-dependent adaptor complex that accelerates CtrA degradation in vitro by ClpXP. The adaptor complex includes CpdR, a single-domain response regulator; PopA, a cdG-binding protein; and RcdA, a protein whose activity cannot be predicted. When CpdR is unphosphorylated and when PopA is bound to cdG, they work together with RcdA in an all-or-none manner to reduce the Km of CtrA proteolysis 10-fold. We further identified a set of amino acids in the receiver domain of CtrA that modulate its adaptor-mediated degradation in vitro and in vivo. Complex formation between PopA and CtrA depends on these amino acids, which reside on alpha-helix 1 of the CtrA receiver domain, and on cdG binding by PopA. These results reveal that each accessory factor plays an essential biochemical role in the regulated proteolysis of CtrA and demonstrate, to our knowledge, the first example of a multiprotein, cdG-dependent proteolytic adaptor.

  2. Cell cycle-dependent adaptor complex for ClpXP-mediated proteolysis directly integrates phosphorylation and second messenger signals

    PubMed Central

    Smith, Stephen C.; Joshi, Kamal K.; Zik, Justin J.; Trinh, Katherine; Kamajaya, Aron; Chien, Peter; Ryan, Kathleen R.

    2014-01-01

    The cell-division cycle of Caulobacter crescentus depends on periodic activation and deactivation of the essential response regulator CtrA. Although CtrA is critical for transcription during some parts of the cell cycle, its activity must be eliminated before chromosome replication because CtrA also blocks the initiation of DNA replication. CtrA activity is down-regulated both by dephosphorylation and by proteolysis, mediated by the ubiquitous ATP-dependent protease ClpXP. Here we demonstrate that proteins needed for rapid CtrA proteolysis in vivo form a phosphorylation-dependent and cyclic diguanylate (cdG)-dependent adaptor complex that accelerates CtrA degradation in vitro by ClpXP. The adaptor complex includes CpdR, a single-domain response regulator; PopA, a cdG-binding protein; and RcdA, a protein whose activity cannot be predicted. When CpdR is unphosphorylated and when PopA is bound to cdG, they work together with RcdA in an all-or-none manner to reduce the Km of CtrA proteolysis 10-fold. We further identified a set of amino acids in the receiver domain of CtrA that modulate its adaptor-mediated degradation in vitro and in vivo. Complex formation between PopA and CtrA depends on these amino acids, which reside on alpha-helix 1 of the CtrA receiver domain, and on cdG binding by PopA. These results reveal that each accessory factor plays an essential biochemical role in the regulated proteolysis of CtrA and demonstrate, to our knowledge, the first example of a multiprotein, cdG-dependent proteolytic adaptor. PMID:25197043

  3. Genetically Modified T-cell Immunotherapy in Treating Patients With Relapsed or Refractory Acute Myeloid Leukemia

    ClinicalTrials.gov

    2016-08-10

    Adult Acute Myeloid Leukemia in Remission; Donor; Early Relapse of Acute Myeloid Leukemia; Late Relapse of Acute Myeloid Leukemia; Recurrent Adult Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia

  4. Structure-Activity Relationship Studies of Mitogen Activated Protein Kinase Interacting Kinase (MNK) 1 and 2 and BCR-ABL1 Inhibitors Targeting Chronic Myeloid Leukemic Cells.

    PubMed

    Cherian, Joseph; Nacro, Kassoum; Poh, Zhi Ying; Guo, Samantha; Jeyaraj, Duraiswamy A; Wong, Yun Xuan; Ho, Melvyn; Yang, Hai Yan; Joy, Joma Kanikadu; Kwek, Zekui Perlyn; Liu, Boping; Wee, John Liang Kuan; Ong, Esther H Q; Choong, Meng Ling; Poulsen, Anders; Lee, May Ann; Pendharkar, Vishal; Ding, Li Jun; Manoharan, Vithya; Chew, Yun Shan; Sangthongpitag, Kanda; Lim, Sharon; Ong, S Tiong; Hill, Jeffrey; Keller, Thomas H

    2016-04-14

    Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented. PMID:27011159

  5. Deficient IFN Signaling by Myeloid Cells Leads to MAVS-Dependent Virus-Induced Sepsis

    PubMed Central

    Pinto, Amelia K.; Ramos, Hilario J.; Wu, Xiaobo; Shrestha, Bimmi; Gorman, Matthew; Kim, Kristin Y.; Suthar, Mehul S.; Atkinson, John P.; Gale Jr, Michael; Diamond, Michael S.

    2014-01-01

    The type I interferon (IFN) signaling response limits infection of many RNA and DNA viruses. To define key cell types that require type I IFN signaling to orchestrate immunity against West Nile virus (WNV), we infected mice with conditional deletions of the type I IFN receptor (IFNAR) gene. Deletion of the Ifnar gene in subsets of myeloid cells resulted in uncontrolled WNV replication, vasoactive cytokine production, sepsis, organ damage, and death that were remarkably similar to infection of Ifnar−/− mice completely lacking type I IFN signaling. In Mavs−/−×Ifnar−/− myeloid cells and mice lacking both Ifnar and the RIG-I-like receptor adaptor gene Mavs, cytokine production was muted despite high levels of WNV infection. Thus, in myeloid cells, viral infection triggers signaling through MAVS to induce proinflammatory cytokines that can result in sepsis and organ damage. Viral pathogenesis was caused in part by massive complement activation, as liver damage was minimized in animals lacking complement components C3 or factor B or treated with neutralizing anti-C5 antibodies. Disease in Ifnar−/− and CD11c Cre+Ifnarf/f mice also was facilitated by the proinflammatory cytokine TNF-α, as blocking antibodies diminished complement activation and prolonged survival without altering viral burden. Collectively, our findings establish the dominant role of type I IFN signaling in myeloid cells in restricting virus infection and controlling pathological inflammation and tissue injury. PMID:24743949

  6. Platelet activation via the collagen receptor GPVI is not altered in platelets from chronic myeloid leukaemia patients despite the presence of the constitutively phosphorylated adapter protein CrkL.

    PubMed

    Best, D; Pasquet, S; Littlewood, T J; Brunskill, S J; Pallister, C J; Watson, S P

    2001-03-01

    In this study, we show that the adapter proteins CrkL and Cbl undergo increases in tyrosine phosphorylation and form an intracellular complex in platelets stimulated with the snake venom toxin convulxin, a selective agonist at the collagen receptor glycoprotein VI (GPVI). Constitutive tyrosine phosphorylation of CrkL has previously been reported in platelets from chronic myeloid leukaemia (CML) patients. This was confirmed in the present study, and shown to result in a weak constitutive association of CrkL with Cbl and a number of other unidentified tyrosine-phosphorylated proteins. There was no further increase in phosphorylation of CrkL in CML platelets in response to GPVI activation, whereas phosphorylation of Cbl and its association with CrkL were potentiated. In addition, this was accompanied by a small increase in p42/ 44 mapkinase (MAPK) activity in CML platelets. The functional consequence of the presence of constitutively phosphorylated proteins in CML platelets was investigated by measurement of aminophospholipid exposure and alpha-granule secretion. This revealed little alteration in the concentration-response curves for either in CML platelets stimulated via GPVI, although maximal levels of P-selectin were depressed. Despite the minimal effect on platelet activation in CML patients, we cannot exclude a role for CrkL or Cbl in signal transduction pathways stimulated via GPVI. PMID:11260061

  7. Myeloid derived suppressor cells

    PubMed Central

    Waldron, Todd J.; Quatromoni, Jon G.; Karakasheva, Tatiana A.; Singhal, Sunil; Rustgi, Anil K.

    2013-01-01

    The goal of achieving measurable response with cancer immunotherapy requires counteracting the immunosuppressive characteristics of tumors. One of the mechanisms that tumors utilize to escape immunosurveillance is the activation of myeloid derived suppressor cells (MDSCs). Upon activation by tumor-derived signals, MDSCs inhibit the ability of the host to mount an anti-tumor immune response via their capacity to suppress both the innate and adaptive immune systems. Despite their relatively recent discovery and characterization, anti-MDSC agents have been identified, which may improve immunotherapy efficacy. PMID:23734336

  8. SH2B1beta adaptor is a key enhancer of RET tyrosine kinase signaling.

    PubMed

    Donatello, S; Fiorino, A; Degl'Innocenti, D; Alberti, L; Miranda, C; Gorla, L; Bongarzone, I; Rizzetti, M G; Pierotti, M A; Borrello, M G

    2007-10-01

    The RET gene encodes two main isoforms of a receptor tyrosine kinase (RTK) implicated in various human diseases. Activating germ-line point mutations are responsible for multiple endocrine neoplasia type 2-associated medullary thyroid carcinomas, inactivating germ-line mutations for Hirschsprung's disease, while somatic rearrangements (RET/PTCs) are specific to papillary thyroid carcinomas. SH2B1beta, a member of the SH2B adaptors family, and binding partner for several RTKs, has been recently described to interact with proto-RET. Here, we show that both RET isoforms and its oncogenic derivatives bind to SH2B1beta through the SRC homology 2 (SH2) domain and a kinase activity-dependent mechanism. As a result, RET phosphorylates SH2B1beta, which in turn enhances its autophosphorylation, kinase activity, and downstream signaling. RET tyrosine residues 905 and 981 are important determinants for functional binding of the adaptor, as removal of both autophosphorylation sites displaces its recruitment. Binding of SH2B1beta appears to protect RET from dephosphorylation by protein tyrosine phosphatases, and might represent a likely mechanism contributing to its upregulation. Thus, overexpression of SH2B1beta, by enhancing phosphorylation/activation of RET transducers, potentiates the cellular differentiation and the neoplastic transformation thereby induced, and counteracts the action of RET inhibitors. Overall, our results identify SH2B1beta as a key enhancer of RET physiologic and pathologic activities.

  9. The Hypoxic Regulator of Sterol Synthesis Nro1 Is a Nuclear Import Adaptor

    SciTech Connect

    T Yeh; C Lee; L Amzel; P Espenshade; M Bianchet

    2011-12-31

    Fission yeast protein Sre1, the homolog of the mammalian sterol regulatory element-binding protein (SREBP), is a hypoxic transcription factor required for sterol homeostasis and low-oxygen growth. Nro1 regulates the stability of the N-terminal transcription factor domain of Sre1 (Sre1N) by inhibiting the action of the prolyl 4-hydroxylase-like Ofd1 in an oxygen-dependent manner. The crystal structure of Nro1 determined at 2.2 {angstrom} resolution shows an all-{alpha}-helical fold that can be divided into two domains: a small N-terminal domain, and a larger C-terminal HEAT-repeat domain. Follow-up studies showed that Nro1 defines a new class of nuclear import adaptor that functions both in Ofd1 nuclear localization and in the oxygen-dependent inhibition of Ofd1 to control the hypoxic response.

  10. Doa1 is a MAD adaptor for Cdc48

    PubMed Central

    Zhang, Ting

    2016-01-01

    Dislocation of polypeptides from the mitochondrial outer membrane by the p97/Cdc48–Ufd1–Npl4 adenosine triphosphatase complex is essential for mitochondria-associated degradation and Parkin-mediated mitophagy. In this issue, Wu et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201510098) identify Doa1 as a pivotal adaptor that recruits substrates to Cdc48 for processing. PMID:27044894

  11. The AP-2 Adaptor β2 Appendage Scaffolds Alternate Cargo Endocytosis

    PubMed Central

    Keyel, Peter A.; Thieman, James R.; Roth, Robyn; Erkan, Elif; Everett, Eric T.; Watkins, Simon C.; Heuser, John E.

    2008-01-01

    The independently folded appendages of the large α and β2 subunits of the endocytic adaptor protein (AP)-2 complex coordinate proper assembly and operation of endocytic components during clathrin-mediated endocytosis. The β2 subunit appendage contains a common binding site for β-arrestin or the autosomal recessive hypercholesterolemia (ARH) protein. To determine the importance of this interaction surface in living cells, we used small interfering RNA-based gene silencing. The effect of extinguishing β2 subunit expression on the internalization of transferrin is considerably weaker than an AP-2 α subunit knockdown. We show the mild sorting defect is due to fortuitous substitution of the β2 chain with the closely related endogenous β1 subunit of the AP-1 adaptor complex. Simultaneous silencing of both β1 and β2 subunit transcripts recapitulates the strong α subunit RNA interference (RNAi) phenotype and results in loss of ARH from endocytic clathrin coats. An RNAi-insensitive β2-yellow fluorescent protein (YFP) expressed in the β1 + β2-silenced background restores cellular AP-2 levels, robust transferrin internalization, and ARH colocalization with cell surface clathrin. The importance of the β appendage platform subdomain over clathrin for precise deposition of ARH at clathrin assembly zones is revealed by a β2-YFP with a disrupted ARH binding interface, which does not restore ARH colocalization with clathrin. We also show a β-arrestin 1 mutant, which engages coated structures in the absence of any G protein-coupled receptor stimulation, colocalizes with β2-YFP and clathrin even in the absence of an operational clathrin binding sequence. These findings argue against ARH and β-arrestin binding to a site upon the β2 appendage platform that is later obstructed by polymerized clathrin. We conclude that ARH and β-arrestin depend on a privileged β2 appendage site for proper cargo recruitment to clathrin bud sites. PMID:18843039

  12. General Information about Childhood Acute Myeloid Leukemia and Other Myeloid Malignancies

    MedlinePlus

    ... Other Myeloid Malignancies Treatment (PDQ®)–Patient Version General Information About Childhood Acute Myeloid Leukemia and Other Myeloid ... the PDQ Pediatric Treatment Editorial Board . Clinical Trial Information A clinical trial is a study to answer ...

  13. De Novo Acute Myeloid Leukemia in Adults: Suppression of MicroRNA-223 is Independent of LMO2 Protein Expression BUT Associate With Adverse Cytogenetic Profile and Undifferentiated Blast Morphology.

    PubMed

    Akhter, Ariz; Patel, Jay L; Farooq, Fahad; Qureshi, Abid; Taher-Rad, Meer-Shahbani; Elyamany, Ghaleb; Al-Zahrani, Ali M; Rashid-Kolvear, Fariborz; Mansoor, Adnan

    2015-01-01

    MicroRNA (MIR) signatures are critical to pathobiology and prognosis of acute myeloid leukemia (AML). MIR223 is expressed at low levels in progenitor cells, whereas high expression is induced by granulocytic differentiation. Novel-targeted therapies through epigenetic manipulation of MIR223 regulators are being explored in AML but correlative data between established clinical prognostic markers and MIR223 expression in AML is lacking. MIR223 has inverse relationship with LMO2 protein expression and our group has recently reported a close association between LMO2 protein expression and chromosomal findings in AML patients. In this study, we examined the expression of MIR223 in a large cohort of AML patients and correlated it with LMO2 protein expression, cytogenetic data, degree of differentiation [French-American and British (FAB)/World Health Organization classifications], and overall survival. MIR223 expression was upregulated in only a subset of patients (37%). Suppression of MIR223 was more frequent among patients with aneuploid karyotype compared with diploid karyotype (P=0.005). In AML, not otherwise specified category, AML with maturation (FAB-M2) showed higher levels of MIR223 when compared with either AML without maturation (FAB M0/M1) (P=0.001); AML with monoblastic differentiation (FAB M4/M5) (P=0.004) or AML with myelodysplasia-related changes (P=0.011). Among cytogenetic risk groups, suppression of MIR223 was universal (>95%) in high-risk group when compared with intermediate-risk group (P=0.004). No correlation between MIR223 and LMO2 protein expression was identified. In conclusion, we have shown that suppression of MIR223 expression, as compared with controls, is associated with lack of differentiation and adverse cytogenetic profile, but unrelated with LMO2 protein expression or overall survival. PMID:25710580

  14. De Novo Acute Myeloid Leukemia in Adults: Suppression of MicroRNA-223 is Independent of LMO2 Protein Expression BUT Associate With Adverse Cytogenetic Profile and Undifferentiated Blast Morphology.

    PubMed

    Akhter, Ariz; Patel, Jay L; Farooq, Fahad; Qureshi, Abid; Taher-Rad, Meer-Shahbani; Elyamany, Ghaleb; Al-Zahrani, Ali M; Rashid-Kolvear, Fariborz; Mansoor, Adnan

    2015-01-01

    MicroRNA (MIR) signatures are critical to pathobiology and prognosis of acute myeloid leukemia (AML). MIR223 is expressed at low levels in progenitor cells, whereas high expression is induced by granulocytic differentiation. Novel-targeted therapies through epigenetic manipulation of MIR223 regulators are being explored in AML but correlative data between established clinical prognostic markers and MIR223 expression in AML is lacking. MIR223 has inverse relationship with LMO2 protein expression and our group has recently reported a close association between LMO2 protein expression and chromosomal findings in AML patients. In this study, we examined the expression of MIR223 in a large cohort of AML patients and correlated it with LMO2 protein expression, cytogenetic data, degree of differentiation [French-American and British (FAB)/World Health Organization classifications], and overall survival. MIR223 expression was upregulated in only a subset of patients (37%). Suppression of MIR223 was more frequent among patients with aneuploid karyotype compared with diploid karyotype (P=0.005). In AML, not otherwise specified category, AML with maturation (FAB-M2) showed higher levels of MIR223 when compared with either AML without maturation (FAB M0/M1) (P=0.001); AML with monoblastic differentiation (FAB M4/M5) (P=0.004) or AML with myelodysplasia-related changes (P=0.011). Among cytogenetic risk groups, suppression of MIR223 was universal (>95%) in high-risk group when compared with intermediate-risk group (P=0.004). No correlation between MIR223 and LMO2 protein expression was identified. In conclusion, we have shown that suppression of MIR223 expression, as compared with controls, is associated with lack of differentiation and adverse cytogenetic profile, but unrelated with LMO2 protein expression or overall survival.

  15. Structural Analysis of the Interaction between Dishevelled2 and Clathrin AP-2 Adaptor, A Critical Step in Noncanonical Wnt Signaling

    SciTech Connect

    Yu, Anan; Xing, Yi; Harrison, Stephen C.; Kirchhausen, Tomas

    2010-10-14

    Wnt association with its receptor, Frizzled (Fz), and recruitment by the latter of an adaptor, Dishevelled (Dvl), initiates signaling through at least two distinct pathways (canonical and noncanonical). Endocytosis and compartmentalization help determine the signaling outcome. Our previous work has shown that Dvl2 links at least one Frizzled family member (Fz4) to clathrin-mediated endocytosis by interacting with the {mu}2 subunit of the AP-2 clathrin adaptor, through both a classical endocytic tyrosine motif and a so-called DEP domain. We report here the crystal structure of a chimeric protein that mimics the Dvl2-{mu}2 complex. The DEP domain binds at one end of the elongated, C-terminal domain of {mu}2. This domain:domain interface shows that parts of the {mu}2 surface distinct from the tyrosine-motif site can help recruit specific receptors or adaptors into a clathrin coated pit. Mutation of residues at the DEP-{mu}2 contact or in the tyrosine motif reduce affinity of Dvl2 for {mu}2 and block efficient internalization of Fz4 in response to ligation by Wnt5a. The crystal structure has thus allowed us to identify the specific interaction that leads to Frizzled uptake and to downstream, noncanonical signaling events.

  16. Isolation of pathogen-induced Chinese cabbage genes by subtractive hybridization employing selective adaptor ligation.

    PubMed

    Ryang, Seung Ho; Chung, Sam Young; Lee, Sung Hee; Cha, Jae Soon; Yong Kim, Hak; Cho, Tae Ju

    2002-12-01

    We have developed a subtractive cloning method in which target sequences are effectively enriched by selective adaptor ligation and PCR after hybridization. In this method both tester and driver DNAs are digested with RsaI, ligated with the linker DNA containing a KpnI recognition site, and amplified by PCR. The tester DNA samples are divided into two aliquots, each digested with either RsaI or KpnI. The two DNA samples are then combined and hybridized with an excess of the driver DNA retaining the linker. After hybridization, the DNA mixture is ligated to a new adaptor compatible only with double-stranded tester/tester DNAs. Therefore, only the tester/tester is selectively amplified in subsequent PCR. This also leads to complete elimination of the tester DNA hybridized with driver DNA from the tester DNA population. Although our protocol employs enzymatic treatments, the efficiency of the enzymatic treatments does not affect the subtraction efficiency. This new subtractive enrichment method was applied to isolate Chinese cabbage defense-related genes induced by Pseudomonas syringae pv. tomato (Pst), which elicits a hypersensitive response in Chinese cabbage. After two or three rounds of subtractive hybridization, the sequences of enriched DNAs were determined and examined by BLAST analysis. Northern blot hybridization showed that 12 of the 19 genes analyzed were strongly induced by Pst treatment. Among the 12 Pst-induced genes five represent pathogenesis-related genes encoding PR1a, two chitinases, a thaumatin-like protein, and a PR4 protein. Other Pst-induced genes include two cytochrome P450 genes responsible for glucosinolate biosynthesis, a disease resistance gene homolog, and several genes encoding proteins with unknown functions.

  17. What Is Chronic Myeloid Leukemia?

    MedlinePlus

    ... leukemia? Next Topic Normal bone marrow and blood What is chronic myeloid leukemia? Cancer starts when cells ... their treatment is the same as for adults. What is leukemia? Leukemia is a cancer that starts ...

  18. Crizotinib resistance in acute myeloid leukemia with inv(2)(p23q13)/RAN binding protein 2 (RANBP2) anaplastic lymphoma kinase (ALK) fusion and monosomy 7.

    PubMed

    Takeoka, Kayo; Okumura, Atsuko; Maesako, Yoshitomo; Akasaka, Takashi; Ohno, Hitoshi

    2015-03-01

    This is the first report on the development of a p.G1269A mutation within the kinase domain (KD) of ALK after crizotinib treatment in RANBP2-ALK acute myeloid leukemia (AML). An elderly woman with AML with an inv(2)(p23q13)/RANBP2-ALK and monosomy 7 was treated with crizotinib. After a short-term hematological response and the restoration of normal hematopoiesis, she experienced a relapse of AML. Fluorescence in situ hybridization using the ALK break-apart probe confirmed the inv(2)(p23q13), while G-banded karyotyping revealed the deletion of a segment of the short arm of chromosome 1 [del(1)(p13p22)] after crizotinib therapy. The ALK gene carried a heterozygous mutation at the nucleotide position g.716751G>C within exon 25, causing the p.G1269A amino acid substitution within the ALK-KD. Reverse transcriptase PCR revealed that the mutated ALK allele was selectively transcribed and the mutation occurred in the ALK allele rearranged with RANBP2. As both the del(1)(p13p22) at the cytogenetic level and p.G1269A at the nucleotide level newly appeared after crizotinib treatment, it is likely that they were secondarily acquired alterations involved in crizotinib resistance. Although secondary genetic abnormalities in ALK are most frequently described in non-small cell lung cancers harboring an ALK alteration, this report suggests that an ALK-KD mutation can occur independently of the tumor cell type or fusion partner after crizotinib treatment.

  19. Detection of minimal residual disease in NPM1-mutated acute myeloid leukemia by next-generation sequencing.

    PubMed

    Salipante, Stephen J; Fromm, Jonathan R; Shendure, Jay; Wood, Brent L; Wu, David

    2014-11-01

    Detection of minimal residual disease predicts adverse outcome in patients with acute myeloid leukemia. Currently, minimal residual disease may be detected by RQ-PCR or flow cytometry, both of which have practical and diagnostic limitations. Here, we describe a next-generation sequencing assay for minimal residual disease detection in NPM1-mutated acute myeloid leukemia, which encompasses ∼60% of patients with normal karyotype acute myeloid leukemia. Exon 12 of NPM1 was PCR amplified using sequencing adaptor-linked primers and deep sequenced to enable detection of low-prevalence, acute myeloid leukemia-specific activating mutations. We benchmarked our results against flow cytometry, the standard of care for acute myeloid leukemia minimal residual disease diagnosis at our institution. The performance of both approaches was evaluated using defined dilutions of an NPM1 mutation-positive cell line and longitudinal clinical samples from acute myeloid leukemia patients. Using defined control material, we found this assay sensitive to approximately 0.001% mutant cells, outperforming flow cytometry by an order of magnitude. Next-generation sequencing was precise and semiquantitative over four orders of magnitude. In 22 longitudinal samples from six acute myeloid leukemia patients, next-generation sequencing detected minimal residual disease in all samples deemed negative by flow cytometry. Further, in one-third of patients, sequencing detected alternate NPM1 mutations in addition to the patient's index mutation, consistent with tumor heterogeneity. Next-generation sequencing provides information without prior knowledge of NPM1 mutation subtype or validation of allele-specific probes as required for RQ-PCR assays, and without generation and interpretation of complex multidimensional flow cytometry data. This approach may complement current technologies to enhance patient-specific clinical decision-making.

  20. STING Requires the Adaptor TRIF to Trigger Innate Immune Responses to Microbial Infection.

    PubMed

    Wang, Xin; Majumdar, Tanmay; Kessler, Patricia; Ozhegov, Evgeny; Zhang, Ying; Chattopadhyay, Saurabh; Barik, Sailen; Sen, Ganes C

    2016-09-14

    The intracellular microbial nucleic acid sensors, TLR3 and STING, recognize pathogen molecules and signal to activate the interferon pathway. The TIR-domain containing protein TRIF is the sole adaptor of TLR3. Here, we report an essential role for TRIF in STING signaling: various activators of STING could not induce genes in the absence of TRIF. TRIF and STING interacted directly, through their carboxy-terminal domains, to promote STING dimerization, intermembrane translocation, and signaling. Herpes simplex virus (HSV), which triggers the STING signaling pathway and is controlled by it, replicated more efficiently in the absence of TRIF, and HSV-infected TRIF(-/-) mice displayed pronounced pathology. Our results indicate that defective STING signaling may be responsible for the observed genetic association between TRIF mutations and herpes simplex encephalitis in patients. PMID:27631700

  1. Decitabine and Bortezomib in Treating Patients With Acute Myeloid Leukemia

    ClinicalTrials.gov

    2014-11-06

    Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Recurrent Adult Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

  2. Decitabine in Treating Patients With Previously Untreated Acute Myeloid Leukemia

    ClinicalTrials.gov

    2016-05-18

    Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

  3. Hydroxylated Dimeric Naphthoquinones Increase the Generation of Reactive Oxygen Species, Induce Apoptosis of Acute Myeloid Leukemia Cells and Are Not Substrates of the Multidrug Resistance Proteins ABCB1 and ABCG2

    PubMed Central

    Lapidus, Rena G.; Carter-Cooper, Brandon A.; Sadowska, Mariola; Choi, Eun Yong; Wonodi, Omasiri; Muvarak, Nidal; Natarajan, Karthika; Pidugu, Lakshmi S.; Jaiswal, Anil; Toth, Eric A.; Rassool, Feyruz V.; Etemadi, Arash; Sausville, Edward A.; Baer, Maria R.; Emadi, Ashkan

    2016-01-01

    Selective targeting of the oxidative state, which is a tightly balanced fundamental cellular property, is an attractive strategy for developing novel anti-leukemic chemotherapeutics with potential applications in the treatment of acute myeloid leukemia (AML), a molecularly heterogeneous disease. Dimeric naphthoquinones (BiQs) with the ability to undergo redox cycling and to generate reactive oxygen species (ROS) in cancer cells are a novel class of compounds with unique characteristics that make them excellent candidates to be tested against AML cells. We evaluated the effect of two BiQ analogues and one monomeric naphthoquinone in AML cell lines and primary cells from patients. All compounds possess one halogen and one hydroxyl group on the quinone cores. Dimeric, but not monomeric, naphthoquinones demonstrated significant anti-AML activity in the cell lines and primary cells from patients with favorable therapeutic index compared to normal hematopoietic cells. BiQ-1 effectively inhibited clonogenicity and induced apoptosis as measured by Western blotting and Annexin V staining and mitochondrial membrane depolarization by flow cytometry. BiQ-1 significantly enhances intracellular ROS levels in AML cells and upregulates expression of key anti-oxidant protein, Nrf2. Notably, systemic exposure to BiQ-1 was well tolerated in mice. In conclusion, we propose that BiQ-induced therapeutic augmentation of ROS in AML cells with dysregulation of antioxidants kill leukemic cells while normal cells remain relatively intact. Further studies are warranted to better understand this class of potential chemotherapeutics. PMID:26797621

  4. A C-terminal mutant of CCAAT-enhancer-binding protein α (C/EBPα-Cm) downregulates Csf1r, a potent accelerator in the progression of acute myeloid leukemia with C/EBPα-Cm.

    PubMed

    Togami, Katsuhiro; Kitaura, Jiro; Uchida, Tomoyuki; Inoue, Daichi; Nishimura, Koutarou; Kawabata, Kimihito C; Nagase, Reina; Horikawa, Sayuri; Izawa, Kumi; Fukuyama, Tomofusa; Nakahara, Fumio; Oki, Toshihiko; Harada, Yuka; Harada, Hironori; Aburatani, Hiroyuki; Kitamura, Toshio

    2015-04-01

    Two types of CCAAT-enhancer-binding protein α (C/EBPα) mutants are found in acute myeloid leukemia (AML) patients: N-terminal frame-shift mutants (C/EBPα-N(m)) generating p30 as a dominant form and C-terminal basic leucine zipper domain mutants (C/EBPα-C(m)). We have previously shown that C/EBPα-K304_R323dup belonging to C/EBPα-C(m), but not C/EBPα-T60fsX159 belonging to C/EBPα-N(m), alone induced AML in mouse bone marrow transplantation (BMT) models. Here we show that various C/EBPα-C(m) mutations have a similar, but not identical, potential in myeloid leukemogenesis. Notably, like C/EBPα-K304_R323dup, any type of C/EBPα-C(m) tested (C/EBPα-S299_K304dup, K313dup, or N321D) by itself induced AML, albeit with different latencies after BMT; C/EBPα-N321D induced AML with the shortest latency. By analyzing the gene expression profiles of C/EBPα-N321D- and mock-transduced c-kit(+)Sca-1(+)Lin(-) cells, we identified Csf1r as a gene downregulated by C/EBPα-N321D. In addition, leukemic cells expressing C/EBPα-C(m) exhibited low levels of colony stimulating factor 1 receptor in mice. On the other hand, transduction with C/EBPα-N(m) did not influence Csf1r expression in c-kit(+)Sca-1(+)Lin(-) cells, implying a unique role for C/EBPα-C(m) in downregulating Csf1r. Importantly, Csf1r overexpression collaborated with C/EBPα-N321D to induce fulminant AML with leukocytosis in mouse BMT models to a greater extent than did C/EBPα-N321D alone. Collectively, these results suggest that C/EBPα-C(m)-mediated downregulation of Csf1r has a negative, rather than a positive, impact on the progression of AML involving C/EBPα-C(m), which might possibly be accelerated by additional genetic and/or epigenetic alterations inducing Csf1r upregulation.

  5. Apoptotic Efficacy of Etomoxir in Human Acute Myeloid Leukemia Cells. Cooperation with Arsenic Trioxide and Glycolytic Inhibitors, and Regulation by Oxidative Stress and Protein Kinase Activities

    PubMed Central

    Estañ, María Cristina; Calviño, Eva; Calvo, Susana; Guillén-Guío, Beatriz; Boyano-Adánez, María del Carmen; de Blas, Elena; Rial, Eduardo; Aller, Patricio

    2014-01-01

    Fatty acid synthesis and oxidation are frequently exacerbated in leukemia cells, and may therefore represent a target for therapeutic intervention. In this work we analyzed the apoptotic and chemo-sensitizing action of the fatty acid oxidation inhibitor etomoxir in human acute myeloid leukemia cells. Etomoxir caused negligible lethality at concentrations up to 100 µM, but efficaciously cooperated to cause apoptosis with the anti-leukemic agent arsenic trioxide (ATO, Trisenox), and with lower efficacy with other anti-tumour drugs (etoposide, cisplatin), in HL60 cells. Etomoxir-ATO cooperation was also observed in NB4 human acute promyelocytic cells, but not in normal (non-tumour) mitogen-stimulated human peripheral blood lymphocytes. Biochemical determinations in HL60 cells indicated that etomoxir (25–200 µM) dose-dependently inhibited mitochondrial respiration while slightly stimulating glycolysis, and only caused marginal alterations in total ATP content and adenine nucleotide pool distribution. In addition, etomoxir caused oxidative stress (increase in intracellular reactive oxygen species accumulation, decrease in reduced glutathione content), as well as pro-apoptotic LKB-1/AMPK pathway activation, all of which may in part explain the chemo-sensitizing capacity of the drug. Etomoxir also cooperated with glycolytic inhibitors (2-deoxy-D-glucose, lonidamine) to induce apoptosis in HL60 cells, but not in NB4 cells. The combined etomoxir plus 2-deoxy-D-glucose treatment did not increase oxidative stress, caused moderate decrease in net ATP content, increased the AMP/ATP ratio with concomitant drop in energy charge, and caused defensive Akt and ERK kinase activation. Apoptosis generation by etomoxir plus 2-deoxy-D-glucose was further increased by co-incubation with ATO, which is apparently explained by the capacity of ATO to attenuate Akt and ERK activation. In summary, co-treatment with etomoxir may represent an interesting strategy to increase the apoptotic

  6. Vosaroxin and Infusional Cytarabine in Treating Patients With Untreated Acute Myeloid Leukemia

    ClinicalTrials.gov

    2016-03-10

    Acute Myeloid Leukemia; Acute Myeloid Leukemia Arising From Previous Myelodysplastic Syndrome; Acute Myeloid Leukemia With Multilineage Dysplasia; Myeloid Sarcoma; Secondary Acute Myeloid Leukemia; Therapy-Related Acute Myeloid Leukemia; Therapy-Related Myelodysplastic Syndrome

  7. Evolutionary Genomics Suggests That CheV Is an Additional Adaptor for Accommodating Specific Chemoreceptors within the Chemotaxis Signaling Complex.

    PubMed

    Ortega, Davi R; Zhulin, Igor B

    2016-02-01

    Escherichia coli and Salmonella enterica are models for many experiments in molecular biology including chemotaxis, and most of the results obtained with one organism have been generalized to another. While most components of the chemotaxis pathway are strongly conserved between the two species, Salmonella genomes contain some chemoreceptors and an additional protein, CheV, that are not found in E. coli. The role of CheV was examined in distantly related species Bacillus subtilis and Helicobacter pylori, but its role in bacterial chemotaxis is still not well understood. We tested a hypothesis that in enterobacteria CheV functions as an additional adaptor linking the CheA kinase to certain types of chemoreceptors that cannot be effectively accommodated by the universal adaptor CheW. Phylogenetic profiling, genomic context and comparative protein sequence analyses suggested that CheV interacts with specific domains of CheA and chemoreceptors from an orthologous group exemplified by the Salmonella McpC protein. Structural consideration of the conservation patterns suggests that CheV and CheW share the same binding spot on the chemoreceptor structure, but have some affinity bias towards chemoreceptors from different orthologous groups. Finally, published experimental results and data newly obtained via comparative genomics support the idea that CheV functions as a "phosphate sink" possibly to off-set the over-stimulation of the kinase by certain types of chemoreceptors. Overall, our results strongly suggest that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex.

  8. Evolutionary genomics suggests that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex

    DOE PAGES

    Ortega, Davi R.; Zhulin, Igor B.; Punta, Marco

    2016-02-04

    Escherichia coli and Salmonella enterica are models for many experiments in molecular biology including chemotaxis, and most of the results obtained with one organism have been generalized to another. While most components of the chemotaxis pathway are strongly conserved between the two species, Salmonella genomes contain some chemoreceptors and an additional protein, CheV, that are not found in E. coli. The role of CheV was examined in distantly related species Bacillus subtilis and Helicobacter pylori, but its role in bacterial chemotaxis is still not well understood. We tested a hypothesis that in enterobacteria CheV functions as an additional adaptor linkingmore » the CheA kinase to certain types of chemoreceptors that cannot be effectively accommodated by the universal adaptor CheW. Phylogenetic profiling, genomic context and comparative protein sequence analyses suggested that CheV interacts with specific domains of CheA and chemoreceptors from an orthologous group exemplified by the Salmonella McpC protein. Structural consideration of the conservation patterns suggests that CheV and CheW share the same binding spot on the chemoreceptor structure, but have some affinity bias towards chemoreceptors from different orthologous groups. Finally, published experimental results and data newly obtained via comparative genomics support the idea that CheV functions as a "phosphate sink" possibly to off-set the over-stimulation of the kinase by certain types of chemoreceptors. Altogether, our results strongly suggest that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex.« less

  9. The adaptor molecules LAT and SLP-76 are specifically targeted by Yersinia to inhibit T cell activation

    PubMed Central

    Gerke, Christiane; Falkow, Stanley; Chien, Yueh-hsiu

    2005-01-01

    T cell responses are critical to the survival of Yersinia-infected animals. Yersinia have the ability to directly suppress T lymphocyte activation through the virulence factor YopH, a tyrosine phosphatase. Using single cell video microscopy and FACS analysis, here we show that even an average of one Yersinia per T cell is sufficient to inhibit or alter T cell responses. This efficient inhibition is traced to specific targeting by YopH of the adaptor proteins, linker for activation of T cells (LAT) and SH2-domain–containing leukocyte protein of 76 kD (SLP-76), which are crucial for T cell antigen receptor (TCR) signaling. A catalytically inactive YopH translocated via the type III secretory pathway from the bacteria into T cells primarily binds to LAT and SLP-76. Furthermore, among the proteins of the TCR signaling pathway, the tyrosine phosphorylation levels of LAT and SLP-76 are the most affected in T cells exposed to low numbers of Yersinia pseudotuberculosis. This is the first example showing that a pathogen targets these adaptor proteins in the TCR signaling pathway, suggesting a novel mechanism by which pathogens may efficiently alter T cell–mediated immune responses. PMID:15699071

  10. Analysis of Arf1 GTPase-dependent membrane binding and remodeling using the exomer secretory vesicle cargo adaptor

    PubMed Central

    Paczkowski, Jon E.; Fromme, J. Christopher

    2016-01-01

    Summary Protein-protein and protein-membrane interactions play a critical role in shaping biological membranes through direct physical contact with the membrane surface. This is particularly evident in many steps of membrane trafficking, in which proteins deform the membrane and induce fission to form transport carriers. The small GTPase Arf1 and related proteins have the ability to remodel membranes by insertion of an amphipathic helix into the membrane. Arf1 and the exomer cargo adaptor coordinate cargo sorting into subset of secretory vesicle carriers in the model organism Saccharomyces cerevisiae. Here, we detail the assays we used to explore the cooperative action of Arf1 and exomer to bind and remodel membranes. We expect these methods are broadly applicable to other small GTPase/effector systems where investigation of membrane binding and remodeling is of interest. PMID:27632000

  11. Analysis of Arf1 GTPase-Dependent Membrane Binding and Remodeling Using the Exomer Secretory Vesicle Cargo Adaptor.

    PubMed

    Paczkowski, Jon E; Fromme, J Christopher

    2016-01-01

    Protein-protein and protein-membrane interactions play a critical role in shaping biological membranes through direct physical contact with the membrane surface. This is particularly evident in many steps of membrane trafficking, in which proteins deform the membrane and induce fission to form transport carriers. The small GTPase Arf1 and related proteins have the ability to remodel membranes by insertion of an amphipathic helix into the membrane. Arf1 and the exomer cargo adaptor coordinate cargo sorting into subset of secretory vesicle carriers in the model organism Saccharomyces cerevisiae. Here, we detail the assays we used to explore the cooperative action of Arf1 and exomer to bind and remodel membranes. We expect these methods are broadly applicable to other small GTPase/effector systems where investigation of membrane binding and remodeling is of interest. PMID:27632000

  12. General Information about Adult Acute Myeloid Leukemia

    MedlinePlus

    ... Acute Myeloid Leukemia Treatment (PDQ®)–Patient Version General Information About Adult Acute Myeloid Leukemia Go to Health ... the PDQ Adult Treatment Editorial Board . Clinical Trial Information A clinical trial is a study to answer ...

  13. Treatment Options for Adult Acute Myeloid Leukemia

    MedlinePlus

    ... Treatment Childhood AML Treatment Research Adult Acute Myeloid Leukemia Treatment (PDQ®)–Patient Version General Information About Adult Acute Myeloid Leukemia Go to Health Professional Version Key Points Adult ...

  14. Stages of Adult Acute Myeloid Leukemia

    MedlinePlus

    ... Treatment Childhood AML Treatment Research Adult Acute Myeloid Leukemia Treatment (PDQ®)–Patient Version General Information About Adult Acute Myeloid Leukemia Go to Health Professional Version Key Points Adult ...

  15. Treatment Option Overview (Adult Acute Myeloid Leukemia)

    MedlinePlus

    ... Treatment Childhood AML Treatment Research Adult Acute Myeloid Leukemia Treatment (PDQ®)–Patient Version General Information About Adult Acute Myeloid Leukemia Go to Health Professional Version Key Points Adult ...

  16. GVHD-associated, inflammasome-mediated loss of function in adoptively transferred myeloid-derived suppressor cells.

    PubMed

    Koehn, Brent H; Apostolova, Petya; Haverkamp, Jessica M; Miller, Jeffrey S; McCullar, Valarie; Tolar, Jakub; Munn, David H; Murphy, William J; Brickey, Willie June; Serody, Jonathan S; Gabrilovich, Dmitry I; Bronte, Vincenzo; Murray, Peter J; Ting, Jenny P-Y; Zeiser, Robert; Blazar, Bruce R

    2015-09-24

    Myeloid-derived suppressor cells (MDSCs) are a naturally occurring immune regulatory population associated with inhibition of ongoing inflammatory responses. In vitro generation of MDSCs from bone marrow has been shown to enhance survival in an acute model of lethal graft-versus-host disease (GVHD). However, donor MDSC infusion only partially ameliorates GVHD lethality. In order to improve the potential therapeutic benefit and ultimately survival outcomes, we set out to investigate the fate of MDSCs after transfer in the setting of acute GVHD (aGVHD). MDSCs transferred to lethally irradiated recipients of allogeneic donor hematopoietic grafts are exposed to an intense inflammatory environment associated with aGVHD, which we now show directly undermines their suppressive capacity. Under a conditioning regimen and GVHD inflammatory settings, MDSCs rapidly lose suppressor function and their potential to inhibit GVHD lethality, which is associated with their induced conversion toward a mature inflammasome-activated state. We find even brief in vitro exposure to inflammasome-activating mediators negates the suppressive potential of cultured murine and human-derived MDSCs. Consistent with a role for the inflammasome, donor MDSCs deficient in the adaptor ASC (apoptosis-associated speck-like protein containing a CARD), which assembles inflammasome complexes, conferred improved survival of mice developing GVHD compared with wild-type donor MDSCs. These data suggest the use of MDSCs as a therapeutic approach for preventing GVHD and other systemic inflammatory conditions will be more effective when combined with approaches limiting in vivo MDSC inflammasome activation, empowering MDSCs to maintain their suppressive potential.

  17. GVHD-associated, inflammasome-mediated loss of function in adoptively transferred myeloid-derived suppressor cells

    PubMed Central

    Koehn, Brent H.; Apostolova, Petya; Haverkamp, Jessica M.; Miller, Jeffrey S.; McCullar, Valarie; Tolar, Jakub; Munn, David H.; Murphy, William J.; Brickey, Willie June; Serody, Jonathan S.; Gabrilovich, Dmitry I.; Bronte, Vincenzo; Murray, Peter J.; Ting, Jenny P.-Y.; Zeiser, Robert

    2015-01-01

    Myeloid-derived suppressor cells (MDSCs) are a naturally occurring immune regulatory population associated with inhibition of ongoing inflammatory responses. In vitro generation of MDSCs from bone marrow has been shown to enhance survival in an acute model of lethal graft-versus-host disease (GVHD). However, donor MDSC infusion only partially ameliorates GVHD lethality. In order to improve the potential therapeutic benefit and ultimately survival outcomes, we set out to investigate the fate of MDSCs after transfer in the setting of acute GVHD (aGVHD). MDSCs transferred to lethally irradiated recipients of allogeneic donor hematopoietic grafts are exposed to an intense inflammatory environment associated with aGVHD, which we now show directly undermines their suppressive capacity. Under a conditioning regimen and GVHD inflammatory settings, MDSCs rapidly lose suppressor function and their potential to inhibit GVHD lethality, which is associated with their induced conversion toward a mature inflammasome-activated state. We find even brief in vitro exposure to inflammasome-activating mediators negates the suppressive potential of cultured murine and human-derived MDSCs. Consistent with a role for the inflammasome, donor MDSCs deficient in the adaptor ASC (apoptosis-associated speck-like protein containing a CARD), which assembles inflammasome complexes, conferred improved survival of mice developing GVHD compared with wild-type donor MDSCs. These data suggest the use of MDSCs as a therapeutic approach for preventing GVHD and other systemic inflammatory conditions will be more effective when combined with approaches limiting in vivo MDSC inflammasome activation, empowering MDSCs to maintain their suppressive potential. PMID:26265697

  18. TGFα expression in myeloid malignancies

    PubMed Central

    Mirzai, Bob; Fuller, Kathy; Erber, Wendy N

    2016-01-01

    Background Transforming growth factor α (TGFα) is a peptide growth factor known to be expressed in normal haemopoiesis. It is also expressed in a range of epithelial neoplasms but has not been assessed in haemopoietic malignancies. We have performed an immunohistochemical evaluation of TGFα in acute and chronic myeloid malignancies. Methods TGFα expression was semiquantitatively assessed in 69 normal bone marrow trephines and 157 cases of myeloid malignancy using an immunohistochemical approach. Results Blast cells of myeloid origin in acute myeloid leukaemia (AML), myelodysplasia and accelerated and blast phases of chronic myeloid leukaemia (CML) were TGFα positive. In acute promyelocytic leukaemia the neoplastic cells had significantly weaker TGFα expression than seen in other forms of AML. The blast cells in CML-accelerated and blast phases were positive with similar expression to AML. Conclusions TGFα is expressed in neoplastic myeloblasts and could, therefore, be used as blast cell biomarker in diagnostic haematopathology. In addition, TGFα immunohistochemistry may be of use in identifying a therapeutic target. PMID:26984929

  19. Origin of Translation - the Hypothesis of Permanently Attached Adaptors

    NASA Astrophysics Data System (ADS)

    Tyagi, Sanjay

    1981-12-01

    A mechanism for prebiotic translation is proposed in which primeval transfer-RNA (adaptors) are assumed to be permanently associated with messenger nucleic acid molecules. Residual ‘fossil’ evidences are found to be present within the base sequences of contemporary tRNAs, suggesting the existence of inter-primal-tRNA interactions necessary for the mechanism. The structure of proposed primal-tRNA is such that it can not only choose its own amino acid in the absence of aminoacyl synthetase, but can also associate nonspecifically with adjacent primal-tRNA molecules attached to the neighbouring codons. Such associations can give rise, through cooperative binding between message and adaptors to the ‘static template surfaces’ which can direct translation of nucleotide sequences into those of amino acids. The origins of ribosomes and contemporary genetic code are suggested by this hypothesis. Proposed structures and processes are thermodynamically compatible. The approximate date of occurence of the proposed system is calculated, which is consistent with the period of occurence of the earliest organisms with ribosomes.

  20. A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly.

    PubMed

    Schmidt, Florian I; Lu, Alvin; Chen, Jeff W; Ruan, Jianbin; Tang, Catherine; Wu, Hao; Ploegh, Hidde L

    2016-05-01

    Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro-caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface. VHHASC not only impairs ASC(CARD) interactions in vitro, but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHHASC leaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHHASC-EGFP into these structures allowed the visualization of endogenous ASC(PYD) filaments for the first time. These data revealed that cross-linking of ASC(PYD) filaments via ASC(CARD) mediates the assembly of ASC foci. PMID:27069117

  1. An adaptor for C++ callbacks with C and Fortran libraries

    NASA Astrophysics Data System (ADS)

    Broeckhove, J.; Vanmechelen, K.

    2013-03-01

    Object-oriented programming using C++ is increasingly being adopted in the development of scientific codes. A recurrent issue in this regard is the interaction of newly developed codes with existing legacy libraries written in C or Fortran. Often, one needs to pass raw function pointers to such libraries' procedures for callback purposes. This is problematic as it conflicts with one of the cornerstones of object-oriented programming: the association of functions and data through objects. Currently ad hoc approaches are used to deal with this issue, but these are error-prone and lack reusability. We present a generic adaptor that is able to wrap any callable C++ entity and provide a raw function pointer that is compatible with C or Fortran library routines. This allows for an object-oriented style of programming, while interfacing with legacy libraries in a straightforward manner. Catalogue identifier: AENU_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AENU_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence /licence.html No. of lines in distributed program, including test data, etc.: 76802 No. of bytes in distributed program, including test data, etc.: 915389 Distribution format: tar.gz Programming language: C++. Computer: All. Operating system: All. Classification: 6.5, 4.14. Nature of problem: Object-oriented programming using C++ is increasingly being adopted in the development of scientific codes. A recurrent issue in this regard is the interaction of newly developed codes with existing legacy libraries written in C or Fortran. Often, one needs to pass raw function pointers to such libraries' procedures for callback purposes. This is problematic as it conflicts with one of the cornerstones of object-oriented programming: the association of functions and data through objects. Currently ad hoc approaches are used to deal with this

  2. A Big-Five Personality Profile of the Adaptor and Innovator.

    ERIC Educational Resources Information Center

    Kwang, Ng Aik; Rodrigues, Daphne

    2002-01-01

    A study explored the relationship between two creative types (adaptor and innovator) and the Big Five personality traits (extraversion, agreeableness, conscientiousness, neuroticism, and openness to experience), in 164 teachers in Singapore. Adaptors were significantly more conscientious than innovators, while innovators were significantly more…

  3. Sox4 cooperates with CREB in myeloid transformation

    PubMed Central

    Sandoval, Salemiz; Kraus, Christina; Cho, Er-Chieh; Cho, Michelle; Bies, Juraj; Manara, Elena; Accordi, Benedetta; Landaw, Elliot M.; Wolff, Linda; Pigazzi, Martina

    2012-01-01

    The cAMP response element-binding protein (CREB) is a nuclear transcription factor that is critical for normal and neoplastic hematopoiesis. Previous studies have demonstrated that CREB is a proto-oncogene whose overexpression promotes cellular proliferation in hematopoietic cells. Transgenic mice that overexpress CREB in myeloid cells develop a myeloproliferative disease with splenomegaly and aberrant myelopoiesis. However, CREB overexpressing mice do not spontaneously develop acute myeloid leukemia. In this study, we used retroviral insertional mutagenesis to identify genes that accelerate leukemia in CREB transgenic mice. Our mutagenesis screen identified several integration sites, including oncogenes Gfi1, Myb, and Ras. The Sox4 transcription factor was identified by our screen as a gene that cooperates with CREB in myeloid leukemogenesis. We show that the transduction of CREB transgenic mouse bone marrow cells with a Sox4 retrovirus increases survival and self-renewal of cells in vitro. Furthermore, leukemic blasts from the majority of acute myeloid leukemia patients have higher CREB, phosphorylated CREB, and Sox 4 protein expression. Sox4 transduction of mouse bone marrow cells results in increased expression of CREB target genes. We also demonstrate that CREB is a direct target of Sox4 by chromatin immunoprecipitation assays. These results indicate that Sox4 and CREB cooperate and contribute to increased proliferation of hematopoietic progenitor cells. PMID:22627767

  4. The role of Trib1 in myeloid leukaemogenesis and differentiation.

    PubMed

    Nakamura, Takuro

    2015-10-01

    Tribbles homolog 1 (Trib1) was identified as a common integration site of the Homeobox a9 (Hoxa9)/murine ecotropic virus integration site 1 (Meis1) retrovirus in acute myeloid leukaemia (AML). Trib1 is by itself a transforming gene for myeloid cells but also significantly accelerates progression of Hoxa9/Meis1-induced AML. The strong transforming activity of Trib1 depends on its bi-directional function in CCAAT/enhancer-binding protein (C/EBPα) degradation and MAPK/ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK) activation. TRIB1 is also involved in a certain type of human AML and a TRIB1 somatic point mutation R107L was identified in a case of Down syndrome (DS)-related acute megakaryocytic leukaemia. Although Trib1 knockout (KO) did not suppress haematopoiesis in mouse bone marrow significantly, increase in mature granulocytes was observed and promotion of myeloid differentiation was associated with the increased C/EBPα protein. Trib1 thus plays an important role in myeloid cell development and transformation.

  5. Cancer-Associated Myeloid Regulatory Cells

    PubMed Central

    De Vlaeminck, Yannick; González-Rascón, Anna; Goyvaerts, Cleo; Breckpot, Karine

    2016-01-01

    Myeloid cells are critically involved in the pathophysiology of cancers. In the tumor microenvironment (TME), they comprise tumor-associated macrophages (TAMs), neutrophils (TANs), dendritic cells, and myeloid-derived suppressor cells, which are further subdivided into a monocytic subset and a granulocytic subset. Some of these myeloid cells, in particular TAMs and TANs, are divided into type 1 or type 2 cells, according to the paradigm of T helper type 1 or type 2 cells. Type 1-activated cells are generally characterized as cells that aid tumor rejection, while all other myeloid cells are shown to favor tumor progression. Moreover, these cells are often at the basis of resistance to various therapies. Much research has been devoted to study the biology of myeloid cells. This endeavor has proven to be challenging, as the markers used to categorize myeloid cells in the TME are not restricted to particular subsets. Also from a functional and metabolic point of view, myeloid cells share many features. Finally, myeloid cells are endowed with a certain level of plasticity, which further complicates studying them outside their environment. In this article, we challenge the exclusive use of cell markers to unambiguously identify myeloid cell subsets in the TME. We further propose to divide myeloid cells into myeloid regulatory or stimulatory cells according to their pro- or antitumor function, because we contend that for therapeutic purposes it is not targeting the cell subsets but rather targeting their protumor traits; hence, myeloid regulatory cells will push antitumor immunotherapy to the next level. PMID:27065074

  6. Wilms tumor 1 mutations in the pathogenesis of acute myeloid leukemia

    PubMed Central

    Rampal, Raajit; Figueroa, Maria E.

    2016-01-01

    Wilms tumor 1 (WT1) has long been implicated in acute myeloid leukemia. It has been described to be both overexpressed and mutated in different forms of acute myeloid leukemia, and overexpression has been reported to play a prognostic role in this disease. However, the precise mechanism through which WT1 may play a role in leukemogenesis has remained elusive. In recent years, new evidence has emerged that points towards a novel role of WT1 mutations in the deregulation of epigenetic programs in leukemic cells through its interaction with TET proteins. Herein we review the current status of the field and its therapeutic and prognostic implications in acute myeloid leukemia. PMID:27252512

  7. Role of the Ada adaptor complex in gene activation by the glucocorticoid receptor.

    PubMed Central

    Henriksson, A; Almlöf, T; Ford, J; McEwan, I J; Gustafsson, J A; Wright, A P

    1997-01-01

    We have shown that the Ada adaptor complex is important for the gene activation capacity of the glucocorticoid receptor in yeast. The recently isolated human Ada2 protein also increases the potency of the receptor protein in mammalian cells. The Ada pathway is of key significance for the tau1 core transactivation domain (tau1c) of the receptor, which requires Ada for activity in vivo and in vitro. Ada2 can be precipitated from nuclear extracts by a glutathione S-transferase-tau1 fusion protein coupled to agarose beads, and a direct interaction between Ada2 and tau1c can be shown by using purified proteins. This interaction is strongly reduced by a mutation in tau1c that reduces transactivation activity. Mutations affecting the Ada complex do not reverse transcriptional squelching by the tau1 domain, as they do for the VP16 transactivation domain, and thus these powerful acidic activators differ in at least some important aspects of gene activation. Mutations that reduce the activity of the tau1c domain in wild-type yeast strains cause similar reductions in ada mutants that contain little or no Ada activity. Thus, gene activation mechanisms, in addition to the Ada pathway, are involved in the activity of the tau1c domain. PMID:9154805

  8. Probing heterobivalent binding to the endocytic AP-2 adaptor complex by DNA-based spatial screening.

    PubMed

    Diezmann, F; von Kleist, L; Haucke, V; Seitz, O

    2015-08-01

    The double helical DNA scaffold offers a unique set of properties, which are particularly useful for studies of multivalency in biomolecular interactions: (i) multivalent ligand displays can be formed upon nucleic acid hybridization in a self-assembly process, which facilitates spatial screening (ii) valency and spatial arrangement of the ligand display can be precisely controlled and (iii) the flexibility of the ligand display can be adjusted by integrating nick sites and unpaired template regions. Herein we describe the use of DNA-based spatial screening for the characterization of the adaptor complex 2 (AP-2), a central interaction hub within the endocytic protein network in clathrin-mediated endocytosis. AP-2 is comprised of a core domain and two, so-called appendage domains, the α- and the β2-ear, which associate with cytoplasmatic proteins required for the formation or maturation of clathrin/AP-2 coated pits. Each appendage domain has two binding grooves which recognize distinct peptide motives with micromolar affinity. This provides opportunities for enhanced interactions with protein molecules that contain two (or more) different peptide motives. To determine whether a particular, spatial arrangement of binding motifs is required for high affinity binding we probed the distance-affinity relationships by means of DNA-programmed spatial screening with self-assembled peptide-DNA complexes. By using trimolecular and tetramolecular assemblies two different peptides were positioned in 2-22 nucleotide distance. The binding data obtained with both recombinant protein in well-defined buffer systems and native AP-2 in brain extract suggests that the two binding sites of the AP-2 α-appendage can cooperate to provide up to 40-fold enhancement of affinity compared to the monovalent interaction. The distance between the two recognized peptide motives was less important provided that the DNA duplex segments were connected by flexible, single strand segments. By

  9. Two calcium-binding proteins associated with specific stages of myeloid cell differentiation are expressed by subsets of macrophages in inflammatory tissues.

    PubMed Central

    Zwadlo, G; Brüggen, J; Gerhards, G; Schlegel, R; Sorg, C

    1988-01-01

    Using a monoclonal antibody to macrophage migration inhibition factor (MIF), two proteins were isolated from supernatants of Concanavalin A-stimulated human peripheral blood mononuclear cells which seem to have complexed to a third component carrying the MIF activity. They are therefore designated MIF-related proteins or MRP-8 and MRP-14 according to their apparent molecular weights. Partial amino acid sequences have been determined and their cDNA have been cloned and expressed in Escherichia coli. Both are calcium-binding proteins and MRP-8 seems to be largely homologous to the cystic fibrosis antigen (Dorin et al., 1987). Antisera were raised in the rabbit against the recombinant proteins and their expression in cells and tissues studied using immunohistological techniques. The proteins are only found in blood granulocytes and monocytes. In culture the number of positive monocytes sharply increased and then declined with time, suggesting that their expression is associated with early stages of monocyte/macrophage differentiation and absent from resident macrophages in all tested tissues. In acute inflammatory reactions, e.g. gingivitis, MRP-8 is never seen in the tissue, whereas MRP-14 is expressed by intravascular monocytes and perivascular macrophages. In contrast, in chronic inflammation, e.g. rheumatoid arthritis, MRP-8 is also expressed by macrophages in the tissue. From this it is concluded that MRP-8 and MRP-14 are expressed sequentially at defined stages of monocyte/macrophage differentiation and that dysregulation of this process in chronic inflammation is mirrored by the presence of MRP-8-positive macrophages in the tissue. Images Fig. 1 PMID:3048809

  10. Gemtuzumab Ozogamicin in Treating Patients With Acute Myeloid Leukemia

    ClinicalTrials.gov

    2013-09-23

    Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Promyelocytic Leukemia (M3); Recurrent Adult Acute Myeloid Leukemia

  11. Coordinated regulation of myeloid cells by tumours.

    PubMed

    Gabrilovich, Dmitry I; Ostrand-Rosenberg, Suzanne; Bronte, Vincenzo

    2012-03-22

    Myeloid cells are the most abundant nucleated haematopoietic cells in the human body and are a collection of distinct cell populations with many diverse functions. The three groups of terminally differentiated myeloid cells - macrophages, dendritic cells and granulocytes - are essential for the normal function of both the innate and adaptive immune systems. Mounting evidence indicates that the tumour microenvironment alters myeloid cells and can convert them into potent immunosuppressive cells. Here, we consider myeloid cells as an intricately connected, complex, single system and we focus on how tumours manipulate the myeloid system to evade the host immune response.

  12. Myeloid Derived Suppressor Cells in Breast Cancer

    PubMed Central

    Markowitz, Joseph; Wesolowski, Robert; Papenfuss, Tracey; Brooks, Taylor R.

    2013-01-01

    Myeloid Derived Suppressor Cells (MDSCs) are a population of immature myeloid cells defined by their suppressive actions on immune cells such as T cells, dendritic cells, and natural killer cells. MDSCs typically are positive for the markers CD33 and CD11b but express low levels of HLADR in humans. In mice, MDSCs are typically positive for both CD11b and Gr1. These cells exert their suppressive activity on the immune system via the production of reactive oxygen species, arginase, and cytokines. These factors subsequently inhibit the activity of multiple protein targets such as the T cell receptor, STAT1, and indoleamine-pyrrole 2,3-dioxygenase. The numbers of MDSCs tend to increase with cancer burden while inhibiting MDSCs improves disease outcome in murine models. MDSCs also inhibit immune cancer therapeutics. In light of the poor prognosis of metastatic breast cancer in women and the correlation of increasing levels of MDSCs with increasing disease burden, the purposes of this review are to 1) discuss why MDSCs may be important in breast cancer, 2) describe model systems used to study MDSCs in vitro and in vivo, 3) discuss mechanisms involved in MDSC induction/function in breast cancer, and 4) present pre-clinical and clinical studies that explore modulation of the MDSC-immune system interaction in breast cancer. MDSCs inhibit the host immune response in breast cancer patients and diminishing MDSC actions may improve therapeutic outcomes. PMID:23828498

  13. Oral Debio1143 (AT406), an antagonist of inhibitor of apoptosis proteins, in combination with daunorubicin and cytarabine in patients with poor-risk acute myeloid leukemia - results of a phase I dose escalation study

    PubMed Central

    Erba, Harry P.; Larson, Richard A.; Luger, Selina M.; Tallman, Martin S.; Brill, Jeffrey M.; Vuagniaux, Gregoire; Rouits, Elisabeth; Sorensen, J. Mel; Zanna, Claudio

    2016-01-01

    Background Treatment of acute myeloid leukemia (AML) remains difficult due to the development of treatment resistance which might be overcome through antagonists of inhibitors of apoptosis proteins (IAPs). Patients and methods This multi-center, open-label, dose escalation study aimed to evaluate tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy of Debio1143 (formerly AT-406), a new IAP antagonist, when given along with a standard "7 plus 3 regimen" of daunorubicin and cytarabine to poor-risk patients with AML during the induction cycle. Consecutive patient cohorts received once daily 100, 200, 300, or 400 mg of oral Debio1143 on treatment days 1–5. Blood samples were collected regularly until hematological recovery or response; bone marrow samples on day 0, 14, and 29; and PK and PD samples on days 1, 3, 5, 8, 10 and 1, 2, 8, respectively. Results Of 29 enrolled patients, 23 completed the study. Most common adverse events of any grade deemed related to treatment were nausea (31% of patients), diarrhea (14%), and febrile neutropenia (14%). Exposure exceeded dose-proportionality, without accumulation over 5 days. Inhibition of cIAP-1 was detectable in CD34/CD117+ cells and blasts. A total of 11 (38%) patients achieved complete remission, the majority in the 100 mg dose cohort. Of these, 6 (56%) relapsed still within the study period. Responders more frequently showed plasma increases of TNFα and IL-8 post-first dose of Debio1143. Conclusion Debio1143 up to 400 mg/day showed good tolerability in combination with daunorubicin and cytarabine; further studies in subsets of patients with AML are warranted. PMID:25842225

  14. Proteasome inhibitor MG-132 enhances histone deacetylase inhibitor SAHA-induced cell death of chronic myeloid leukemia cells by an ROS-mediated mechanism and downregulation of the Bcr-Abl fusion protein

    PubMed Central

    ZHOU, WENJING; ZHU, WEIWEI; MA, LIYA; XIAO, FENG; QIAN, WENBIN

    2015-01-01

    Recently, there has been progress in the treatment of chronic myeloid leukemia (CML). However, novel therapeutic strategies are required in order to address the emerging problem of imatinib resistance. Histone deacetylase inhibitors (HDACi) and proteasome inhibitors are promising alternatives, and may be amenable to integration with current therapeutic approaches. However, the mechanisms underlying the interaction between these two agents remain unclear. The present study assessed the cytotoxic effect of the HDACi, suberoylanilide hydroxamic acid (SAHA), in combination with the proteasome inhibitor, MG-132, in imatinib-sensitive K562 and imatinib-resistant K562G cells, and investigated the mechanism underlying this effect. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and protein expression levels were determined by western blotting. Reactive oxygen species (ROS) generation levels were observed under a fluorescence microscope The results indicated that SAHA and MG-132 act in a synergistic manner to induce cell death in K562 and K562G cells. This effect was associated with Bcr-Abl downregulation and the production of ROS. Notably, the ROS scavenger, N-acetyl-L-cysteine, almost fully reversed the cell death and Bcr-Abl downregulation that was induced by the combination of SAHA and MG-132. By contrast, the pan-caspase inhibitor, z-VAD-fmk, only partially reversed the cell death induced by these two drugs in CML cells. These results indicated that increased intracellular ROS levels are important in the induction of cell death and the downregulation of Bcr-Abl. In conclusion, the present results suggested that combined SAHA and MG-132 may be a promising treatment for CML. PMID:26722260

  15. Phase I/II Trial of AEG35156 X-Linked Inhibitor of Apoptosis Protein Antisense Oligonucleotide Combined With Idarubicin and Cytarabine in Patients With Relapsed or Primary Refractory Acute Myeloid Leukemia

    PubMed Central

    Schimmer, Aaron D.; Estey, Elihu H.; Borthakur, Gautam; Carter, Bing Z.; Schiller, Gary J.; Tallman, Martin S.; Altman, Jessica K.; Karp, Judith E.; Kassis, Jeannine; Hedley, David W.; Brandwein, Joseph; Xu, Wei; Mak, Duncan H.; LaCasse, Eric; Jacob, Christine; Morris, Stephen J.; Jolivet, Jacques; Andreeff, Michael

    2009-01-01

    Purpose X-linked inhibitor of apoptosis protein (XIAP) is an inhibitor of caspases 3 and 9 which are overexpressed in acute myeloid leukemia (AML) and may contribute to chemoresistance. We report on a phase I/II trial of the XIAP antisense oligonucleotide AEG35156 in combination with reinduction chemotherapy. Patients and Methods Twenty-four patients with rapidly relapsed or refractory AML were treated with escalating doses of AEG35156 (12 to 250 mg/m2) as an intravenous solution over 2 hours and 32 patients were treated with the highest planned dose of 350 mg/m2 in combination with idarubicin and high-dose cytarabine reinduction chemotherapy. Correlative studies were conducted to determine the effects of AEG35156 on levels of XIAP mRNA. Results Knockdown of XIAP mRNA during treatment increased with the dose of the antisense. All patients who received 350 mg/m2 of AEG35156 had higher than 30% target knockdown with a median maximal knockdown of 90% (range, 48% to 100%). The overall response rate was higher among the patients receiving the highest dose of AEG35156. In this group, 15 (47%) of 32 patients achieved complete response (CR)/CR with incomplete platelet count recovery (CRp) compared with only one (4%) of 24 receiving 12 to 250 mg/m2 AEG35156. Among the patients receiving 350 mg/m2 of AEG35156 in combination with chemotherapy, 10 (91%) of 11 who were refractory to a single induction chemotherapy regimen achieved CR/CRp after reinduction with AEG35156 and chemotherapy. AEG35156 was well tolerated save for two cases of peripheral neuropathy in patients receiving multiple doses of AEG35156. Conclusion At the highest dose tested, AEG35156 knocks down its target and appears very effective when combined with chemotherapy in patients with AML refractory to a single induction regimen. PMID:19652057

  16. Molecular Pathways: Myeloid Complicity in Cancer

    PubMed Central

    Stromnes, Ingunn M.; Greenberg, Philip D.; Hingorani, Sunil R.

    2014-01-01

    Cancer-induced inflammation results in accumulation of myeloid cells. It has become increasingly evident that tumor-dependent factors condition myeloid cells toward an immunosuppressive and pro-tumorigenic phenotype. These myeloid cells include progenitors and progeny of monocytes, granulocytes, macrophages, and dendritic cells. Myeloid cells are not simply bystanders in malignancy or barometers of disease burden. Reflecting their dynamic and plastic nature, myeloid cells manifesta continuum of cellular differentiation and are intimately involved at all stages of neoplastic progression. They can promote tumorigenesis through both immune-dependent and independent mechanisms and can dictate response to therapies. A greater understanding of the inherent plasticity and relationships among myeloid subsets is needed to inform therapeutic targeting. New clinical trials are being designed to modulate the activities of myeloid cells in cancer, which may be essential to maximize the efficacy of both conventional cytotoxic and immune-based therapies for solid tumors. PMID:25047706

  17. Mudline casing hanger tieback adaptor with adjustable load ring

    SciTech Connect

    Alandy, J.M.

    1987-03-31

    This patent describes a mudline casing hanger tieback adaptor means for use in a mudline suspension system having a subsea wellhead with an inner bore and concentric casing disposed within an outer tubular member comprising: sealing means for sealably engaging the innermost of the concentric casing; engaging means connected to the sealing means for engaging the inner bore of the wellhead; and the engaging means being axially moveable with respect to the wellhead to engage supporting means on the wellhead and thus support the sealing means and provide adjustment means between the wellhead and the sealing means when the engaging means is within the inner bore. A method is described of adapting a partially converted mudline suspension system, the conversion including a wellhead positioned within an outer tubular member forming part of the mudline suspension system.

  18. SEUSS and SEUSS-LIKE Transcriptional Adaptors Regulate Floral and Embryonic Development in Arabidopsis1[C][W][OA

    PubMed Central

    Bao, Fang; Azhakanandam, Sridevi; Franks, Robert G.

    2010-01-01

    Multimeric protein complexes are required during development to regulate transcription and orchestrate cellular proliferation and differentiation. The Arabidopsis (Arabidopsis thaliana) SEUSS (SEU) gene encodes a transcriptional adaptor that shares sequence similarity with metazoan Lim domain-binding transcriptional adaptors. In Arabidopsis, SEU forms a physical complex with the LEUNIG transcriptional coregulator. This complex regulates a number of diverse developmental events, including proper specification of floral organ identity and number and the development of female reproductive tissues derived from the carpel margin meristem. In addition to SEU, there are three Arabidopsis SEUSS-LIKE (SLK) genes that encode putative transcriptional adaptors. To determine the functions of the SLK genes and to investigate the degree of functional redundancy between SEU and SLK genes, we characterized available slk mutant lines in Arabidopsis. Here, we show that mutations in any single SLK gene failed to condition an obvious morphological abnormality. However, by generating higher order mutant plants, we uncovered a degree of redundancy between the SLK genes and between SLK genes and SEU. We report a novel role for SEU and the SLK genes during embryonic development and show that the concomitant loss of both SEU and SLK2 activities conditions severe embryonic and seedling defects characterized by a loss of the shoot apical meristem. Furthermore, we demonstrate that SLK gene function is required for proper development of vital female reproductive tissues derived from the carpel margin. We propose a model that posits that SEU and SLK genes support organ development from meristematic regions through two different pathways: one that facilitates auxin response and thus organ initiation and a second that sustains meristematic potential through the maintenance of SHOOTMERISTEM-LESS and PHABULOSA expression. PMID:20007451

  19. Roles nrf2 plays in myeloid cells and related disorders.

    PubMed

    Kobayashi, Eri; Suzuki, Takafumi; Yamamoto, Masayuki

    2013-01-01

    The Keap1-Nrf2 system protects animals from oxidative and electrophilic stresses. Nrf2 is a transcription factor that induces the expression of genes essential for detoxifying reactive oxygen species (ROS) and cytotoxic electrophiles. Keap1 is a stress sensor protein that binds to and ubiquitinates Nrf2 under unstressed conditions, leading to the rapid proteasomal degradation of Nrf2. Upon exposure to stress, Keap1 is modified and inactivated, which allows Nrf2 to accumulate and activate the transcription of a battery of cytoprotective genes. Antioxidative and detoxification activities are important for many types of cells to avoid DNA damage and cell death. Accumulating lines of recent evidence suggest that Nrf2 is also required for the primary functions of myeloid cells, which include phagocytosis, inflammation regulation, and ROS generation for bactericidal activities. In fact, results from several mouse models have shown that Nrf2 expression in myeloid cells is required for the proper regulation of inflammation, antitumor immunity, and atherosclerosis. Moreover, several molecules generated upon inflammation activate Nrf2. Although ROS detoxification mediated by Nrf2 is assumed to be required for anti-inflammation, the entire picture of the Nrf2-mediated regulation of myeloid cell primary functions has yet to be elucidated. In this review, we describe the Nrf2 inducers characteristic of myeloid cells and the contributions of Nrf2 to diseases.

  20. Induction of Nerve Injury-Induced Protein 1 (Ninjurin 1) in Myeloid Cells in Rat Brain after Transient Focal Cerebral Ischemia

    PubMed Central

    Lee, Hye-Kyung; Lee, Hahnbie; Luo, Lidan

    2016-01-01

    Nerve injury-induced protein-1 (Ninjurin-1, Ninj1) was initially identified as a novel adhesion molecule in rat sciatic nerve and to be up-regulated in neurons and Schwann cells of distal nerve segments after nerve transection or crush injury. Recently, Ninj1 was found to act as a modulator of cell migration, angiogenesis, and apoptosis. Accumulating evidence indicates that innate immune response plays beneficial and deleterious roles in brain ischemia, and the trans-endothelial migration of blood-derived immune cells is key initiator of this response. In the present study, we examined the expression profile and cellular distribution of Ninj1 in rat brain after transient focal cerebral ischemia. Ninj1 expression was found to be significantly induced in cortical penumbras 1 day after 60 min of middle cerebral artery occlusion (MCAO) and to increase gradually for 8 days and then declined. In infarction cores of cortices, patterns of Ninj1 expression were similar to those observed in cortical penumbras, except induction was maintained for 10 days. At 1 day post-MCAO, Ninj1 inductions were detected mainly in neutrophils and endothelial cells in both infarction cores and penumbras, but reactive macrophages were the major cellular expressers of Ninj1 at 4 days post-MCAO. Expressional induction in reactive macrophages was maintained in infarction cores after 12 days post-MCAO but not in penumbras. These dynamic expressions of Ninj1 in different immune cells at different times suggest that this protein performs various, critical roles in the modulation of acute and delayed immune responses in the postischemic brain. PMID:27122992

  1. Competitive and Cooperative Interactions Mediate RNA Transfer from Herpesvirus Saimiri ORF57 to the Mammalian Export Adaptor ALYREF

    PubMed Central

    Tunnicliffe, Richard B.; Hautbergue, Guillaume M.; Wilson, Stuart A.; Kalra, Priti; Golovanov, Alexander P.

    2014-01-01

    The essential herpesvirus adaptor protein HVS ORF57, which has homologs in all other herpesviruses, promotes viral mRNA export by utilizing the cellular mRNA export machinery. ORF57 protein specifically recognizes viral mRNA transcripts, and binds to proteins of the cellular transcription-export (TREX) complex, in particular ALYREF. This interaction introduces viral mRNA to the NXF1 pathway, subsequently directing it to the nuclear pore for export to the cytoplasm. Here we have used a range of techniques to reveal the sites for direct contact between RNA and ORF57 in the absence and presence of ALYREF. A binding site within ORF57 was characterized which recognizes specific viral mRNA motifs. When ALYREF is present, part of this ORF57 RNA binding site, composed of an α-helix, binds preferentially to ALYREF. This competitively displaces viral RNA from the α-helix, but contact with RNA is still maintained by a flanking region. At the same time, the flexible N-terminal domain of ALYREF comes into contact with the viral RNA, which becomes engaged in an extensive network of synergistic interactions with both ALYREF and ORF57. Transfer of RNA to ALYREF in the ternary complex, and involvement of individual ORF57 residues in RNA recognition, were confirmed by UV cross-linking and mutagenesis. The atomic-resolution structure of the ORF57-ALYREF interface was determined, which noticeably differed from the homologous ICP27-ALYREF structure. Together, the data provides the first site-specific description of how viral mRNA is locked by a herpes viral adaptor protein in complex with cellular ALYREF, giving herpesvirus access to the cellular mRNA export machinery. The NMR strategy used may be more generally applicable to the study of fuzzy protein-protein-RNA complexes which involve flexible polypeptide regions. PMID:24550725

  2. [Cytogenetic abnormalities and gene mutations in myeloid leukemia].

    PubMed

    Kato, Naoko; Kitamura, Toshio

    2009-10-01

    Myeloid leukemia is a clinically and genetically heterogeneous disease. Cytogenetic studies have revealed specific chromosomal abnormalities, such as translocations, and inversions. Fusion proteins derived from these abnormalities were identified in various subtypes of leukemia. Because most of these fusion proteins were not sufficient to induce leukemia by themselves in mouse models, additional oncogenic events have been thought to be necessary for leukemogenesis. Recently, a hypothesis called "two-hit model" for leukemia has been proposed. Two broad classes of mutations that proliferative or survival advantage of hematopoietic progenitors and impaired differentiation are required for inducing leukemia. In this article, we summarize some typical chromosomal abnormalities or gene mutations associated with myeloid leukemia on the basis of this hypothesis.

  3. The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation.

    PubMed

    Das, Rupali; Bassiri, Hamid; Guan, Peng; Wiener, Susan; Banerjee, Pinaki P; Zhong, Ming-Chao; Veillette, André; Orange, Jordan S; Nichols, Kim E

    2013-04-25

    The adaptor molecule signaling lymphocytic activation molecule-associated protein (SAP) plays critical roles during invariant natural killer T (iNKT) cell ontogeny. As a result, SAP-deficient humans and mice lack iNKT cells. The strict developmental requirement for SAP has made it difficult to discern its possible involvement in mature iNKT cell functions. By using temporal Cre recombinase-mediated gene deletion to ablate SAP expression after completion of iNKT cell development, we demonstrate that SAP is essential for T-cell receptor (TCR)-induced iNKT cell cytotoxicity against T-cell and B-cell leukemia targets in vitro and iNKT-cell-mediated control of T-cell leukemia growth in vivo. These findings are not restricted to the murine system: silencing RNA-mediated suppression of SAP expression in human iNKT cells also significantly impairs TCR-induced cytolysis. Mechanistic studies reveal that iNKT cell killing requires the tyrosine kinase Fyn, a known SAP-binding protein. Furthermore, SAP expression is required within iNKT cells to facilitate their interaction with T-cell targets and induce reorientation of the microtubule-organizing center to the immunologic synapse (IS). Collectively, these studies highlight a novel and essential role for SAP during iNKT cell cytotoxicity and formation of a functional IS.

  4. Allostery Mediates Ligand Binding to Grb2 Adaptor in a Mutually Exclusive Manner

    PubMed Central

    McDonald, Caleb B.; El Hokayem, Jimmy; Zafar, Nawal; Balke, Jordan E.; Bhat, Vikas; Mikles, David C.; Deegan, Brian J.; Seldeen, Kenneth L.; Farooq, Amjad

    2012-01-01

    Allostery plays a key role in dictating the stoichiometry and thermodynamics of multi-protein complexes driving a plethora of cellular processes central to health and disease. Herein, using various biophysical tools, we demonstrate that although Sos1 nucleotide exchange factor and Gab1 docking protein recognize two non-overlapping sites within the Grb2 adaptor, allostery promotes the formation of two distinct pools of Grb2-Sos1 and Grb2-Gab1 binary signaling complexes in concert in lieu of a composite Sos1-Grb2-Gab1 ternary complex. Of particular interest is the observation that the binding of Sos1 to the nSH3 domain within Grb2 sterically blocks the binding of Gab1 to the cSH3 domain and vice versa in a mutually exclusive manner. Importantly, the formation of both the Grb2-Sos1 and Grb2-Gab1 binary complexes is governed by a stoichiometry of 2:1, whereby the respective SH3 domains within Grb2 homodimer bind to Sos1 and Gab1 via multivalent interactions. Collectively, our study sheds new light on the role of allostery in mediating cellular signaling machinery. PMID:23334917

  5. The clathrin adaptor AP-1 complex and Arf1 regulate planar cell polarity in vivo.

    PubMed

    Carvajal-Gonzalez, Jose Maria; Balmer, Sophie; Mendoza, Meg; Dussert, Aurore; Collu, Giovanna; Roman, Angel-Carlos; Weber, Ursula; Ciruna, Brian; Mlodzik, Marek

    2015-04-07

    A key step in generating planar cell polarity (PCP) is the formation of restricted junctional domains containing Frizzled/Dishevelled/Diego (Fz/Dsh/Dgo) or Van Gogh/Prickle (Vang/Pk) complexes within the same cell, stabilized via Flamingo (Fmi) across cell membranes. Although models have been proposed for how these complexes acquire and maintain their polarized localization, the machinery involved in moving core PCP proteins around cells remains unknown. We describe the AP-1 adaptor complex and Arf1 as major regulators of PCP protein trafficking in vivo. AP-1 and Arf1 disruption affects the accumulation of Fz/Fmi and Vang/Fmi complexes in the proximo-distal axis, producing severe PCP phenotypes. Using novel tools, we demonstrate a direct and specific Arf1 involvement in Fz trafficking in vivo. Moreover, we uncover a conserved Arf1 PCP function in vertebrates. Our data support a model whereby the trafficking machinery plays an important part during PCP establishment, promoting formation of polarized PCP-core complexes in vivo.

  6. The clathrin adaptor AP-1 complex and Arf1 regulate planar cell polarity in vivo

    PubMed Central

    Mendoza, Meg; Dussert, Aurore; Collu, Giovanna; Roman, Angel-Carlos; Weber, Ursula; Ciruna, Brian; Mlodzik, Marek

    2015-01-01

    A key step in generating planar cell polarity (PCP) is the formation of restricted junctional domains containing Frizzled/Dishevelled/Diego (Fz/Dsh/Dgo) or Van Gogh/Prickle (Vang/Pk) complexes within the same cell, stabilized via Flamingo (Fmi) across cell membranes. Although models have been proposed for how these complexes acquire and maintain their polarized localization, the machinery involved in moving core PCP proteins around cells remains unknown. We describe the AP-1 adaptor complex and Arf1 as major regulators of PCP protein trafficking in vivo. AP-1 and Arf1 disruption affects the accumulation of Fz/Fmi and Vang/Fmi complexes in the proximo–distal axis, producing severe PCP phenotypes. Using novel tools, we demonstrate a direct and specific Arf1 involvement in Fz trafficking in vivo. Moreover, we uncover a conserved Arf1 PCP function in vertebrates. Our data support a model whereby the trafficking machinery plays an important part during PCP establishment, promoting formation of polarized PCP-core complexes in vivo. PMID:25849195

  7. Allostery mediates ligand binding to Grb2 adaptor in a mutually exclusive manner.

    PubMed

    McDonald, Caleb B; El Hokayem, Jimmy; Zafar, Nawal; Balke, Jordan E; Bhat, Vikas; Mikles, David C; Deegan, Brian J; Seldeen, Kenneth L; Farooq, Amjad

    2013-02-01

    Allostery plays a key role in dictating the stoichiometry and thermodynamics of multi-protein complexes driving a plethora of cellular processes central to health and disease. Herein, using various biophysical tools, we demonstrate that although Sos1 nucleotide exchange factor and Gab1 docking protein recognize two non-overlapping sites within the Grb2 adaptor, allostery promotes the formation of two distinct pools of Grb2-Sos1 and Grb2-Gab1 binary signaling complexes in concert in lieu of a composite Sos1-Grb2-Gab1 ternary complex. Of particular interest is the observation that the binding of Sos1 to the nSH3 domain within Grb2 sterically blocks the binding of Gab1 to the cSH3 domain and vice versa in a mutually exclusive manner. Importantly, the formation of both the Grb2-Sos1 and Grb2-Gab1 binary complexes is governed by a stoichiometry of 2:1, whereby the respective SH3 domains within Grb2 homodimer bind to Sos1 and Gab1 via multivalent interactions. Collectively, our study sheds new light on the role of allostery in mediating cellular signaling machinery. PMID:23334917

  8. Preventing farnesylation of the dynein adaptor Spindly contributes to the mitotic defects caused by farnesyltransferase inhibitors

    PubMed Central

    Holland, Andrew J.; Reis, Rita M.; Niessen, Sherry; Pereira, Cláudia; Andres, Douglas A.; Spielmann, H. Peter; Cleveland, Don W.; Desai, Arshad; Gassmann, Reto

    2015-01-01

    The clinical interest in farnesyltransferase inhibitors (FTIs) makes it important to understand how these compounds affect cellular processes involving farnesylated proteins. Mitotic abnormalities observed after treatment with FTIs have so far been attributed to defects in the farnesylation of the outer kinetochore proteins CENP-E and CENP-F, which are involved in chromosome congression and spindle assembly checkpoint signaling. Here we identify the cytoplasmic dynein adaptor Spindly as an additional component of the outer kinetochore that is modified by farnesyltransferase (FTase). We show that farnesylation of Spindly is essential for its localization, and thus for the proper localization of dynein and its cofactor dynactin, to prometaphase kinetochores and that Spindly kinetochore recruitment is more severely affected by FTase inhibition than kinetochore recruitment of CENP-E and CENP-F. Molecular replacement experiments show that both Spindly and CENP-E farnesylation are required for efficient chromosome congression. The identification of Spindly as a new mitotic substrate of FTase provides insight into the causes of the mitotic phenotypes observed with FTase inhibitors. PMID:25808490

  9. The clathrin adaptor Numb regulates intestinal cholesterol absorption through dynamic interaction with NPC1L1.

    PubMed

    Li, Pei-Shan; Fu, Zhen-Yan; Zhang, Ying-Yu; Zhang, Jin-Hui; Xu, Chen-Qi; Ma, Yi-Tong; Li, Bo-Liang; Song, Bao-Liang

    2014-01-01

    Hypercholesterolemia, typically due to excessive cholesterol uptake, is a major risk factor for cardiovascular disease, which is responsible for ∼50% of all deaths in developed societies. Although it has been shown that intestinal cholesterol absorption is mediated by vesicular endocytosis of the Niemann-Pick C1-like 1 (NPC1L1) protein, the mechanism of sterol-stimulated NPC1L1 internalization is still mysterious. Here, we identified an endocytic peptide signal, YVNXXF (where X stands for any amino acid), in the cytoplasmic C-terminal tail of NPC1L1. Cholesterol binding on the N-terminal domain of NPC1L1 released the YVNXXF-containing region of NPC1L1 from association with the plasma membrane and enabled Numb binding. We also found that Numb, a clathrin adaptor, specifically recognized this motif and recruited clathrin for internalization. Disrupting the NPC1L1-Numb interaction decreased cholesterol uptake. Ablation of Numb in mouse intestine significantly reduced dietary cholesterol absorption and plasma cholesterol level. Together, these data show that Numb is a pivotal protein for intestinal cholesterol absorption and may provide a therapeutic target for hypercholesterolemia.

  10. Genetics Home Reference: core binding factor acute myeloid leukemia

    MedlinePlus

    ... acute myeloid leukemia core binding factor acute myeloid leukemia Enable Javascript to view the expand/collapse boxes. ... Close All Description Core binding factor acute myeloid leukemia (CBF-AML) is one form of a cancer ...

  11. Myeloid Sarcoma: Current Approach and Therapeutic Options

    PubMed Central

    Avni, Batia; Koren-Michowitz, Maya

    2011-01-01

    Myeloid sarcoma is a rare disease that can present as an isolated extramedullary leukemic tumor, concurrently with or at relapse of acute myeloid leukemia. Owing to the rarity of this disorder, most of the literature comprises small retrospective studies and case reports. The aim of this review is to summarize the current published data regarding the clinical presentation, morphological, cytogenetic and molecular features, prognosis and treatment of myeloid sarcoma. PMID:23556098

  12. Increasing the efficiency of SAGE adaptor ligation by directed ligation chemistry

    PubMed Central

    So, Austin P.; Turner, Robin F. B.; Haynes, Charles A.

    2004-01-01

    The ability of Serial Analysis of Gene Expression (SAGE) to provide a quantitative picture of global gene expression relies not only on the depth and accuracy of sequencing into the SAGE library, but also on the efficiency of each step required to generate the SAGE library from the starting mRNA material. The first critical step is the ligation of adaptors containing a Type IIS recognition sequence to the anchored 3′ end cDNA population that permits the release of short sequence tags (SSTs) from defined sites within the 3′ end of each transcript. Using an in vitro transcript as a template, we observed that only a small fraction of anchored 3′ end cDNA are successfully ligated with added SAGE adaptors under typical reaction conditions currently used in the SAGE protocol. Although the introduction of ∼500-fold molar excess of adaptor or the inclusion of 15% (w/v) PEG-8000 increased the yield of the adaptor-modified product, complete conversion to the desired adaptor:cDNA hetero-ligation product is not achieved. An alternative method of ligation, termed as directed ligation, is described which exploits a favourable mass-action condition created by the presence of NlaIII during ligation in combination with a novel SAGE adaptor containing a methylated base within the ligation site. Using this strategy, we were able to achieve near complete conversion of the anchored 3′ end cDNA into the desired adaptor-modified product. This new protocol therefore greatly increases the probability that a SST will be generated from every transcript, greatly enhancing the fidelity of SAGE. Directed ligation also provides a powerful means to achieve near-complete ligation of any appropriately designed adaptor to its respective target. PMID:15247329

  13. Progress in acute myeloid leukemia.

    PubMed

    Kadia, Tapan M; Ravandi, Farhad; O'Brien, Susan; Cortes, Jorge; Kantarjian, Hagop M

    2015-03-01

    Significant progress has been made in the treatment of acute myeloid leukemia (AML). Steady gains in clinical research and a renaissance of genomics in leukemia have led to improved outcomes. The recognition of tremendous heterogeneity in AML has allowed individualized treatments of specific disease entities within the context of patient age, cytogenetics, and mutational analysis. The following is a comprehensive review of the current state of AML therapy and a roadmap of our approach to these distinct disease entities. PMID:25441110

  14. Automated Adaptor Generation for Behavioral Mismatching Services Based on Pushdown Model Checking

    NASA Astrophysics Data System (ADS)

    Lin, Hsin-Hung; Aoki, Toshiaki; Katayama, Takuya

    In this paper, we introduce an approach of service adaptation for behavior mismatching services using pushdown model checking. This approach uses pushdown systems as model of adaptors so that capturing non-regular behavior in service interactions is possible. Also, the use of pushdown model checking integrates adaptation and verification. This guarantees that an adaptor generated by our approach not only solves behavior mismatches but also satisfies usual verification properties if specified. Unlike conventional approaches, we do not count on specifications of adaptor contracts but take only information from behavior interfaces of services and perform fully automated adaptor generation. Three requirements relating to behavior mismatches, unbounded messages, and branchings are retrieved from behavior interfaces and used to build LTL properties for pushdown model checking. Properties for unbounded messages, i.e., messages sent and received arbitrary multiple times, are especially addressed since it characterizes non-regular behavior in service composition. This paper also shows some experimental results from a prototype tool and provides directions for building BPEL adaptors from behavior interface of generated adaptor. The results show that our approach does solve behavior mismatches and successfully capture non-regular behavior in service composition under the scale of real service applications.

  15. Biophysical basis of the binding of WWOX tumor suppressor to WBP1 and WBP2 adaptors.

    PubMed

    McDonald, Caleb B; Buffa, Laura; Bar-Mag, Tomer; Salah, Zaidoun; Bhat, Vikas; Mikles, David C; Deegan, Brian J; Seldeen, Kenneth L; Malhotra, Arun; Sudol, Marius; Aqeilan, Rami I; Nawaz, Zafar; Farooq, Amjad

    2012-09-01

    The WW-containing oxidoreductase (WWOX) tumor suppressor participates in a diverse array of cellular activities by virtue of its ability to recognize WW-binding protein 1 (WBP1) and WW-binding protein 2 (WBP2) signaling adaptors among a wide variety of other ligands. Herein, using a multitude of biophysical techniques, we provide evidence that while the WW1 domain of WWOX binds to PPXY motifs within WBP1 and WBP2 in a physiologically relevant manner, the WW2 domain exhibits no affinity toward any of these PPXY motifs. Importantly, our data suggest that while R25/W44 residues located within the binding pocket of a triple-stranded β-fold of WW1 domain are critical for the recognition of PPXY ligands, they are replaced by the chemically distinct E66/Y85 duo at structurally equivalent positions within the WW2 domain, thereby accounting for its failure to bind PPXY ligands. Predictably, not only does the introduction of E66R/Y85W double substitution within the WW2 domain result in gain of function but the resulting engineered domain, hereinafter referred to as WW2_RW, also appears to be a much stronger binding partner of WBP1 and WBP2 than the wild-type WW1 domain. We also show that while the WW1 domain is structurally disordered and folds upon ligand binding, the WW2 domain not only adopts a fully structured conformation but also aids stabilization and ligand binding to WW1 domain. This salient observation implies that the WW2 domain likely serves as a chaperone to augment the physiological function of WW1 domain within WWOX. Collectively, our study lays the groundwork for understanding the molecular basis of a key protein-protein interaction pertinent to human health and disease. PMID:22634283

  16. A dual-band adaptor for infrared imaging.

    PubMed

    McLean, A G; Ahn, J-W; Maingi, R; Gray, T K; Roquemore, A L

    2012-05-01

    A novel imaging adaptor providing the capability to extend a standard single-band infrared (IR) camera into a two-color or dual-band device has been developed for application to high-speed IR thermography on the National Spherical Tokamak Experiment (NSTX). Temperature measurement with two-band infrared imaging has the advantage of being mostly independent of surface emissivity, which may vary significantly in the liquid lithium divertor installed on NSTX as compared to that of an all-carbon first wall. In order to take advantage of the high-speed capability of the existing IR camera at NSTX (1.6-6.2 kHz frame rate), a commercial visible-range optical splitter was extensively modified to operate in the medium wavelength and long wavelength IR. This two-band IR adapter utilizes a dichroic beamsplitter, which reflects 4-6 μm wavelengths and transmits 7-10 μm wavelength radiation, each with >95% efficiency and projects each IR channel image side-by-side on the camera's detector. Cutoff filters are used in each IR channel, and ZnSe imaging optics and mirrors optimized for broadband IR use are incorporated into the design. In-situ and ex-situ temperature calibration and preliminary data of the NSTX divertor during plasma discharges are presented, with contrasting results for dual-band vs. single-band IR operation.

  17. Regulation of natural cytotoxicity by the adaptor SAP and the Src-related kinase Fyn

    PubMed Central

    Bloch-Queyrat, Coralie; Fondanèche, Marie-Claude; Chen, Riyan; Yin, Luo; Relouzat, Francis; Veillette, André; Fischer, Alain; Latour, Sylvain

    2005-01-01

    SAP is an adaptor protein that is expressed in NK and T cells. It is mutated in humans who have X-linked lymphoproliferative (XLP) disease. By interacting with SLAM family receptors, SAP enables tyrosine phosphorylation signaling of these receptors by its ability to recruit the Src-related kinase, Fyn. Here, we analyzed the role of SAP in NK cell functions using the SAP-deficient mouse model. Our results showed that SAP was required for the ability of NK cells to eliminate tumor cells in vitro and in vivo. This effect strongly correlated with expression of CD48 on tumor cells, the ligand of 2B4, a SLAM-related receptor expressed in NK cells. In keeping with earlier reports that studied human NK cells, we showed that SAP was necessary for the ability of 2B4 to trigger cytotoxicity and IFN-γ secretion. In the absence of SAP, 2B4 function was shifted toward inhibition of NK cell–mediated cytotoxicity. By analyzing mice lacking Fyn, we showed that similarly to SAP, Fyn was strictly required for 2B4 function. Taken together, these results provide evidence that the 2B4-SAP-Fyn cascade defines a potent activating pathway of natural cytotoxicity. They also could help to explain the high propensity of patients who have XLP disease to develop lymphoproliferative disorders. PMID:15998796

  18. The Clathrin Adaptor Complex AP-1 Binds HIV-1 and MLV Gag and Facilitates Their Budding

    PubMed Central

    Camus, Grégory; Segura-Morales, Carolina; Molle, Dorothee; Lopez-Vergès, Sandra; Begon-Pescia, Christina; Cazevieille, Chantal; Schu, Peter; Bertrand, Edouard

    2007-01-01

    Retroviral assembly is driven by Gag, and nascent viral particles escape cells by recruiting the machinery that forms intralumenal vesicles of multivesicular bodies. In this study, we show that the clathrin adaptor complex AP-1 is involved in retroviral release. The absence of AP-1μ obtained by genetic knock-out or by RNA interference reduces budding of murine leukemia virus (MLV) and HIV-1, leading to a delay of viral propagation in cell culture. In contrast, overexpression of AP-1μ enhances release of HIV-1 Gag. We show that the AP-1 complex facilitates retroviral budding through a direct interaction between the matrix and AP-1μ. Less MLV Gag is found associated with late endosomes in cells lacking AP-1, and our results suggest that AP-1 and AP-3 could function on the same pathway that leads to Gag release. In addition, we find that AP-1 interacts with Tsg101 and Nedd4.1, two cellular proteins known to be involved in HIV-1 and MLV budding. We propose that AP-1 promotes Gag release by transporting it to intracellular sites of active budding, and/or by facilitating its interactions with other cellular partners. PMID:17538020

  19. A Common Variant in the Adaptor Mal Regulates Interferon Gamma Signaling.

    PubMed

    Ní Cheallaigh, Clíona; Sheedy, Frederick J; Harris, James; Muñoz-Wolf, Natalia; Lee, Jinhee; West, Kim; McDermott, Eva Palsson; Smyth, Alicia; Gleeson, Laura E; Coleman, Michelle; Martinez, Nuria; Hearnden, Claire H A; Tynan, Graham A; Carroll, Elizabeth C; Jones, Sarah A; Corr, Sinéad C; Bernard, Nicholas J; Hughes, Mark M; Corcoran, Sarah E; O'Sullivan, Mary; Fallon, Ciara M; Kornfeld, Hardy; Golenbock, Douglas; Gordon, Stephen V; O'Neill, Luke A J; Lavelle, Ed C; Keane, Joseph

    2016-02-16

    Humans that are heterozygous for the common S180L polymorphism in the Toll-like receptor (TLR) adaptor Mal (encoded by TIRAP) are protected from a number of infectious diseases, including tuberculosis (TB), whereas those homozygous for the allele are at increased risk. The reason for this difference in susceptibility is not clear. We report that Mal has a TLR-independent role in interferon-gamma (IFN-γ) receptor signaling. Mal-dependent IFN-γ receptor (IFNGR) signaling led to mitogen-activated protein kinase (MAPK) p38 phosphorylation and autophagy. IFN-γ signaling via Mal was required for phagosome maturation and killing of intracellular Mycobacterium tuberculosis (Mtb). The S180L polymorphism, and its murine equivalent S200L, reduced the affinity of Mal for the IFNGR, thereby compromising IFNGR signaling in macrophages and impairing responses to TB. Our findings highlight a role for Mal outside the TLR system and imply that genetic variation in TIRAP may be linked to other IFN-γ-related diseases including autoimmunity and cancer. PMID:26885859

  20. Functional similarity and physical association between GCN5 and ADA2: putative transcriptional adaptors.

    PubMed Central

    Marcus, G A; Silverman, N; Berger, S L; Horiuchi, J; Guarente, L

    1994-01-01

    A selection for yeast mutants resistant to GAL4-VP16-induced toxicity previously identified two genes, ADA2 and ADA3, which may function as adaptors for some transcriptional activation domains and thereby facilitate activation. Here we identify two new genes by the same selection, one of which is identical to GCN5. We show that gcn5 mutants share properties with ada mutants, including slow growth, temperature sensitivity and reduced activation by the VP16 and GCN4 activation domains. Double mutant studies suggest that ADA2 and GCN5 function together in a complex or pathway. Moreover, we demonstrate that GCN5 binds to ADA2 both by the two-hybrid assay in vivo and by co-immunoprecipitation in vitro. This suggests that ADA2 and GCN5 are part of a heteromeric complex that mediates transcriptional activation. Finally, we demonstrate the functional importance of the bromodomain of GCN5, a sequence found in other global transcription factors such as the SWI/SNF complex and the TATA binding protein-associated factors. This domain is not required for the interaction between GCN5 and ADA2 and thus may mediate a more general activity of transcription factors. Images PMID:7957049

  1. Transmembrane adaptor molecules: a new category of lymphoid-cell markers.

    PubMed

    Tedoldi, Sara; Paterson, Jennifer C; Hansmann, Martin-Leo; Natkunam, Yasodha; Rüdiger, Thomas; Angelisova, Pavla; Du, Ming Q; Roberton, Helen; Roncador, Giovanna; Sanchez, Lydia; Pozzobon, Michela; Masir, Noraidah; Barry, Richard; Pileri, Stefano; Mason, David Y; Marafioti, Teresa; Horejsí, Václav

    2006-01-01

    Transmembrane adaptor proteins (of which 7 have been identified so far) are involved in receptor signaling in immune cells. They have only a short extracellular region, with most of the molecule comprising a substantial intracytoplasmic region carrying multiple tyrosine residues that can be phosphorylated by Src- or Syk-family kinases. In this paper, we report an immunohistologic study of 6 of these molecules in normal and neoplastic human tissue sections and show that they are restricted to subpopulations of lymphoid cells, being present in either T cells (LAT, LIME, and TRIM), B cells (NTAL), or subsets of both cell types (PAG and SIT). Their expression in neoplastic lymphoid cells broadly reflects that of normal lymphoid tissue, including the positivity of plasma cells and myeloma/plasmacytoma for LIME, NTAL, PAG, and SIT. However, this study also revealed some reactions that may be of diagnostic/prognostic value. For example, lymphocytic lymphoma and mantle-cell lymphoma showed similar profiles but differed clearly from follicle-center lymphoma, whereas PAG tended to be selectively expressed in germinal center-derived subsets of diffuse large B-cell lymphoma. These molecules represent a potentially important addition to the panel of immunophenotypic markers detectable in routine biopsies that can be used in hematopathologic studies. PMID:16160011

  2. A Common Variant in the Adaptor Mal Regulates Interferon Gamma Signaling.

    PubMed

    Ní Cheallaigh, Clíona; Sheedy, Frederick J; Harris, James; Muñoz-Wolf, Natalia; Lee, Jinhee; West, Kim; McDermott, Eva Palsson; Smyth, Alicia; Gleeson, Laura E; Coleman, Michelle; Martinez, Nuria; Hearnden, Claire H A; Tynan, Graham A; Carroll, Elizabeth C; Jones, Sarah A; Corr, Sinéad C; Bernard, Nicholas J; Hughes, Mark M; Corcoran, Sarah E; O'Sullivan, Mary; Fallon, Ciara M; Kornfeld, Hardy; Golenbock, Douglas; Gordon, Stephen V; O'Neill, Luke A J; Lavelle, Ed C; Keane, Joseph

    2016-02-16

    Humans that are heterozygous for the common S180L polymorphism in the Toll-like receptor (TLR) adaptor Mal (encoded by TIRAP) are protected from a number of infectious diseases, including tuberculosis (TB), whereas those homozygous for the allele are at increased risk. The reason for this difference in susceptibility is not clear. We report that Mal has a TLR-independent role in interferon-gamma (IFN-γ) receptor signaling. Mal-dependent IFN-γ receptor (IFNGR) signaling led to mitogen-activated protein kinase (MAPK) p38 phosphorylation and autophagy. IFN-γ signaling via Mal was required for phagosome maturation and killing of intracellular Mycobacterium tuberculosis (Mtb). The S180L polymorphism, and its murine equivalent S200L, reduced the affinity of Mal for the IFNGR, thereby compromising IFNGR signaling in macrophages and impairing responses to TB. Our findings highlight a role for Mal outside the TLR system and imply that genetic variation in TIRAP may be linked to other IFN-γ-related diseases including autoimmunity and cancer.

  3. The Clathrin Adaptor Gga2p Is a Phosphatidylinositol 4-phosphate Effector at the Golgi Exit

    PubMed Central

    Demmel, Lars; Gravert, Maike; Ercan, Ebru; Habermann, Bianca; Müller-Reichert, Thomas; Kukhtina, Viktoria; Haucke, Volker; Baust, Thorsten; Sohrmann, Marc; Kalaidzidis, Yannis; Klose, Christian; Beck, Mike; Peter, Matthias

    2008-01-01

    Phosphatidylinositol 4-phosphate (PI(4)P) is a key regulator of membrane transport required for the formation of transport carriers from the trans-Golgi network (TGN). The molecular mechanisms of PI(4)P signaling in this process are still poorly understood. In a search for PI(4)P effector molecules, we performed a screen for synthetic lethals in a background of reduced PI(4)P and found the gene GGA2. Our analysis uncovered a PI(4)P-dependent recruitment of the clathrin adaptor Gga2p to the TGN during Golgi-to-endosome trafficking. Gga2p recruitment to liposomes is stimulated both by PI(4)P and the small GTPase Arf1p in its active conformation, implicating these two molecules in the recruitment of Gga2p to the TGN, which ultimately controls the formation of clathrin-coated vesicles. PI(4)P binding occurs through a phosphoinositide-binding signature within the N-terminal VHS domain of Gga2p resembling a motif found in other clathrin interacting proteins. These data provide an explanation for the TGN-specific membrane recruitment of Gga2p. PMID:18287542

  4. VCAM-1 and VAP-1 recruit myeloid cells that promote pulmonary metastasis in mice.

    PubMed

    Ferjančič, Špela; Gil-Bernabé, Ana M; Hill, Sally A; Allen, Philip D; Richardson, Peter; Sparey, Tim; Savory, Edward; McGuffog, Jane; Muschel, Ruth J

    2013-04-18

    Pulmonary metastasis is a frequent cause of poor outcome in cancer patients. The formation of pulmonary metastasis is greatly facilitated by recruitment of myeloid cells, which are crucial for tumor cell survival and extravasation. During inflammation, homing of myeloid cells is mediated by endothelial activation, raising the question of a potential role for endothelial activation in myeloid cell recruitment during pulmonary metastasis. Here, we show that metastatic tumor cell attachment causes the induction of the endothelial activation markers vascular cell adhesion molecule-1 (VCAM-1) and vascular adhesion protein-1 (VAP-1). Induction of VCAM-1 is dependent on tumor cell-clot formation, decreasing upon induction of tissue factor pathway inhibitor or treatment with hirudin. Furthermore, inhibition of endothelial activation with a VCAM-1 blocking antibody or a VAP-1 small molecule inhibitor leads to reduced myeloid cell recruitment and diminished tumor cell survival and metastasis without affecting tumor cell adhesion. Simultaneous inhibition of VCAM-1 and VAP-1 does not result in further reduction in myeloid cell recruitment and tumor cell survival, suggesting that both act through closely related mechanisms. These results establish VCAM-1 and VAP-1 as mediators of myeloid cell recruitment in metastasis and identify VAP-1 as a potential target for therapeutic intervention to combat early metastasis.

  5. Pirin downregulation is a feature of AML and leads to impairment of terminal myeloid differentiation.

    PubMed

    Licciulli, S; Cambiaghi, V; Scafetta, G; Gruszka, A M; Alcalay, M

    2010-02-01

    Terminal differentiation of blood cells requires the concerted action of a series of transcription factors that are expressed at specific stages of maturation and function in a cell-type and dosage-dependent manner. Leukemogenic oncoproteins block differentiation by subverting the normal transcriptional status of hematopoietic precursor cells. Pirin (PIR) is a putative transcriptional regulator whose expression is silenced in cells bearing the acute myeloid leukemia-1 eight-twenty-one (AML1/ETO) and promyelocytic leukemia/retinoic acid receptor (PML/RAR) leukemogenic fusion proteins. A role for PIR in myeloid differentiation has not to date been reported. In this study we show that PIR expression is significantly repressed in a large proportion of acute myeloid leukemias (AMLs), regardless of subtype or underlying karyotypic abnormalities. We show that PIR expression increases during in vitro myeloid differentiation of primary hematopoietic precursor cells, and that ablation of PIR in the U937 myelomonocytic cell line or in murine primary hematopoietic precursor cells results in impairment of terminal myeloid differentiation. Gene expression profiling of U937 cells after knockdown of PIR revealed increased expression of genes associated with the early phases of hematopoiesis, in particular, homeobox A (HOXA) genes. Our results suggest that PIR is required for terminal myeloid maturation, and its downregulation may contribute to the differentiation arrest associated with AML. PMID:20010624

  6. Mycobacterium tuberculosis Rv2882c Protein Induces Activation of Macrophages through TLR4 and Exhibits Vaccine Potential

    PubMed Central

    Back, Yong Woo; Park, Hye-Soo; Bae, Hyun Shik; Choi, Chul Hee; Kim, Hwa-Jung

    2016-01-01

    Macrophages constitute the first line of defense against Mycobacterium tuberculosis and are critical in linking innate and adaptive immunity. Therefore, the identification and characterization of mycobacterial proteins that modulate macrophage function are essential for understanding tuberculosis pathogenesis. In this study, we identified the novel macrophage-activating protein, Rv2882c, from M. tuberculosis culture filtrate proteins. Recombinant Rv2882c protein activated macrophages to secrete pro-inflammatory cytokines and express co-stimulatory and major histocompatibility complex molecules via Toll-like receptor 4, myeloid differentiation primary response protein 88, and Toll/IL-1 receptor-domain-containing adaptor inducing IFN-beta. Mitogen-activated protein kinases and NF-κB signaling pathways were involved in Rv2882c-induced macrophage activation. Further, Rv2882c-treated macrophages induced expansion of the effector/memory T cell population and Th1 immune responses. In addition, boosting Bacillus Calmette-Guerin vaccination with Rv2882c improved protective efficacy against M. tuberculosis in our model system. These results suggest that Rv2882c is an antigen that could be used for tuberculosis vaccine development. PMID:27711141

  7. Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells

    PubMed Central

    Goyama, Susumu; Schibler, Janet; Cunningham, Lea; Zhang, Yue; Rao, Yalan; Nishimoto, Nahoko; Nakagawa, Masahiro; Olsson, Andre; Wunderlich, Mark; Link, Kevin A.; Mizukawa, Benjamin; Grimes, H. Leighton; Kurokawa, Mineo; Liu, P. Paul; Huang, Gang; Mulloy, James C.

    2013-01-01

    RUNX1 is generally considered a tumor suppressor in myeloid neoplasms. Inactivating RUNX1 mutations have frequently been found in patients with myelodysplastic syndrome (MDS) and cytogenetically normal acute myeloid leukemia (AML). However, no somatic RUNX1 alteration was found in AMLs with leukemogenic fusion proteins, such as core-binding factor (CBF) leukemia and MLL fusion leukemia, raising the possibility that RUNX1 could actually promote the growth of these leukemia cells. Using normal human cord blood cells and those expressing leukemogenic fusion proteins, we discovered a dual role of RUNX1 in myeloid leukemogenesis. RUNX1 overexpression inhibited the growth of normal cord blood cells by inducing myeloid differentiation, whereas a certain level of RUNX1 activity was required for the growth of AML1-ETO and MLL-AF9 cells. Using a mouse genetic model, we also showed that the combined loss of Runx1/Cbfb inhibited leukemia development induced by MLL-AF9. RUNX2 could compensate for the loss of RUNX1. The survival effect of RUNX1 was mediated by BCL2 in MLL fusion leukemia. Our study unveiled an unexpected prosurvival role for RUNX1 in myeloid leukemogenesis. Inhibiting RUNX1 activity rather than enhancing it could be a promising therapeutic strategy for AMLs with leukemogenic fusion proteins. PMID:23979164

  8. Inclined selective plane illumination microscopy adaptor for conventional microscopes.

    PubMed

    Cutrale, Francesco; Gratton, Enrico

    2012-11-01

    Driven by the biological sciences, there is an increased need for imaging modalities capable of live cell imaging with high spatial and temporal resolution. To achieve this goal in a comprehensive manner, three-dimensional acquisitions are necessary. Ideal features of a modern microscope system should include high imaging speed, high contrast ratio, low photo-bleaching and photo-toxicity, good resolution in a 3D context, and mosaic acquisition for large samples. Given the importance of collecting data in live sample further increases the technical challenges required to solve these issues. This work presents a practical version of a microscopy method, Selective Plane Illumination Microscopy re-introduced by Huisken et al. (Science2004,305,1007-1009). This method is gaining importance in the biomedical field, but its use is limited by difficulties associated with unconventional microscope design which employs two objectives and a particular kind of sample preparation needed to insert the sample between the objectives. Based on the selective plane illumination principle but with a design similar to the Total Internal Reflection Fluorescence microscope, Dunsby (Dunsby, Opt Express 2008,16,20306-20316) demonstrated the oblique plane microscope (OPM) using a single objective which uses conventional sample preparation protocols. However, the Dunsby instrument was not intended to be part of a commercial microscope. In this work, we describe a system with the advantages of OPM and that can be used as an adaptor to commonly used microscopes, such as IX-71 Olympus, simplifying the construction of the OPM and increasing performance of a conventional microscope. We named our design inclined selective plane illumination microscope (iSPIM).

  9. Ascent Heating Thermal Analysis on Spacecraft Adaptor Fairings

    NASA Technical Reports Server (NTRS)

    Wang, Xiao Yen; Yuko, James; Motil, Brian

    2011-01-01

    When the Crew Exploration Vehicle (CEV) is launched, the spacecraft adaptor (SA) fairings that cover the CEV service module (SM) are exposed to aero heating. Thermal analysis is performed to compute the fairing temperatures and to investigate whether the temperatures are within the material limits for nominal ascent aeroheating case. The ascent heating is analyzed by using computational fluid dynamics (CFD) and engineering codes at Marshall Space Flight Center. The aeroheating environment data used for this work is known as Thermal Environment 3 (TE3) heating data. One of the major concerns is with the SA fairings covering the CEV SM and the SM/crew launch vehicle (CLV) flange interface. The TE3 heating rate is a function of time, wall temperature, and the spatial locations. The implementation of the TE3 heating rate as boundary conditions in the thermal analysis becomes challenging. The ascent heating thermal analysis on SA fairings and SM/CLV flange interface are performed using two commercial software packages: Cullimore & Ring (C&R) Thermal Desktop (TD) 5.1 and MSC Patran 2007r1 b. TD is the pre-and post-processor for SINDA, which is a finite-difference-based solver. In TD, the geometry is built and meshed, the boundary conditions are defined, and then SINDA is used to compute temperatures. MSC Pthermal is a finite-element- based thermal solver. MSC Patran is the pre- and post-processor for Pthermal. Regarding the boundary conditions, the convection, contact resistance, and heat load can be imposed in different ways in both programs. These two software packages are used to build the thermal model for the same analysis to validate each other and show the differences in the modeling details.

  10. Goose Toll-like receptor 7 (TLR7), myeloid differentiation factor 88 (MyD88) and antiviral molecules involved in anti-H5N1 highly pathogenic avian influenza virus response.

    PubMed

    Wei, Liangmeng; Jiao, Peirong; Yuan, Runyu; Song, Yafen; Cui, Pengfei; Guo, Xuchen; Zheng, Bofang; Jia, Weixin; Qi, Wenbao; Ren, Tao; Liao, Ming

    2013-05-15

    In mammals, Toll-like receptor 7 (TLR7) is an important membrane-bound receptor triggered by antiviral compounds and single-stranded RNA. It is implicated in the immune response to viruses such as influenza virus. It was not known whether geese, a natural host for avian influenza viruses, possess a homologue of mammalian TLR7 for recognizing avian influenza virus. In this study, we cloned the full-length of goose TLR7 and partial sequences of its adaptor protein, myeloid differentiation factor 88 (MyD88), some antiviral molecules such as RNA-dependent protein kinase (PKR) and 2',5'-oligoadenylate synthetase (OAS). Goose TLR7 has a protein secondary structure identical to that of mammals, consisting of several leucine-rich domains, a transmembrane domain, and Toll/interleukin-1 receptor domain. To further understand whether the MyD88-dependent pathway of TLR7 is involved in the antiviral innate immune response against highly pathogenic avian influenza virus (HPAIV) infection in geese, we inoculated geese with an H5N1 HPAIV isolated from ducks in 2004. The virus, A/Duck/Guangdong/212/2004, replicated in various tissues resulting in 40% mortality. Quantitative real-time PCR analysis showed upregulation of mRNA transcripts for TLR7, MyD88, PKR and OAS in the lungs of geese at 1, 2 and 3 days post-inoculation. Therefore, the MyD88-dependent pathway of TLR7 was involved in the early stage of antiviral innate immune response in geese during H5N1 HPAIV infection.

  11. Stat3 and G-CSF-induced myeloid differentiation.

    PubMed

    Chakraborty, A; Tweardy, D J

    1998-08-01

    Granulocyte colony-stimulating factor (G-CSF) is the cytokine critical for directing neutrophilic granulocyte differentiation. Early G-CSF signaling events in myeloid cells involves activation of STATs, proteins that serve the dual function of signal transduction and activation of transcription, especially the activation of Stat3. A dominant-negative mutant construct of Stat3 inhibited G-CSF-mediated neutrophilic differentiation indicating that Stat3 is a essential component for driving the G-CSF-mediated differentiation program in myeloid cells. Three isoforms of Stat3 have been identified, alpha(p92), beta(p83) and gamma(p72) each derived from a single gene. Stat3alpha is the predominant isoform expressed in most cells. Stat3beta is derived from Stat3alpha by alternative RNA splicing. Stat3gamma is derived from Stat3alpha by limited proteolysis. Mapping of Stat3alpha and Stat3beta activation in M1 murine myeloid leukemia cells revealed that their optimal activation required G-CSFR constructs containing both Y704 and Y744. These amino acid residues has previously been demonstrated to be essential for G-CSF-induced differentiation in this cells. Phosphopeptide affinity and phosphopeptide inhibition studies indicate that Stat3alpha and Stat3beta are recruited to the G-CSF receptor complex through their interaction with the receptor at phosphotyrosines Y704 and Y744. Y744 is followed at the +3 position by Cys (C). This sequence YXXC, represents a novel motif implicated in the recruitment and activation of Stat3alpha, Stat3beta and Stat3gamma by the hG-CSFR. Structurally, Stat3alpha, Stat3beta and Stat3gamma differ from each other in their C-terminal transactivation domain. In the beta isoform, the Stat3alpha transactivation domain is replaced by 7 amino acid residues which enable Stat3beta to interact with c-Jun. In the gamma isoform, the Stat3alpha transactivation domain is removed by limited proteolysis creating a dominant negative isoform. In immature human

  12. Dasatinib, Cytarabine, and Idarubicin in Treating Patients With High-Risk Acute Myeloid Leukemia

    ClinicalTrials.gov

    2016-04-08

    Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Recurrent Adult Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

  13. Tipifarnib in Treating Older Patients With Acute Myeloid Leukemia

    ClinicalTrials.gov

    2015-03-19

    Adult Acute Megakaryoblastic Leukemia; Adult Acute Monoblastic Leukemia; Adult Acute Monocytic Leukemia; Adult Acute Myeloid Leukemia With Inv(16)(p13.1q22); CBFB-MYH11; Adult Acute Myeloid Leukemia With Maturation; Adult Acute Myeloid Leukemia With Minimal Differentiation; Adult Acute Myeloid Leukemia With t(16;16)(p13.1;q22); CBFB-MYH11; Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); RUNX1-RUNX1T1; Adult Acute Myeloid Leukemia With t(9;11)(p22;q23); MLLT3-MLL; Adult Acute Myeloid Leukemia Without Maturation; Adult Acute Myelomonocytic Leukemia; Adult Erythroleukemia; Adult Pure Erythroid Leukemia; Alkylating Agent-Related Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

  14. Therapy-related Myeloid Leukemia

    PubMed Central

    Godley, Lucy A.; Larson, Richard A.

    2008-01-01

    Therapy-related myelodysplastic syndrome and acute myeloid leukemia (t-MDS/t-AML) are thought to be the direct consequence of mutational events induced by chemotherapy, radiation therapy, immunosuppressive therapy, or a combination of these modalities, given for a pre-existing condition. The outcomes for these patients have been poor historically compared to people who develop de novo AML. The spectrum of cytogenetic abnormalities in t-AML is similar to de novo AML, but the frequency of unfavorable cytogenetics, such as a complex karyotype or deletion or loss of chromosomes 5 and/or 7, is considerably higher in t-AML. Survival varies according to cytogenetic risk group in t-AML patients, with better outcomes being observed in those with favorable-risk karyotypes. Treatment recommendations should be based on performance status and karyotype. A deeper understanding of the factors that predispose patients to the development of therapy-related myeloid leukemia would help clinicians monitor patients more carefully after treatment for a primary condition. Ultimately, this knowledge could influence initial treatment strategies with the goal of decreasing the incidence of this serious complication. PMID:18692692

  15. Ipilimumab and Decitabine in Treating Patients With Relapsed or Refractory Myelodysplastic Syndrome or Acute Myeloid Leukemia

    ClinicalTrials.gov

    2016-09-12

    Chimerism; Hematopoietic Cell Transplantation Recipient; Previously Treated Myelodysplastic Syndrome; RAEB-1; RAEB-2; Recurrent Adult Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

  16. MicroRNAs in Myeloid Hematological Malignancies

    PubMed Central

    Ciccone, Maria; Calin, George Adrian

    2015-01-01

    MicroRNAs are 19-24 nucleotides noncoding RNAs which silence modulate the expression of target genes by binding to the messenger RNAs. Myeloid malignancies include a broad spectrum of acute and chronic disorders originating from from the clonal transformation of a hematopoietic stem cell. Specific genetic abnormalities may define myeloid malignancies, such as translocation t(9;22) that represent the hallmark of chronic myeloid leukemia. Although next-generation sequencing pro-vided new insights in the genetic characterization and pathogenesis of myeloid neoplasms, the molecular mechanisms underlying myeloid neoplasms are lacking in most cases. Recently, several studies have demonstrated that the expression levels of specific miRNAs may vary among patients with myeloid malignancies compared with healthy individuals and partially unveiled how miRNAs participate in the leukemic transformation process. Finally, in vitro experiments and pre-clinical model provided preliminary data of the safety and efficacy of miRNA inhibitory molecules, opening new avenue in the treatment of myeloid hematological malignancies. PMID:27047254

  17. Cytomegalovirus immune evasion of myeloid lineage cells.

    PubMed

    Brinkmann, Melanie M; Dağ, Franziska; Hengel, Hartmut; Messerle, Martin; Kalinke, Ulrich; Čičin-Šain, Luka

    2015-06-01

    Cytomegalovirus (CMV) evades the immune system in many different ways, allowing the virus to grow and its progeny to spread in the face of an adverse environment. Mounting evidence about the antiviral role of myeloid immune cells has prompted the research of CMV immune evasion mechanisms targeting these cells. Several cells of the myeloid lineage, such as monocytes, dendritic cells and macrophages, play a role in viral control, but are also permissive for CMV and are naturally infected by it. Therefore, CMV evasion of myeloid cells involves mechanisms that qualitatively differ from the evasion of non-CMV-permissive immune cells of the lymphoid lineage. The evasion of myeloid cells includes effects in cis, where the virus modulates the immune signaling pathways within the infected myeloid cell, and those in trans, where the virus affects somatic cells targeted by cytokines released from myeloid cells. This review presents an overview of CMV strategies to modulate and evade the antiviral activity of myeloid cells in cis and in trans.

  18. Molecular pathways: myeloid complicity in cancer.

    PubMed

    Stromnes, Ingunn M; Greenberg, Philip D; Hingorani, Sunil R

    2014-10-15

    Cancer-induced inflammation results in accumulation of myeloid cells. These myeloid cells include progenitors and progeny of monocytes, granulocytes, macrophages, and dendritic cells. It has become increasingly evident that tumor-dependent factors can condition myeloid cells toward an immunosuppressive and protumorigenic phenotype. Thus, myeloid cells are not simply bystanders in malignancy or barometers of disease burden. Reflecting their dynamic and plastic nature, myeloid cells manifest a continuum of cellular differentiation and are intimately involved at all stages of neoplastic progression. They can promote tumorigenesis through both immune-dependent and -independent mechanisms and can dictate response to therapies. A greater understanding of the inherent plasticity and relationships among myeloid subsets is needed to inform therapeutic targeting. New clinical trials are being designed to modulate the activities of myeloid cells in cancer, which may be essential to maximize the efficacy of both conventional cytotoxic and immune-based therapies for solid tumors. Clin Cancer Res; 20(20); 5157-70. ©2014 AACR.

  19. Molecular pathways: myeloid complicity in cancer.

    PubMed

    Stromnes, Ingunn M; Greenberg, Philip D; Hingorani, Sunil R

    2014-10-15

    Cancer-induced inflammation results in accumulation of myeloid cells. These myeloid cells include progenitors and progeny of monocytes, granulocytes, macrophages, and dendritic cells. It has become increasingly evident that tumor-dependent factors can condition myeloid cells toward an immunosuppressive and protumorigenic phenotype. Thus, myeloid cells are not simply bystanders in malignancy or barometers of disease burden. Reflecting their dynamic and plastic nature, myeloid cells manifest a continuum of cellular differentiation and are intimately involved at all stages of neoplastic progression. They can promote tumorigenesis through both immune-dependent and -independent mechanisms and can dictate response to therapies. A greater understanding of the inherent plasticity and relationships among myeloid subsets is needed to inform therapeutic targeting. New clinical trials are being designed to modulate the activities of myeloid cells in cancer, which may be essential to maximize the efficacy of both conventional cytotoxic and immune-based therapies for solid tumors. Clin Cancer Res; 20(20); 5157-70. ©2014 AACR. PMID:25047706

  20. Metalloproteinases: A functional pathway for myeloid cells

    PubMed Central

    Chou, Jonathan; Chan, Matilda F.; Werb, Zena

    2015-01-01

    Myeloid cells have diverse roles in regulating immunity, inflammation, and extracellular matrix (ECM) turnover. To accomplish these tasks, myeloid cells carry an arsenal of metalloproteinases, which include the matrix metalloproteinases (MMPs) and the adamalysins. These enzymes have diverse substrate repertoires, and are thus involved in mediating proteolytic cascades, cell migration and cell signaling. Dysregulation of metalloproteinases contributes to pathogenic processes, including inflammation, fibrosis and cancer. Metalloproteinases also have important non-proteolytic functions in controlling cytoskeletal dynamics during macrophage fusion and enhancing transcription to promote anti-viral immunity. This review highlights the diverse contributions of metalloproteinases to myeloid cell functions. PMID:27227311

  1. Proteomic discovery of MNT as a novel interacting partner of E3 ubiquitin ligase E6AP and a key mediator of myeloid differentiation

    PubMed Central

    Kapoor, Isha; Kanaujiya, Jitendra; Kumar, Yogesh; Thota, Jagadeshwar Reddy; Bhatt, Madan L.B.; Chattopadhyay, Naibedya; Sanyal, Sabyasachi; Trivedi, Arun Kumar

    2016-01-01

    Perturbed stability of regulatory proteins is a major cause of transformations leading to cancer, including several leukemia subtypes. Here, for the first time we demonstrate that E6-associated protein (E6AP), an E3 ubiquitin ligase negatively targets MAX binding protein MNT for ubiquitin-mediated proteasome degradation and impedes ATRA mediated myeloid cell differentiation. MNT is a member of the Myc/Max/Mad network of transcription factor that regulates cell proliferation, differentiation, cellular transformation and tumorigenesis. Wild-type E6AP promoted proteasome dependent degradation of MNT, while catalytically inactive E6AP having cysteine replaced with alanine at amino-acid 843 position (E6APC843A) rather stabilized it. Further, these proteins physically associated with each other both in non-myeloid (HEK293T) and myeloid cells. MNT overexpression induced G0-G1 growth arrest and promoted myeloid differentiation while its knockdown mitigated even ATRA induced differentiation suggesting MNT to be crucial for myeloid differentiation. We further showed that ATRA inhibited E6AP and stabilized MNT expression by protecting it from E6AP mediated ubiquitin-proteasome degradation. Notably, E6AP knockdown in HL60 cells restored MNT expression and promoted myeloid differentiation. Taken together, our data demonstrated that E6AP negatively regulates granulocytic differentiation by targeting MNT for degradation which is required for growth arrest and subsequent myeloid differentiation by various differentiation inducing agents. PMID:26506232

  2. Imatinib in Chronic Myeloid Leukemia: an Overview

    PubMed Central

    Sacha, Tomasz

    2014-01-01

    Imatinib was the first signal transduction inhibitor (STI), used in a clinical setting. It prevents a BCR-ABL protein from exerting its role in the oncogenic pathway in chronic myeloid leukemia (CML). Imatinib directly inhibits the constitutive tyrosine kinase activity. Imatinib binds to BCR-ABL kinase domain by preventing the transfer of a phosphate group to tyrosine on the protein substrate and the subsequent activation of phosphorylated protein. As the result, the transmission of proliferative signals to the nucleus is blocked and leukemic cell apoptosis is induced. The FDA has approved imatinib as first-line treatment for newly diagnosed CML in December 2002 following an International Randomized Study (IRIS), initiated in June 2000, comparing imatinib at a single daily dose 400 mg to IFN alpha plus cytarabine in newly diagnosed patients with CML in CP. Results from this study show the outstanding effectiveness of imatinib and its superiority with respect to the rates of complete hematological response (CHR), major and complete cytogenetic response (MCyR, CCyR). Patients randomized to imatinib arm at 8 – year data cut off continue to have a durable hematologic and cytogenetic responses, low progression rates to AP or BC, and remarkable survival outcomes. An overall survival (OS) rate is 85% for patients receiving imatinib (93% when only CML-related deaths and those prior to stem cell transplantation are considered). The results have been confirmed in the last years by several groups. According these cumulative results the rates of CCyR achieved after one year of therapy with imatinib at standard dose ranged from 49% to 77%, and the proportion of patients who achieved major molecular response (MMR) after one year ranged between 18% and 58%. Discontinuation of imatinib has been also tried in patients in MMR, a molecular relapse occurs in about one third of patients, generally within 6 months from imatinib cessation. PMID:24455116

  3. Genetic Deletion of the Clathrin Adaptor GGA3 Reduces Anxiety and Alters GABAergic Transmission

    PubMed Central

    Albrecht, David; Lomoio, Selene; Haydon, Philip G.; Moss, Stephen J.; Tesco, Giuseppina

    2016-01-01

    Golgi-localized γ-ear-containing ARF binding protein 3 (GGA3) is a monomeric clathrin adaptor that has been shown to regulate the trafficking of the Beta-site APP-cleaving enzyme (BACE1), which is required for production of the Alzheimer’s disease (AD)-associated amyloid βpeptide. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that depletion of GGA3 results in increased BACE1 levels and activity owing to impaired lysosomal trafficking and degradation. We further demonstrated the role of GGA3 in the regulation of BACE1 in vivo by showing that BACE1 levels are increased in the brain of GGA3 null mice. We report here that GGA3 deletion results in novelty-induced hyperactivity and decreased anxiety-like behaviors. Given the pivotal role of GABAergic transmission in the regulation of anxiety-like behaviors, we performed electrophysiological recordings in hippocampal slices and found increased phasic and decreased tonic inhibition in the dentate gyrus granule cells (DGGC). Moreover, we found that the number of inhibitory synapses is increased in the dentate gyrus of GGA3 null mice in further support of the electrophysiological data. Thus, the increased GABAergic transmission is a leading candidate mechanism underlying the reduced anxiety-like behaviors observed in GGA3 null mice. All together these findings suggest that GGA3 plays a key role in GABAergic transmission. Since BACE1 levels are elevated in the brain of GGA3 null mice, it is possible that at least some of these phenotypes are a consequence of increased processing of BACE1 substrates. PMID:27192432

  4. Genetic Deletion of the Clathrin Adaptor GGA3 Reduces Anxiety and Alters GABAergic Transmission.

    PubMed

    Walker, Kendall R; Modgil, Amit; Albrecht, David; Lomoio, Selene; Haydon, Philip G; Moss, Stephen J; Tesco, Giuseppina

    2016-01-01

    Golgi-localized γ-ear-containing ARF binding protein 3 (GGA3) is a monomeric clathrin adaptor that has been shown to regulate the trafficking of the Beta-site APP-cleaving enzyme (BACE1), which is required for production of the Alzheimer's disease (AD)-associated amyloid βpeptide. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that depletion of GGA3 results in increased BACE1 levels and activity owing to impaired lysosomal trafficking and degradation. We further demonstrated the role of GGA3 in the regulation of BACE1 in vivo by showing that BACE1 levels are increased in the brain of GGA3 null mice. We report here that GGA3 deletion results in novelty-induced hyperactivity and decreased anxiety-like behaviors. Given the pivotal role of GABAergic transmission in the regulation of anxiety-like behaviors, we performed electrophysiological recordings in hippocampal slices and found increased phasic and decreased tonic inhibition in the dentate gyrus granule cells (DGGC). Moreover, we found that the number of inhibitory synapses is increased in the dentate gyrus of GGA3 null mice in further support of the electrophysiological data. Thus, the increased GABAergic transmission is a leading candidate mechanism underlying the reduced anxiety-like behaviors observed in GGA3 null mice. All together these findings suggest that GGA3 plays a key role in GABAergic transmission. Since BACE1 levels are elevated in the brain of GGA3 null mice, it is possible that at least some of these phenotypes are a consequence of increased processing of BACE1 substrates. PMID:27192432

  5. Acute myeloid leukemia presenting as galactorrhea

    PubMed Central

    Nambiar, K. Rakul; Devi, R. Nandini

    2016-01-01

    Acute myeloid leukemia (AML) presents with symptoms related to pancytopenia (weakness, infections, bleeding diathesis) and organ infiltration with leukemic cells. Galactorrhea is an uncommon manifestation of AML. We report a case of AML presenting with galactorrhea. PMID:27695173

  6. Acute myeloid leukemia presenting as galactorrhea

    PubMed Central

    Nambiar, K. Rakul; Devi, R. Nandini

    2016-01-01

    Acute myeloid leukemia (AML) presents with symptoms related to pancytopenia (weakness, infections, bleeding diathesis) and organ infiltration with leukemic cells. Galactorrhea is an uncommon manifestation of AML. We report a case of AML presenting with galactorrhea.

  7. Recognizing familial myeloid leukemia in adults

    PubMed Central

    Nickels, Eric M.; Soodalter, Jesse; Churpek, Jane E.

    2013-01-01

    Germline testing for familial cases of myeloid leukemia in adults is becoming more common with the recognition of multiple genetic syndromes predisposing people to bone marrow disease. Currently, Clinical Laboratory Improvement Amendments approved testing exists for several myeloid leukemia predisposition syndromes: familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML), caused by mutations in RUNX1; familial AML with mutated CEBPA; familial myelodysplastic syndrome and acute leukemia with mutated GATA2; and the inherited bone marrow failure syndromes, including dyskeratosis congenita, a disease of abnormal telomere maintenance. With the recognition of additional families with a genetic component to their leukemia, new predisposition alleles will likely be identified. We highlight how to recognize and manage these cases as well as outline the characteristics of the major known syndromes. We look forward to future research increasing our understanding of the scope of inherited myeloid leukemia syndromes. PMID:23926458

  8. Measurement of myeloid cell immune suppressive activity.

    PubMed

    Dolcetti, Luigi; Peranzoni, Elisa; Bronte, Vincenzo

    2010-11-01

    This unit presents simple methods to assess the immunosuppressive properties of immunoregulatory cells of myeloid origin, such as myeloid-derived suppressor cells (MDSCs), both in vitro and in vivo. These methods are general and could be adapted to test the impact of different suppressive populations on T cell activation, proliferation, and cytotoxic activity; moreover they could be useful to assess the influence exerted on immune suppressive pathways by genetic modifications, chemical inhibitors, and drugs.

  9. Cutaneous myeloid sarcoma of the penile foreskin.

    PubMed

    Afrose, Ruquiya; Nebhnani, Deepa; Wadhwa, Neelam

    2015-01-01

    Myeloid sarcoma, considered to herald the onset of a blast crisis in the setting of chronic myeloproliferative neoplasm/dysplasia, typically presents during the course of the disorder. Cutaneous involvement is uncommon and lesions on genital skin are seldom seen. We present a case of a well-differentiated myeloid sarcoma in the penile foreskin in an apparently healthy 29-year-old male presenting with phimosis. The unusual composition of the inflammatory cell infiltrate, and characteristic sparing of dermal blood vessels, nerves and smooth muscle fibres led to the correct diagnosis. Absence of commonly observed changes in the circumcision skin like those of balanitis xerotica was also helpful. Detailed hematological work up revealed a previously undiagnosed chronic myeloid leukemia in chronic phase. The patient also had simultaneous priapism, another rare presentation of chronic myeloid leukemia. One year hence, the patient is in hematological remission with no evidence of extramedullary disease. Although priapism has been described as a rare presenting symptom in chronic myeloid leukemia, the present case is unique as this is the first time a cutaneous myeloid sarcoma has been documented in the penile foreskin. PMID:24913300

  10. Essential role of spi-1-like (spi-1l) in zebrafish myeloid cell differentiation.

    PubMed

    Bukrinsky, Alex; Griffin, Kevin J P; Zhao, Yan; Lin, Shuo; Banerjee, Utpal

    2009-02-26

    The ETS protein Spi-1/Pu.1 plays a pivotal and widespread role throughout hematopoiesis in many species. This study describes the identification, characterization, and functional analysis of a new zebrafish spi transcription factor spi-1-like (spi-1l) that is expressed in primitive myeloid cells, erythro-myelo progenitor cells, and in the adult kidney. Spi-1l functions genetically downstream of etsrp, scl, and spi-1/pu.1 in myeloid differentiation. Spi-1l is coexpressed in a subset of spi-1/pu.1 cells and its function is necessary and sufficient for macrophage and granulocyte differentiation. These results establish a critical role for spi-1l in zebrafish myeloid cell differentiation.

  11. Restoration of CCAAT enhancer binding protein α P42 induces myeloid differentiation and overcomes all-trans retinoic acid resistance in human acute promyelocytic leukemia NB4-R1 cells

    PubMed Central

    WANG, LIMENGMENG; XIAO, HAOWEN; ZHANG, XING; LIAO, WEICHAO; FU, SHAN; HUANG, HE

    2015-01-01

    All-trans retinoic acid (ATRA) is one of the first line agents in differentiation therapy for acute promyelocytic leukemia (APL). However, drug resistance is a major problem influencing the efficacy of ATRA. Identification of mechanisms of ATRA resistance are urgenly needed. In the present study, we found that expression of C/EBPα, an important transcription factor for myeloid differentiation, was significantly suppressed in ATRA resistant APL cell line NB4-R1 compared with ATRA sensitive NB4 cells. Moreover, two forms of C/EBPα were unequally suppressed in NB4-R1 cells. Suppression of the full-length form P42 was more pronounced than the truncated form P30. Inhibition of PI3K/Akt/mTOR pathway was also observed in NB4-R1 cells. Moreover, C/EBPα expression was reduced by PI3K inhibitor LY294002 and mTOR inhibitor RAD001 in NB4 cells, suggesting that inactivation of the PI3K/Akt/mTOR pathway was responsible for C/EBPα suppression in APL cells. We restored C/EBPα P42 and P30 by lentivirus vectors in NB4-R1 cells, respectively, and found C/EBPα P42, but not P30, could increase CD11b, CD14, G-CSFR and GM-CSFR expression, which indicated the occurrence of myeloid differentiation. Further upregulating of CD11b expression and differential morphological changes were found in NB4-R1 cells with restored C/EBPα P42 after ATRA treatment. However, CD11b expression and differential morphological changes could not be induced by ATRA in NB4-R1 cells infected with P30 expressing or control vector. Thus, we inferred that ATRA sensitivity of NB4-R1 cells was enhanced by restoration of C/EBPα P42. In addition, we used histone deacetylase inhibitor trichostatin (TSA) to restore C/EBPα expression in NB4-R1 cells. Similar enhancement of myeloid differentiation and cell growth arrest were detected. Together, the present study demonstrated that suppression of C/EBPα P42 induced by PI3K/Akt/mTOR inhibition impaired the differentiation and ATRA sensitivity of APL cells. Restoring C

  12. Selinexor and Chemotherapy in Treating Patients With Relapsed or Refractory Acute Myeloid Leukemia

    ClinicalTrials.gov

    2016-09-29

    Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Recurrent Adult Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia

  13. S100A8, S100A9 and the S100A8/A9 heterodimer complex specifically bind to human endothelial cells: identification and characterization of ligands for the myeloid-related proteins S100A9 and S100A8/A9 on human dermal microvascular endothelial cell line-1 cells.

    PubMed

    Eue, Ines; König, Simone; Pior, Jolanthe; Sorg, Clemens

    2002-03-01

    The natural ligands of the S100 EF hand proteins S100A8 and A9 [myeloid-related proteins 8 and 14] have long been searched for in order to further the understanding of the role of the S100A8/A9-expressing monocyte subpopulation in progressing inflammatory processes. We demonstrate that S100A8, S100A9 and the S100A8/A9 heterodimeric complex bind to human dermal microvascular endothelial cell line (HMEC)-1 with an increasing binding capacity progressing from S100A8 < or = S100A9 < or = S100A8/A9. Similar results were obtained in the apolipoprotein E knockout mouse model, where preferably recombinant S100A9 but no S100A8 bound to the endothelium of the aorta ascendens. The binding of the S100A8/A9 heterodimer complex to activated HMEC-1 is specific as demonstrated by a dose-responding and satiable binding curve and the competition of FITC-labeled versus unlabeled protein. The protein character of the binding site was proven by treatment with trypsin. S100A8/A9 binding to HMEC-1 is inducible by lipopolysaccharide and tumor necrosis factor-alpha, and in the presence of calcium. A 163-kDa protein was isolated from a cell lysate of activated HMEC-1 cells using an affinity-chromatography protocol. The endothelial cell-associated ligand proteins isolated by the use of the S100A9 monomer and the S100A8/A9 dimer were subjected to mass spectrometry for protein identification. Clearly, alpha(2)-macroglobulin was identified as a binding partner for the S100A9 monomer, whereas no protein could be identified from the database for the ligand of the S100A8/A9 dimer. PMID:11867565

  14. Heme Oxygenase-1 Regulates Myeloid Cell Trafficking in AKI.

    PubMed

    Hull, Travis D; Kamal, Ahmed I; Boddu, Ravindra; Bolisetty, Subhashini; Guo, Lingling; Tisher, Cornelia C; Rangarajan, Sunil; Chen, Bo; Curtis, Lisa M; George, James F; Agarwal, Anupam

    2015-09-01

    Renal ischemia-reperfusion injury is mediated by a complex cascade of events, including the immune response, that occur secondary to injury to renal epithelial cells. We tested the hypothesis that heme oxygenase-1 (HO-1) expression, which is protective in ischemia-reperfusion injury, regulates trafficking of myeloid-derived immune cells in the kidney. Age-matched male wild-type (HO-1(+/+)), HO-1-knockout (HO-1(-/-)), and humanized HO-1-overexpressing (HBAC) mice underwent bilateral renal ischemia for 10 minutes. Ischemia-reperfusion injury resulted in significantly worse renal structure and function and increased mortality in HO-1(-/-) mice. In addition, there were more macrophages (CD45(+) CD11b(hi)F4/80(lo)) and neutrophils (CD45(+) CD11b(hi) MHCII(-) Gr-1(hi)) in HO-1(-/-) kidneys than in sham and HO-1(+/+) control kidneys subjected to ischemia-reperfusion. However, ischemic injury resulted in a significant decrease in the intrarenal resident dendritic cell (DC; CD45(+)MHCII(+)CD11b(lo)F4/80(hi)) population in HO-1(-/-) kidneys compared with controls. Syngeneic transplant experiments utilizing green fluorescent protein-positive HO-1(+/+) or HO-1(-/-) donor kidneys and green fluorescent protein-negative HO-1(+/+) recipients confirmed increased migration of the resident DC population from HO-1(-/-) donor kidneys, compared to HO-1(+/+) donor kidneys, to the peripheral lymphoid organs. This effect on renal DC migration was corroborated in myeloid-specific HO-1(-/-) mice subjected to bilateral ischemia. These mice also displayed impaired renal recovery and increased fibrosis at day 7 after injury. These results highlight an important role for HO-1 in orchestrating the trafficking of myeloid cells in AKI, which may represent a key pathway for therapeutic intervention.

  15. Randomly broken fragment PCR with 5' end-directed adaptor for genome walking.

    PubMed

    Xu, Wentao; Shang, Ying; Zhu, Pengyu; Zhai, Zhifang; He, Jing; Huang, Kunlun; Luo, Yunbo

    2013-01-01

    Many genome walking methods based on polymerase chain reaction (PCR) are available, including those with and without restriction enzyme modification. Nevertheless, these methods suffer from low reproducibility, inefficiency, and non-specificity. Here, we present a traceable and efficient PCR strategy: randomly broken fragment PCR with 5' end-directed adaptor for genome walking. The genome is first fragmented randomly. After blunting ends, the fragments are ligated to the 5' end-directed adaptors. Semi-nested PCR is then performed. Thus, we can obtain an unknown sequence by cloning the fragments of interest, followed by sequencing. This method effectively bypasses the above-mentioned obstacles and offers the advances: 1) genome fragmentation without using restriction enzymes; 2) enhancement of primer specificity and the prevention of self-ligation between the adaptors by employing a 5' end-directed adaptor. All of the steps in this new method are straightforward, and the unknown sequence can be definitively obtained by merely applying the method once.

  16. Proteomic Cornerstones of Hematopoietic Stem Cell Differentiation: Distinct Signatures of Multipotent Progenitors and Myeloid Committed Cells*

    PubMed Central

    Klimmeck, Daniel; Hansson, Jenny; Raffel, Simon; Vakhrushev, Sergey Y.; Trumpp, Andreas; Krijgsveld, Jeroen

    2012-01-01

    Regenerative tissues such as the skin epidermis, the intestinal mucosa or the hematopoietic system are organized in a hierarchical manner with stem cells building the top of this hierarchy. Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors which differentiate into lineage committed progenitors and subsequently mature cells. In this report, we applied an in-depth quantitative proteomic approach to analyze and compare the full proteomes of ex vivo isolated and FACS-sorted populations highly enriched for either multipotent hematopoietic stem/progenitor cells (HSPCs, LinnegSca-1+c-Kit+) or myeloid committed precursors (LinnegSca-1−c-Kit+). By employing stable isotope dimethyl labeling and high-resolution mass spectrometry, more than 5000 proteins were quantified. From biological triplicate experiments subjected to rigorous statistical evaluation, 893 proteins were found differentially expressed between multipotent and myeloid committed cells. The differential protein content in these cell populations points to a distinct structural organization of the cytoskeleton including remodeling activity. In addition, we found a marked difference in the expression of metabolic enzymes, including a clear shift of specific protein isoforms of the glycolytic pathway. Proteins involved in translation showed a collective higher expression in myeloid progenitors, indicating an increased translational activity. Strikingly, the data uncover a unique signature related to immune defense mechanisms, centering on the RIG-I and type-1 interferon response systems, which are installed in multipotent progenitors but not evident in myeloid committed cells. This suggests that specific, and so far unrecognized, mechanisms protect these immature cells before they mature. In conclusion, this study indicates that the transition of hematopoietic stem/progenitors toward myeloid commitment is accompanied by a profound change in processing of

  17. Decitabine, Cytarabine, and Daunorubicin Hydrochloride in Treating Patients With Acute Myeloid Leukemia

    ClinicalTrials.gov

    2016-07-20

    Adult Acute Basophilic Leukemia; Adult Acute Eosinophilic Leukemia; Adult Acute Monoblastic Leukemia; Adult Acute Monocytic Leukemia; Adult Acute Myeloid Leukemia With Maturation; Adult Acute Myeloid Leukemia With t(9;11)(p22;q23); MLLT3-MLL; Adult Acute Myeloid Leukemia Without Maturation; Adult Acute Myelomonocytic Leukemia; Alkylating Agent-Related Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

  18. Dephosphorylation of the adaptor LAT and phospholipase C-γ by SHP-1 inhibits natural killer cell cytotoxicity.

    PubMed

    Matalon, Omri; Fried, Sophia; Ben-Shmuel, Aviad; Pauker, Maor H; Joseph, Noah; Keizer, Danielle; Piterburg, Marina; Barda-Saad, Mira

    2016-01-01

    Natural killer (NK) cells discriminate between healthy cells and virally infected or transformed self-cells by tuning activating and inhibitory signals received through cell surface receptors. Inhibitory receptors inhibit NK cell function by recruiting and activating the tyrosine phosphatase Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-1 (SHP-1) to the plasma membrane. However, to date, the guanine nucleotide exchange factor VAV1 is the only direct SHP-1 substrate identified in NK cells. We reveal that the adaptor protein linker for activation of T cells (LAT) as well as phospholipase C-γ1 (PLC-γ1) and PLC-γ2 are SHP-1 substrates. Dephosphorylation of Tyr(132) in LAT by SHP-1 in NK cells abrogated the recruitment of PLC-γ1 and PLC-γ2 to the immunological synapse between the NK cell and a cancer cell target, which reduced NK cell degranulation and target cell killing. Furthermore, the ubiquitylation of LAT by the E3 ubiquitin ligases c-Cbl and Cbl-b, which was induced by LAT phosphorylation, led to the degradation of LAT in response to the engagement of inhibitory receptors on NK cells, which abrogated NK cell cytotoxicity. Knockdown of the Cbl proteins blocked LAT ubiquitylation, which promoted NK cell function. Expression of a ubiquitylation-resistant mutant LAT blocked inhibitory receptor signaling, enabling cells to become activated. Together, these data identify previously uncharacterized SHP-1 substrates and inhibitory mechanisms that determine the response of NK cells.

  19. Histone deacetylases: a common molecular target for differentiation treatment of acute myeloid leukemias?

    PubMed

    Minucci, S; Nervi, C; Lo Coco, F; Pelicci, P G

    2001-05-28

    Recent discoveries have identified key molecular events in the pathogenesis of acute promyelocytic leukemia (APL), caused by chromosomal rearrangements of the transcription factor RAR (resulting in a fusion protein with the product of other cellular genes, such as PML). Oligomerization of RAR, through a self-association domain present in PML, imposes an altered interaction with transcriptional co-regulators (NCoR/SMRT). NCoR/SMRT are responsible for recruitment of histone deacetylases (HDACs), which is required for transcriptional repression of PML-RAR target genes, and for the transforming potential of the fusion protein. Oligomerization and altered recruitment of HDACs are also responsible for transformation by the fusion protein AML1-ETO, extending these mechanisms to other forms of acute myeloid leukemias (AMLs) and suggesting that HDAC is a common target for myeloid leukemias. Strikingly, AML1-ETO expression blocks retinoic acid (RA) signaling in hematopoietic cells, suggesting that interference with the RA pathway (genetically altered in APL) by HDAC recruitment may be a common theme in AMLs. Treatment of APLs with RA, and of other AMLs with RA plus HDAC inhibitors (HDACi), results in myeloid differentiation. Thus, activation of the RA signaling pathway and inhibition of HDAC activity might represent a general strategy for the differentiation treatment of myeloid leukemias.

  20. Myeloid sarcomas of the head and neck in pediatric patients with myeloid leukemia.

    PubMed

    Cuthbertson, David W; Punia, Jyotinder Nain; Owczarzak, Vicki L

    2016-09-01

    Myeloid sarcoma is a rare extramedullary tumor composed of malignant myeloid cells that occur in the presence of myeloid leukemia. We report a case series of pediatric head and neck myeloid sarcomas representative of the epidemiology, symptomatology, laboratorial correlations, prognoses, and treatment of extramedullary leukemia. Presented are 3 cases involving patients ranging from 17 months to 11 years of age. Two patients were successfully treated with chemotherapy, and in the third patient, a large lytic lesion was treated palliatively with proton beam therapy. Knowledge and recognition of myeloid sarcomas is important as they can be locally invasive, and they may also be used as a diagnostic tool or a prognostic indicator for leukemia. PMID:27657319

  1. EAT-2, a SAP-like adaptor, controls NK cell activation through phospholipase Cγ, Ca++, and Erk, leading to granule polarization.

    PubMed

    Pérez-Quintero, Luis-Alberto; Roncagalli, Romain; Guo, Huaijian; Latour, Sylvain; Davidson, Dominique; Veillette, André

    2014-04-01

    Ewing's sarcoma-associated transcript 2 (EAT-2) is an Src homology 2 domain-containing intracellular adaptor related to signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), the X-linked lymphoproliferative gene product. Both EAT-2 and SAP are expressed in natural killer (NK) cells, and their combined expression is essential for NK cells to kill abnormal hematopoietic cells. SAP mediates this function by coupling SLAM family receptors to the protein tyrosine kinase Fyn and the exchange factor Vav, thereby promoting conjugate formation between NK cells and target cells. We used a variety of genetic, biochemical, and imaging approaches to define the molecular and cellular mechanisms by which EAT-2 controls NK cell activation. We found that EAT-2 mediates its effects in NK cells by linking SLAM family receptors to phospholipase Cγ, calcium fluxes, and Erk kinase. These signals are triggered by one or two tyrosines located in the carboxyl-terminal tail of EAT-2 but not found in SAP. Unlike SAP, EAT-2 does not enhance conjugate formation. Rather, it accelerates polarization and exocytosis of cytotoxic granules toward hematopoietic target cells. Hence, EAT-2 promotes NK cell activation by molecular and cellular mechanisms distinct from those of SAP. These findings explain the cooperative and essential function of these two adaptors in NK cell activation.

  2. SUMO modification of TBK1 at the adaptor-binding C-terminal coiled-coil domain contributes to its antiviral activity.

    PubMed

    Saul, Vera V; Niedenthal, Rainer; Pich, Andreas; Weber, Friedemann; Schmitz, M Lienhard

    2015-01-01

    The non-canonical IKK kinase TBK1 serves as an important signal transmitter of the antiviral interferon response, but is also involved in the regulation of further processes such as autophagy. The activity of TBK1 is regulated by posttranslational modifications comprising phosphorylation and ubiquitination. This study identifies SUMOylation as a novel posttranslational TBK1 modification. TBK1 kinase activity is required to allow the attachment of SUMO1 or SUMO2/3 proteins. Since TBK1 does not bind to the E2 enzyme Ubc9, this modification most likely proceeds via trans-SUMOylation. Mass spectrometry allowed identifying K694 as the SUMO acceptor site, a residue located in the C-terminal coiled-coil domain which is exclusively responsible for the association with the adaptor proteins NAP1, Sintbad and TANK. SUMO modification at K694 contributes to the antiviral function of TBK1 and accordingly the viral protein Gam1 antagonizes this posttranslational modification. PMID:25409927

  3. ChIP-seq Analysis of Human Chronic Myeloid Leukemia Cells.

    PubMed

    Anders, Lars; Li, Zhaodong

    2016-01-01

    Many transcription factors, chromatin-associated proteins and regulatory DNA elements are genetically and/or epigenetically altered in cancer, including Chronic Myeloid Leukemia (CML). This leads to deregulation of transcription that is often causally linked to the tumorigenic state. Chromatin-immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) is the key technology to study transcription as it allows in vivo whole-genome mapping of epigenetic modifications and interactions of proteins with DNA or chromatin. However, numerous DNA/chromatin-binding proteins, including EZH2, remain difficult to "ChIP," thus yielding genome-wide binding maps of only suboptimal quality. Here, we describe a ChIP-seq protocol optimized for high-quality protein-genome binding maps that have proven especially useful for studying difficult to 'ChIP' transcription regulatory factors in Chronic Myeloid Leukemia (CML) and related malignancies. PMID:27581144

  4. Targeting DNA vaccines to myeloid cells using a small peptide.

    PubMed

    Ye, Chunting; Choi, Jang Gi; Abraham, Sojan; Shankar, Premlata; Manjunath, N

    2015-01-01

    Targeting DNA vaccines to dendritic cells (DCs) greatly enhances immunity. Although several approaches have been used to target protein Ags to DCs, currently there is no method that targets DNA vaccines directly to DCs. Here, we show that a small peptide derived from the rabies virus glycoprotein fused to protamine residues (RVG-P) can target DNA to myeloid cells, including DCs, which results in enhanced humoral and T-cell responses. DCs targeted with a DNA vaccine encoding the immunodominant vaccinia B8R gene via RVG-P were able to restimulate vaccinia-specific memory T cells in vitro. Importantly, a single i.v. injection of B8R gene bound to RVG-P was able to prime a vaccinia-specific T-cell response that was able to rapidly clear a subsequent vaccinia challenge in mice. Moreover, delivery of DNA in DCs was enough to induce DC maturation and efficient Ag presentation without the need for adjuvants. Finally, immunization of mice with a DNA-vaccine encoding West Nile virus (WNV) prM and E proteins via RVG-P elicited high titers of WNV-neutralizing Abs that protected mice from lethal WNV challenge. Thus, RVG-P provides a reagent to target DNA vaccines to myeloid cells and elicit robust T-cell and humoral immune responses.

  5. What's New in Adult Acute Myeloid Leukemia Research and Treatment?

    MedlinePlus

    ... Topic Additional resources for acute myeloid leukemia What’s new in acute myeloid leukemia research and treatment? Researchers ... benefit from current treatments. Researchers are studying many new chemo drugs for use in AML, including: Sapacitabine, ...

  6. What's New in Chronic Myeloid Leukemia Research and Treatment?

    MedlinePlus

    ... Topic Additional resources for chronic myeloid leukemia What`s new in chronic myeloid leukemia research and treatment? Studies ... such as cyclosporine or hydroxychloroquine, with a TKI. New drugs for CML Because researchers now know the ...

  7. Genetics Home Reference: cytogenetically normal acute myeloid leukemia

    MedlinePlus

    ... one form of a cancer of the blood-forming tissue (bone marrow) called acute myeloid leukemia. In ... 1 link) PubMed Sources for This Page Döhner H. Implication of the molecular characterization of acute myeloid ...

  8. What Are the Risk Factors for Chronic Myeloid Leukemia?

    MedlinePlus

    ... of an atomic bomb blast or nuclear reactor accident) increases the risk of getting CML Age : The ... Myeloid (CML)? Causes, Risk Factors, and Prevention Early Detection, Diagnosis, and Staging Treating Leukemia - Chronic Myeloid (CML) ...

  9. Immunotherapy for acute myeloid leukemia.

    PubMed

    Jurcic, Joseph G

    2005-09-01

    Immunotherapeutic strategies have become part of standard cancer treatment. Chimeric and humanized antibodies have demonstrated activity against a variety of tumors. Although the humanized anti-CD33 antibody HuM195 has only modest activity against overt acute myeloid leukemia (AML), it can eliminate minimal residual disease in acute promyelocytic leukemia. High-dose radioimmunotherapy with b-particle-emitting isotopes targeting CD33, CD45, and CD66 can potentially allow intensification of antileukemic therapy before hematopoietic stem cell transplantation. Conversely, a-particle immunotherapy with isotopes such as bismuth-213 or actinium-225 offers the possibility of selective tumor cell kill while sparing surrounding normal tissues. Targeted chemotherapy with the anti-CD33- calicheamicin construct gemtuzumab ozogamicin has produced remissions in relapsed AML and appears promising when used in combination with standard chemotherapy for newly diagnosed AML. T-cell recognition of peptide antigens presented on the cell surface in combination with major histocompatibility complex antigen provides another potentially promising approach for the treatment of AML. PMID:16091194

  10. Early endothelial progenitor cells as a source of myeloid cells to improve the pre-vascularisation of bone constructs.

    PubMed

    Shi, Y; Kramer, G; Schröder, A; Kirkpatrick, C J; Seekamp, A; Schmidt, H; Fuchs, S

    2014-01-25

    According to present knowledge, blood derived endothelial progenitor cells (EPC) might act as proangiogenic myeloid cells, which play a fundamental role in the regulation of angiogenesis and blood vessel reorganisation. In this context, we have evaluated the contribution of endogenous myeloid cells in co-cultures of blood derived outgrowth endothelial cells (OEC) and osteogenic cells. In addition, we investigated the role of EPC as a potential source of myeloid cells in the formation of vascular structures in an in vitro model consisting of mesenchymal stem cells (MSC) and OEC. For this purpose, we added EPCs to co-cultures of MSC and OECs. Vascular structures and the co-localisation of myeloid cells were analysed by confocal laser microscopy (CLSM) for endothelial and myeloid markers and quantitative image analysis. The molecular effects of myeloid cells were evaluated by quantitative real time PCR, ELISA and protein arrays from cell culture supernatants and lysates. Endogenous myeloid cells were significantly co-localised with angiogenic structures in co-cultures of OEC and osteogenic cells. The active addition of EPC to co-cultures of OEC and MSC resulted in a statistically approved increase in the formation of prevascular structures at early stages of the co-culture process. In addition, we observed an increase of endothelial markers, indicating beneficial effects of EPC or myeloid cells on endothelial cell growth. Furthermore, real time PCR indicated high expression levels of CD68, CD11b and CD163 in co-cultures of EPC and MSC indicating that EPC act at least partly as macrophage like-cells.

  11. Molecular Pathology: Prognostic and Diagnostic Genomic Markers for Myeloid Neoplasms.

    PubMed

    Kuo, Frank C

    2016-09-01

    Application of next-generation sequencing (NGS) on myeloid neoplasms has expanded our knowledge of genomic alterations in this group of diseases. Genomic alterations in myeloid neoplasms are complex, heterogeneous, and not specific to a disease entity. NGS-based panel testing of myeloid neoplasms can complement existing diagnostic modalities and is gaining acceptance in the clinics and diagnostic laboratories. Prospective, randomized trials to evaluate the prognostic significance of genomic markers in myeloid neoplasms are under way in academic medical centers. PMID:27523973

  12. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

    SciTech Connect

    Gustafsson, Karin; Heffner, Garrett; Wenzel, Pamela L.; Curran, Matthew; Grawé, Jan; McKinney-Freeman, Shannon L.; Daley, George Q.; Welsh, Michael

    2013-07-15

    The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time. -- Highlights: • Shb is an adaptor protein operating downstream of tyrosine kinase receptors. • Shb deficiency reduces hematopoietic stem cell proliferation. • The proliferative effect of Shb occurs via increased

  13. Phase I Combination of Midostaurin, Bortezomib, and Chemo in Relapsed/Refractory Acute Myeloid Leukemia

    ClinicalTrials.gov

    2016-07-04

    Acute Myeloid Leukemia; Acute Myeloid Leukemia With Multilineage Dysplasia Following; Myelodysplastic Syndrome; Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Recurrent Adult Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia

  14. AR-42 and Decitabine in Treating Patients With Acute Myeloid Leukemia

    ClinicalTrials.gov

    2016-04-21

    Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Recurrent Adult Acute Myeloid Leukemia; Recurrent Childhood Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

  15. Using selenomethionyl derivatives to assign sequence in low-resolution structures of the AP2 clathrin adaptor

    PubMed Central

    Kelly, Bernard T.; Graham, Stephen C.; Owen, David J.

    2016-01-01

    Selenomethionine incorporation is a powerful technique for assigning sequence to regions of electron density at low resolution. Genetic introduction of methionine point mutations and the subsequent preparation and crystallization of selenomethionyl derivatives permits unambiguous sequence assignment by enabling the placement of the anomalous scatterers (Se atoms) thus introduced. Here, the use of this approach in the assignment of sequence in a part of the AP2 clathrin adaptor complex that is responsible for clathrin binding is described. AP2 plays a pivotal role in clathrin-mediated endocytosis, a tightly regulated process in which cell-surface transmembrane proteins are internalized from the plasma membrane by incorporation into lipid-enclosed transport vesicles. AP2 binds cargo destined for internalization and recruits clathrin, a large trimeric protein that helps to deform the membrane to produce the transport vesicle. By selenomethionine labelling of point mutants, it was shown that the clathrin-binding site is buried within a deep cleft of the AP2 complex. A membrane-stimulated conformational change in AP2 releases the clathrin-binding site from autoinhibition, thereby linking clathrin recruitment to membrane localization. PMID:26960121

  16. An autonomous CEBPA enhancer specific for myeloid-lineage priming and neutrophilic differentiation

    PubMed Central

    Avellino, Roberto; Havermans, Marije; Erpelinck, Claudia; Sanders, Mathijs A.; Hoogenboezem, Remco; van de Werken, Harmen J. G.; Rombouts, Elwin; van Lom, Kirsten; van Strien, Paulina M. H.; Gebhard, Claudia; Rehli, Michael; Pimanda, John; Beck, Dominik; Erkeland, Stefan; Kuiken, Thijs; de Looper, Hans; Gröschel, Stefan; Touw, Ivo; Bindels, Eric

    2016-01-01

    Neutrophilic differentiation is dependent on CCAAT enhancer-binding protein α (C/EBPα), a transcription factor expressed in multiple organs including the bone marrow. Using functional genomic technologies in combination with clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 genome editing and in vivo mouse modeling, we show that CEBPA is located in a 170-kb topological-associated domain that contains 14 potential enhancers. Of these, 1 enhancer located +42 kb from CEBPA is active and engages with the CEBPA promoter in myeloid cells only. Germ line deletion of the homologous enhancer in mice in vivo reduces Cebpa levels exclusively in hematopoietic stem cells (HSCs) and myeloid-primed progenitor cells leading to severe defects in the granulocytic lineage, without affecting any other Cebpa-expressing organ studied. The enhancer-deleted progenitor cells lose their myeloid transcription program and are blocked in differentiation. Deletion of the enhancer also causes loss of HSC maintenance. We conclude that a single +42-kb enhancer is essential for CEBPA expression in myeloid cells only. PMID:26966090

  17. A Case of Myeloid Sarcoma of Intestine.

    PubMed

    Lim, Sung Won; Lee, Hang Lak; Lee, Kang Nyeong; Jun, Dae Won; Kim, In Young; Kim, Eunjin; Ahn, Hyein; Park, Chan Kum

    2016-09-25

    Myeloid sarcoma (MS) is an extramedullary involvement of immature myeloid proliferation. An isolated MS is defined as a myeloblastic tumor when it arises without any concomitant circulating disease. A diagnosis of MS is established using pathologic features including infiltration of myeloblasts and strong myeloperoxidase expression with negative cytokeratin immunohistochemical staining. We report a rare case of colonic MS without any peripheral blood abnormality. If the affected patient were left untreated, the MS could evolve into acute myeloid leukemia (AML) within one year. Several studies recommend the same regimens of chemotherapy as used for circulating AML to treat isolated MS. We focused on the diagnosis of MS in this study. The correct diagnosis of MS is important for adequate treatment. In conclusion, MS should be considered in the differential diagnosis of intestinal tumor. PMID:27646584

  18. Novel drug therapies in myeloid leukemia.

    PubMed

    Horne, Gillian A; Kinstrie, Ross; Copland, Mhairi

    2015-01-01

    Both acute myeloid leukemia and chronic myeloid leukemia are thought to arise from a subpopulation of primitive cells, termed leukemic stem cells that share properties with somatic stem cells. Leukemic stem cells are capable of continued self-renewal, and are resistant to conventional chemotherapy and are considered to be responsible for disease relapse. In recent years, improved understanding of the underlying mechanisms of myeloid leukemia biology has led to the development of novel and targeted therapies. This review focuses on clinically relevant patent applications and their relevance within the known literature in two areas of prevailing therapeutic interest, namely monoclonal antibody therapy and small molecule inhibitors in disease-relevant signaling pathways. PMID:26030080

  19. Myelopoiesis and Myeloid Leukaemogenesis in the Zebrafish

    PubMed Central

    Forrester, A. Michael; Berman, Jason N.; Payne, Elspeth M.

    2012-01-01

    Over the past ten years, studies using the zebrafish model have contributed to our understanding of vertebrate haematopoiesis, myelopoiesis, and myeloid leukaemogenesis. Novel insights into the conservation of haematopoietic lineages and improvements in our capacity to identify, isolate, and culture such haematopoietic cells continue to enhance our ability to use this simple organism to address disease biology. Coupled with the strengths of the zebrafish embryo to dissect developmental myelopoiesis and the continually expanding repertoire of models of myeloid malignancies, this versatile organism has established its niche as a valuable tool to address key questions in the field of myelopoiesis and myeloid leukaemogenesis. In this paper, we address the recent advances and future directions in the field of myelopoiesis and leukaemogenesis using the zebrafish system. PMID:22851971

  20. Non-cleavable talin rescues defect in the T-cell conjugation of T-cells deficient in the immune adaptor SKAP1

    PubMed Central

    Lim, Daina; Lu, Yuning; Rudd, Christopher E.

    2016-01-01

    While the cytoskeletal protein talin binds to the β-chain of LFA-1, the immune cell adaptor SKAP1 (SKAP-55) binds to the α-chain of the same integrin via RapL. Whereas calpain protease cleavage of talin is important for LFA-1 activation, it has been unclear whether SKAP1 can alter the function of talin or its associated adaptor RIAM in T-cells. In this paper, we report that Skap1−/− T-cells showed a reduction in the translocation of talin and RIAM to the contact interface of T-cells with antigenic beads or dendritic cells (DCs) presenting OVA peptide to OT-1 T-cells. In addition, Skap1−/− T-cells show an altered pattern of talin cleavage, while the expression of a cleavage resistant form of talin (L432G) restored the impaired adhesion of OT1 transgenic Skap1−/− T-cells with DCs. SKAP1 therefore can affect the function of talin in T-cells needed for optimal T-cell/DC conjugation. PMID:26905930

  1. The murine Nck SH2/SH3 adaptors are important for the development of mesoderm-derived embryonic structures and for regulating the cellular actin network.

    PubMed

    Bladt, Friedhelm; Aippersbach, Elke; Gelkop, Sigal; Strasser, Geraldine A; Nash, Piers; Tafuri, Anna; Gertler, Frank B; Pawson, Tony

    2003-07-01

    Mammalian Nck1 and Nck2 are closely related adaptor proteins that possess three SH3 domains, followed by an SH2 domain, and are implicated in coupling phosphotyrosine signals to polypeptides that regulate the actin cytoskeleton. However, the in vivo functions of Nck1 and Nck2 have not been defined. We have mutated the murine Nck1 and Nck2 genes and incorporated beta-galactosidase reporters into the mutant loci. In mouse embryos, the two Nck genes have broad and overlapping expression patterns. They are functionally redundant in the sense that mice deficient for either Nck1 or Nck2 are viable, whereas inactivation of both Nck1 and Nck2 results in profound defects in mesoderm-derived notochord and embryonic lethality at embryonic day 9.5. Fibroblast cell lines derived from Nck1(-/-) Nck2(-/-) embryos have defects in cell motility and in the organization of the lamellipodial actin network. These data suggest that the Nck SH2/SH3 adaptors have important functions in the development of mesodermal structures during embryogenesis, potentially linked to a role in cell movement and cytoskeletal organization.

  2. The Innate Immunity Adaptor SARM Translocates to the Nucleus to Stabilize Lamins and Prevent DNA Fragmentation in Response to Pro-Apoptotic Signaling

    PubMed Central

    Sethman, Chad R.; Hawiger, Jacek

    2013-01-01

    Sterile alpha and armadillo-motif containing protein (SARM), a highly conserved and structurally unique member of the MyD88 family of Toll-like receptor adaptors, plays an important role in innate immunity signaling and apoptosis. Its exact mechanism of intracellular action remains unclear. Apoptosis is an ancient and ubiquitous process of programmed cell death that results in disruption of the nuclear lamina and, ultimately, dismantling of the nucleus. In addition to supporting the nuclear membrane, lamins serve important roles in chromatin organization, epigenetic regulation, transcription, nuclear transport, and mitosis. Mutations and other damage that destabilize nuclear lamins (laminopathies) underlie a number of intractable human diseases. Here, we report that SARM translocates to the nucleus of human embryonic kidney cells by using its amino-terminal Armadillo repeat region. Within the nucleus, SARM forms a previously unreported lattice akin to the nuclear lamina scaffold. Moreover, we show that SARM protects lamins from apoptotic degradation and reduces internucleosomal DNA fragmentation in response to signaling induced by the proinflammatory cytokine Tumor Necrosis Factor alpha. These findings indicate an important link between the innate immunity adaptor SARM and stabilization of nuclear lamins during inflammation-driven apoptosis in human cells. PMID:23923041

  3. Clofarabine, Cytarabine, and G-CSF in Treating Patients With Relapsed or Refractory Acute Myeloid Leukemia

    ClinicalTrials.gov

    2015-05-05

    Acute Myeloid Leukemia; Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Promyelocytic Leukemia (M3); Recurrent Adult Acute Myeloid Leukemia

  4. Vaccine Therapy and Basiliximab in Treating Patients With Acute Myeloid Leukemia in Complete Remission

    ClinicalTrials.gov

    2016-06-27

    Adult Acute Myeloid Leukemia in Remission; Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22)

  5. CPI-613, Cytarabine, and Mitoxantrone Hydrochloride in Treating Patients With Relapsed or Refractory Acute Myeloid Leukemia

    ClinicalTrials.gov

    2016-07-26

    Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Recurrent Adult Acute Myeloid Leukemia

  6. Clofarabine and Cytarabine in Treating Patients With Acute Myeloid Leukemia With Minimal Residual Disease

    ClinicalTrials.gov

    2013-05-07

    Adult Acute Myeloid Leukemia in Remission; Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Recurrent Adult Acute Myeloid Leukemia

  7. Romidepsin in Treating Patients With Relapsed or Refractory Acute Myeloid Leukemia

    ClinicalTrials.gov

    2015-12-03

    Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Promyelocytic Leukemia (M3); Recurrent Adult Acute Myeloid Leukemia

  8. Decitabine With or Without Bortezomib in Treating Older Patients With Acute Myeloid Leukemia

    ClinicalTrials.gov

    2016-03-14

    Acute Myeloid Leukemia Arising From Previous Myelodysplastic Syndrome; Adult Acute Megakaryoblastic Leukemia; Adult Acute Monoblastic Leukemia; Adult Acute Monocytic Leukemia; Adult Acute Myeloid Leukemia With Minimal Differentiation; Adult Acute Myeloid Leukemia With t(9;11)(p22;q23); MLLT3-MLL; Adult Acute Myeloid Leukemia Without Maturation; Adult Erythroleukemia; Adult Pure Erythroid Leukemia; Alkylating Agent-Related Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

  9. Highly sensitive targeted methylome sequencing by post-bisulfite adaptor tagging

    PubMed Central

    Miura, Fumihito; Ito, Takashi

    2015-01-01

    The current gold standard method for methylome analysis is whole-genome bisulfite sequencing (WGBS), but its cost is substantial, especially for the purpose of multi-sample comparison of large methylomes. Shotgun bisulfite sequencing of target-enriched DNA, or targeted methylome sequencing (TMS), can be a flexible, cost-effective alternative to WGBS. However, the current TMS protocol requires a considerable amount of input DNA and hence is hardly applicable to samples of limited quantity. Here we report a method to overcome this limitation by using post-bisulfite adaptor tagging (PBAT), in which adaptor tagging is conducted after bisulfite treatment to circumvent bisulfite-induced loss of intact sequencing templates, thereby enabling TMS of a 100-fold smaller amount of input DNA with far fewer cycles of polymerase chain reaction than in the current protocol. We thus expect that the PBAT-mediated TMS will serve as an invaluable method in epigenomics. PMID:25324297

  10. A rare case of myeloid sarcoma presenting as anal fissure

    PubMed Central

    VECCHIO, R.; INTAGLIATA, E.; FIUMARA, P.F.; VILLARI, L.; MARCHESE, S.; CACCIOLA, E.

    2015-01-01

    Myeloid sarcoma is a tumor composed of myeloblasts occurring at an extramedullary site. It may develop in patients with acute myeloid leukemia, myeloproliferative or myelodysplastic syndrome, sometimes preceding onset of the systemic disease. Frequent sites of myeloid sarcoma are bones or various soft tissues. Gastrointestinal involvement is very rare. We report a unique case of myeloid sarcoma presenting as a painful anal fissure, in a patient with a history of acute myeloid leukemia. The diagnosis was achieved by a surgical excisional biopsy and immunoistochemical staining. PMID:26712260

  11. Drafting the proteome landscape of myeloid-derived suppressor cells.

    PubMed

    Gato, María; Blanco-Luquin, Idoia; Zudaire, Maribel; de Morentin, Xabier Martínez; Perez-Valderrama, Estela; Zabaleta, Aintzane; Kochan, Grazyna; Escors, David; Fernandez-Irigoyen, Joaquín; Santamaría, Enrique

    2016-01-01

    Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that are defined by their myeloid origin, immature state, and ability to potently suppress T-cell responses. They regulate immune responses and the population significantly increases in the tumor microenvironment of patients with glioma and other malignant tumors. For their study, MDSCs are usually isolated from the spleen or directly of tumors from a large number of tumor-bearing mice although promising ex vivo differentiated MDSC production systems have been recently developed. During the last years, proteomics has emerged as a powerful approach to analyze MDSCs proteomes using shotgun-based mass spectrometry (MS), providing functional information about cellular homeostasis and metabolic state at a global level. Here, we will revise recent proteome profiling studies performed in MDSCs from different origins. Moreover, we will perform an integrative functional analysis of the protein compilation derived from these large-scale proteomic studies in order to obtain a comprehensive view of MDSCs biology. Finally, we will also discuss the potential application of high-throughput proteomic approaches to study global proteome dynamics and post-translational modifications (PTMs) during the differentiation process of MDSCs that will greatly boost the identification of novel MDSC-specific therapeutic targets to apply in cancer immunotherapy. PMID:26403437

  12. Adaptor Scaffoldins: An Original Strategy for Extended Designer Cellulosomes, Inspired from Nature

    PubMed Central

    Stern, Johanna; Moraïs, Sarah; Lamed, Raphael

    2016-01-01

    ABSTRACT Designer cellulosomes consist of chimeric cohesin-bearing scaffoldins for the controlled incorporation of recombinant dockerin-containing enzymes. The largest designer cellulosome reported to date is a chimeric scaffoldin that contains 6 cohesins. This scaffoldin represented a technical limit of sorts, since adding another cohesin proved problematic, owing to resultant low expression levels, instability (cleavage) of the scaffoldin polypeptide, and limited numbers of available cohesin-dockerin specificities—the hallmark of designer cellulosomes. Nevertheless, increasing the number of enzymes integrated into designer cellulosomes is critical, in order to further enhance degradation of plant cell wall material. Adaptor scaffoldins comprise an intermediate type of scaffoldin that can both incorporate various enzymes and attach to an additional scaffoldin. Using this strategy, we constructed an efficient form of adaptor scaffoldin that possesses three type I cohesins for enzyme integration, a single type II dockerin for interaction with an additional scaffoldin, and a carbohydrate-binding module for targeting to the cellulosic substrate. In parallel, we designed a hexavalent scaffoldin capable of connecting to the adaptor scaffoldin by the incorporation of an appropriate type II cohesin. The resultant extended designer cellulosome comprised 8 recombinant enzymes—4 xylanases and 4 cellulases—thereby representing a potent enzymatic cocktail for solubilization of natural lignocellulosic substrates. The contribution of the adaptor scaffoldin clearly demonstrated that proximity between the two scaffoldins and their composite set of enzymes is crucial for optimized degradation. After 72 h of incubation, the performance of the extended designer cellulosome was determined to be approximately 70% compared to that of native cellulosomes. PMID:27048796

  13. The adaptor molecule Trif contributes to murine host defense during Leptospiral infection.

    PubMed

    Jayaraman, Priya A; Devlin, Amy A; Miller, Jennifer C; Scholle, Frank

    2016-09-01

    Leptospirosis is a zoonotic disease and is caused by pathogenic species of the Leptospira genus, including Leptospira interrogans (L. interrogans). Humans, domestic and wild animals are susceptible to acute or chronic infection. The innate immune response is a critical defense mechanism against Leptospira interrogans, and has been investigated in mouse models. Murine Toll-like receptors (TLRs) have been shown to be key factors in sensing and responding to L. interrogans infection. Specifically, TLR2, TLR4 and the TLR adaptor molecule MyD88 are essential for host defense against L. interrogans; however, the role of the TLR adaptor molecule TIR-domain-containing adaptor-inducing interferon β (TRIF) in the response to L. interrogans has not been previously determined. In the present study, TRIF was found to play an important role during leptospiral infection. Following challenge with L. interrogans, Trif(-/-) mice exhibited delayed weight gain compared to wild-type mice. Moreover, Trif(-/-) mice exhibited an increase in L. interrogans burden in the kidneys, lungs, and blood at early time points (less than 7days post infection). Multiple components of the innate immune responses were dampened in response to leptospiral infection including transcription and production of cytokines, and the humoral response, which suggested that TRIF contributes to expression and production of cytokines important for the host defense against L. interrogans. PMID:27259371

  14. Fluorescein gonioangiography of the normal canine eye using a dSLR camera adaptor.

    PubMed

    Alario, Anthony F; Pirie, Christopher G

    2015-06-01

    The purpose of this study was to describe fluorescein gonioangiography (FGA) of the normal canine eye using a digital single lens reflex (dSLR) camera adaptor. Dogs were anesthetized using intravenous propofol. Imaging was performed using a Lovac Barkan goniolens, dSLR camera, dSLR camera adaptor, camera lens, and accessory flash. Twelve dogs with a mean age of 2.0 +/- 0.8 years were imaged. No characteristic angiographic phases were observed. Leakage from the peri-limbal capillary network was a common finding and occurred 7.7 +/- 2.2 s post injection in 9 (75%) dogs. In 3 (25%) dogs, filling of the circumferential ciliary artery was observed 10.3 +/- 2.8 s post injection. Dye leakage within the iris base and into the aqueous humor was demonstrated in 4 (33%) and 6 dogs (50%) respectively. No adverse events were noted. This study demonstrates FGA findings in normal canine eyes using a cost effective dSLR camera adaptor. PMID:25823859

  15. Lenalidomide in Treating Older Patients With Acute Myeloid Leukemia

    ClinicalTrials.gov

    2014-07-25

    Adult Acute Basophilic Leukemia; Adult Acute Eosinophilic Leukemia; Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

  16. cDNA cloning, tissue distribution, and chromosomal localization of myelodysplasia/Myeloid Leukemia Factor 2 (MLF2)

    SciTech Connect

    Kuefer, M.U.; Valentine, V.; Behm, F.G.

    1996-07-15

    A fusion gene between nucleophosmin (NPM) and myelodysplasia/myeloid leukemia factor 1 (MLF1) and myelodysplasia/myeloid leukemia factor 1 (MLF1) is formed by a recurrent t(3;5)(q25.1;q34) in myelodysplastic syndrome and acute myeloid leukemia. Here we report the identification of a novel gene, MLF2, which contains an open reading frame of 744 bp encoding a 248-amino-acid protein highly related to the previously identified MLF1 protein (63% similarity, 40% identity). In contrast to the tissue-restricted expression pattern of MLF1, and MLF2 messenger RNA is expressed ubiquitously. The MLF2 gene locus was mapped by fluorescence in situ hybridization to human chromosome 12p13, a chromosomal region frequently involved in translocations and deletions in acute leukemias of lymphoid or myeloid lineage. In a physical map of chromosome 12, MLF2 was found to reside on the yeast artificial chromosome clone 765b9. Southern blotting analysis of malignant cell DNAs prepared from a series of acute lymphoblastic leukemia cases with translocations involving chromosome arm 12p, as well as a group of acute myeloid leukemias with various cytogenetic abnormalities, failed to reveal MLF2 gene rearrangements. 19 refs., 2 figs.

  17. PTEN's regulation of VEGF and VEGFR1 expression and its clinical significance in myeloid leukemia.

    PubMed

    Zhiyong, Cheng; Wentong, Liang; Xiaoyang, Yang; Ling, Pan

    2012-06-01

    Phosphatase and tensin homolog (PTEN) acts as a novel tumor suppressor gene. PTEN protein plays an important role in regulating proliferation, apoptosis, invasion, and migration of many cancer cells. PTEN also modulates angiogenesis mediated by vascular endothelial growth factor (VEGF) via down-regulating PI3K/Akt pathway in many solid tumors. However, the effects of PTEN on VEGF and VEGFR1 (FLT1)-mediated angiogenesis, migration, invasion of leukemia cells, and its clinical significance are still unknown in myeloid leukemia. Therefore, we investigated the effect of PTEN on PI3K/Akt and VEGF/FLT1 pathways by transfecting wild-type PTEN gene to induce high expression of wild-type PTEN gene and protein in chronic myelogenous leukemia cell line K562 cells. We also observed the correlation between the expression levels of PTEN and VEGF/FLT1 and its clinical significance in myeloid leukemia patients. We found that the expression reconstitution of wild-type PTEN had significant effect on inhibiting proliferation, migration, and invasion abilities of K562 cells via down-regulation of Akt phosphorylation and inhibition of VEGF/FLT1 expression. In myeloid leukemia patients, a negative correlation was found between the expression level of PTEN mRNA and that of VEGF and FLT1 mRNA. Down-regulation of PTEN expression accompanied by up-regulation of VEGF and FLT1 mRNA indicated a higher tendency of extramedullary disease in acute myeloid leukemia patients. In conclusion, PTEN could modulate the function of VEGF/VEGFR signaling pathway down-regulation of Akt phosphorylation and that PTEN would be a candidate target to be addressed for inhibiting angiogenesis along with the treatment of myeloid leukemia.

  18. Increase in myeloid-derived suppressor cells (MDSCs) associated with minimal residual disease (MRD) detection in adult acute myeloid leukemia.

    PubMed

    Sun, Hui; Li, Yi; Zhang, Zhi-fen; Ju, Ying; Li, Li; Zhang, Bing-chang; Liu, Bin

    2015-11-01

    Myeloid-derived suppressor cells (MDSCs) are thought to help provide a cellular microenvironments in many solid tumors, in which transformed cells proliferate, acquire new mutations, and evade host immunosurveillance. In the present study, we found that MDSCs (CD33 + CD11b + HLA-DR(low/neg)) in bone marrow were significantly increased in adult acute myeloid leukemia (AML) patients. MDSCs levels in newly diagnosed AML patients correlated well with extramedullary infiltration and plasma D-dimer levels. Remission rates in the MDSCs > 1500 group and MDSCs < 1500 group were 72.73 and 81.25 %, respectively. No significant differences were found between the two groups. MDSC levels in the complete remission group were significantly decreased after chemotherapy, while in the partial remission and non-remission groups, there were no significant differences. The level of MDSCs in the high minimal residual disease (MRD) group was significantly higher than that in the middle and low MRD groups. High levels of Wilms' Tumor-1 (WT-1) protein were strongly correlated with higher bone marrow MDSC levels. In conclusion, we report here a population of immunosuppressive monocytes in the bone marrow of patients with AML characterized by the CD33(high)CD11b + HLA-DR(low/neg) phenotype. These cells appear to impact the clinical course and prognosis of AML. This data may provide potentially important targets for novel therapies.

  19. Structural basis of lentiviral subversion of a cellular protein degradation pathway

    NASA Astrophysics Data System (ADS)

    Schwefel, David; Groom, Harriet C. T.; Boucherit, Virginie C.; Christodoulou, Evangelos; Walker, Philip A.; Stoye, Jonathan P.; Bishop, Kate N.; Taylor, Ian A.

    2014-01-01

    Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells as well as resting CD4+ T cells by reducing the cellular deoxynucleoside 5'-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation. The molecular mechanism by which these viral proteins are able to usurp the host cell's ubiquitination machinery to destroy the cell's protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell's normal protein degradation pathway to inactivate the cellular viral defence system.

  20. Chronic myeloid leukemia: reminiscences and dreams

    PubMed Central

    Mughal, Tariq I.; Radich, Jerald P.; Deininger, Michael W.; Apperley, Jane F.; Hughes, Timothy P.; Harrison, Christine J.; Gambacorti-Passerini, Carlo; Saglio, Giuseppe; Cortes, Jorge; Daley, George Q.

    2016-01-01

    With the deaths of Janet Rowley and John Goldman in December 2013, the world lost two pioneers in the field of chronic myeloid leukemia. In 1973, Janet Rowley, unraveled the cytogenetic anatomy of the Philadelphia chromosome, which subsequently led to the identification of the BCR-ABL1 fusion gene and its principal pathogenetic role in the development of chronic myeloid leukemia. This work was also of major importance to support the idea that cytogenetic changes were drivers of leukemogenesis. John Goldman originally made seminal contributions to the use of autologous and allogeneic stem cell transplantation from the late 1970s onwards. Then, in collaboration with Brian Druker, he led efforts to develop ABL1 tyrosine kinase inhibitors for the treatment of patients with chronic myeloid leukemia in the late 1990s. He also led the global efforts to develop and harmonize methodology for molecular monitoring, and was an indefatigable organizer of international conferences. These conferences brought together clinicians and scientists, and accelerated the adoption of new therapies. The abundance of praise, tributes and testimonies expressed by many serve to illustrate the indelible impressions these two passionate and affable scholars made on so many people’s lives. This tribute provides an outline of the remarkable story of chronic myeloid leukemia, and in writing it, it is clear that the historical triumph of biomedical science over this leukemia cannot be considered without appreciating the work of both Janet Rowley and John Goldman. PMID:27132280

  1. Calcineurin/NFAT signalling inhibits myeloid haematopoiesis.

    PubMed

    Fric, Jan; Lim, Clarice X F; Koh, Esther G L; Hofmann, Benjamin; Chen, Jinmiao; Tay, Hock Soon; Mohammad Isa, Siti Aminah Bte; Mortellaro, Alessandra; Ruedl, Christiane; Ricciardi-Castagnoli, Paola

    2012-04-01

    Nuclear factor of activated T cells (NFAT) comprises a family of transcription factors that regulate T cell development, activation and differentiation. NFAT signalling can also mediate granulocyte and dendritic cell (DC) activation, but it is unknown whether NFAT influences their development from progenitors. Here, we report a novel role for calcineurin/NFAT signalling as a negative regulator of myeloid haematopoiesis. Reconstituting lethally irradiated mice with haematopoietic stem cells expressing an NFAT-inhibitory peptide resulted in enhanced development of the myeloid compartment. Culturing bone marrow cells in media supplemented with Flt3-L in the presence of the calcineurin/NFAT inhibitor Cyclosporin A increased numbers of differentiated DC. Global gene expression analysis of untreated DC and NFAT-inhibited DC revealed differential expression of transcripts that regulate cell cycle and apoptosis. In conclusion, these results provide evidence that calcineurin/NFAT signalling negatively regulates myeloid lineage development. The finding that inhibition of NFAT enhances myeloid development provides a novel insight into understanding how the treatment with drugs targeting calcineurin/NFAT signalling influence the homeostasis of the innate immune system.

  2. Chronic myeloid leukemia: reminiscences and dreams.

    PubMed

    Mughal, Tariq I; Radich, Jerald P; Deininger, Michael W; Apperley, Jane F; Hughes, Timothy P; Harrison, Christine J; Gambacorti-Passerini, Carlo; Saglio, Giuseppe; Cortes, Jorge; Daley, George Q

    2016-05-01

    With the deaths of Janet Rowley and John Goldman in December 2013, the world lost two pioneers in the field of chronic myeloid leukemia. In 1973, Janet Rowley, unraveled the cytogenetic anatomy of the Philadelphia chromosome, which subsequently led to the identification of the BCR-ABL1 fusion gene and its principal pathogenetic role in the development of chronic myeloid leukemia. This work was also of major importance to support the idea that cytogenetic changes were drivers of leukemogenesis. John Goldman originally made seminal contributions to the use of autologous and allogeneic stem cell transplantation from the late 1970s onwards. Then, in collaboration with Brian Druker, he led efforts to develop ABL1 tyrosine kinase inhibitors for the treatment of patients with chronic myeloid leukemia in the late 1990s. He also led the global efforts to develop and harmonize methodology for molecular monitoring, and was an indefatigable organizer of international conferences. These conferences brought together clinicians and scientists, and accelerated the adoption of new therapies. The abundance of praise, tributes and testimonies expressed by many serve to illustrate the indelible impressions these two passionate and affable scholars made on so many people's lives. This tribute provides an outline of the remarkable story of chronic myeloid leukemia, and in writing it, it is clear that the historical triumph of biomedical science over this leukemia cannot be considered without appreciating the work of both Janet Rowley and John Goldman.

  3. Myeloid Cells in Cutaneous Wound Repair.

    PubMed

    Cash, Jenna L; Martin, Paul

    2016-06-01

    Cutaneous wound repair is a complex, dynamic process with the goal of rapidly sealing any breach in the skin's protective barrier. Myeloid cells compose a significant proportion of the inflammatory cells recruited to a wound site and play important roles in decontaminating the injured tissue of any invading microorganisms. Subsequently, myeloid cells are able to influence many aspects of the healing response, in part through their capacity to release a large array of signaling molecules that allow them to communicate with and regulate the behavior of other wound cells and in turn, be themselves exquisitely regulated by the wound microenvironment. Macrophages, for example, appear to play important, temporally changing roles in the initiation of scarring and subsequently in matrix remodeling to resolve fibrosis. In this way, myeloid cells seem to play both positive (e.g., pathogen killing and matrix remodeling) and negative (e.g., scarring) roles in wound repair. Further research is of course needed to elucidate the precise temporal and spatial myeloid cell phenotypes and behaviors and ultimately to design effective strategies to optimize the beneficial functions of these cells while minimizing their detrimental contributions to improve wound healing in the clinic. PMID:27337466

  4. Chronic myeloid leukemia: reminiscences and dreams.

    PubMed

    Mughal, Tariq I; Radich, Jerald P; Deininger, Michael W; Apperley, Jane F; Hughes, Timothy P; Harrison, Christine J; Gambacorti-Passerini, Carlo; Saglio, Giuseppe; Cortes, Jorge; Daley, George Q

    2016-05-01

    With the deaths of Janet Rowley and John Goldman in December 2013, the world lost two pioneers in the field of chronic myeloid leukemia. In 1973, Janet Rowley, unraveled the cytogenetic anatomy of the Philadelphia chromosome, which subsequently led to the identification of the BCR-ABL1 fusion gene and its principal pathogenetic role in the development of chronic myeloid leukemia. This work was also of major importance to support the idea that cytogenetic changes were drivers of leukemogenesis. John Goldman originally made seminal contributions to the use of autologous and allogeneic stem cell transplantation from the late 1970s onwards. Then, in collaboration with Brian Druker, he led efforts to develop ABL1 tyrosine kinase inhibitors for the treatment of patients with chronic myeloid leukemia in the late 1990s. He also led the global efforts to develop and harmonize methodology for molecular monitoring, and was an indefatigable organizer of international conferences. These conferences brought together clinicians and scientists, and accelerated the adoption of new therapies. The abundance of praise, tributes and testimonies expressed by many serve to illustrate the indelible impressions these two passionate and affable scholars made on so many people's lives. This tribute provides an outline of the remarkable story of chronic myeloid leukemia, and in writing it, it is clear that the historical triumph of biomedical science over this leukemia cannot be considered without appreciating the work of both Janet Rowley and John Goldman. PMID:27132280

  5. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects.

    PubMed

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A

    2016-09-19

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes. PMID:27257060

  6. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects

    PubMed Central

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A.

    2016-01-01

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes. PMID:27257060

  7. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects.

    PubMed

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A

    2016-09-19

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes.

  8. Myeloid thrombomodulin lectin-like domain inhibits osteoclastogenesis and inflammatory bone loss

    PubMed Central

    Cheng, Tsung-Lin; Lai, Chao-Han; Shieh, Shyh-Jou; Jou, Yin-Bo; Yeh, Jwu-Lai; Yang, Ai-Lun; Wang, Yan-Hsiung; Wang, Chau-Zen; Chen, Chung-Hwan; Shi, Guey-Yueh; Ho, Mei-Ling; Wu, Hua-Lin

    2016-01-01

    Osteoclastogenesis is an essential process during bone metabolism which can also be promoted by inflammatory signals. Thrombomodulin (TM), a transmembrane glycoprotein, exerts anti-inflammatory activities such as neutralization of proinflammatory high-mobility group box 1 (HMGB1) through TM lectin-like domain. This study aimed to identify the role of myeloid TM (i.e., endogenous TM expression on the myeloid lineage) in osteoclastogenesis and inflammatory bone loss. Using human peripheral blood mononuclear cells and mouse bone marrow-derived macrophages, we observed that the protein levels of TM were dramatically reduced as these cells differentiated into osteoclasts. In addition, osteoclastogenesis and extracellular HMGB1 accumulation were enhanced in primary cultured monocytes from myeloid-specific TM-deficient mice (LysMcre/TMflox/flox) and from TM lectin-like domain deleted mice (TMLeD/LeD) compared with their respective controls. Micro-computerized tomography scans showed that ovariectomy-induced bone loss was more pronounced in TMLeD/LeD mice compared with controls. Finally, the inhibiting effects of recombinant TM lectin-like domain (rTMD1) on bone resorption in vitro, and bone loss in both the ovariectomized model and collagen antibody-induced arthritis model has been detected. These findings suggested that the myeloid TM lectin-like domain may inhibit osteoclastogenesis by reducing HMGB1 signaling, and rTMD1 may hold therapeutic potential for inflammatory bone loss. PMID:27311356

  9. Epigenetic regulators and their impact on therapy in acute myeloid leukemia

    PubMed Central

    Pastore, Friederike; Levine, Ross L.

    2016-01-01

    Genomic studies of hematologic malignancies have identified a spectrum of recurrent somatic alterations that contribute to acute myeloid leukemia initiation and maintenance, and which confer sensitivities to molecularly targeted therapies. The majority of these genetic events are small, site-specific alterations in DNA sequence. In more than two thirds of patients with de novo acute myeloid leukemia mutations epigenetic modifiers are detected. Epigenetic modifiers encompass a large group of proteins that modify DNA at cytosine residues or cause post-translational histone modifications such as methylations or acetylations. Altered functions of these epigenetic modifiers disturb the physiological balance between gene activation and gene repression and contribute to aberrant gene expression regulation found in acute myeloid leukemia. This review provides an overview of the epigenetic modifiers mutated in acute myeloid leukemia, their clinical relevance and how a deeper understanding of their biological function has led to the discovery of new specific targets, some of which are currently tested in mechanism-based clinical trials. PMID:26928248

  10. A Cell-Based High-Throughput Screening for Inducers of Myeloid Differentiation

    PubMed Central

    Radomska, Hanna S.; Jernigan, Finith; Nakayama, Sohei; Jorge, Susan E.; Sun, Lijun; Tenen, Daniel G.; Kobayashi, Susumu S.

    2015-01-01

    Recent progress of genetic studies has dramatically unveiled pathogenesis of acute myeloid leukemia (AML). However, overall survival of AML still remains unsatisfactory and development of novel therapeutics is required. CCAAT/Enhancer Binding Protein α (C/EBPα) is one of crucial transcription factors that induce granulocytic differentiation and its activity is perturbed in human myeloid leukemias. As its re-expression can induce differentiation and subsequent apoptosis of leukemic cells in vitro, we hypothesized that chemical compounds that restore C/EBPα expression and/or activity would lead to myeloid differentiation of leukemic cells. Using a cell-based high-throughput screening, we identified 2-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-3-(2-methoxyphenyl)-4(3H)-quinazolinone as a potent inducer of C/EBPα and myeloid differentiation. Leukemia cell lines and primary blast cells isolated from human AML patients treated with ICCB280 demonstrated evidence of morphological and functional differentiation, as well as massive apoptosis. We performed conformational analyses of the high-throughput screening hit compounds to postulate the spatial requirements for high potency. Our results warrant a development of novel differentiation therapies and significantly impact care of AML patients with unfavorable prognosis in the near future. PMID:26109609

  11. Azacitidine, Mitoxantrone Hydrochloride, and Etoposide in Treating Older Patients With Poor-Prognosis Acute Myeloid Leukemia

    ClinicalTrials.gov

    2015-08-18

    Acute Myeloid Leukemia Arising From Previous Myelodysplastic Syndrome; Adult Acute Basophilic Leukemia; Adult Acute Eosinophilic Leukemia; Adult Acute Megakaryoblastic Leukemia; Adult Acute Monoblastic Leukemia; Adult Acute Monocytic Leukemia; Adult Acute Myeloid Leukemia in Remission; Adult Acute Myeloid Leukemia With Inv(16)(p13.1q22); CBFB-MYH11; Adult Acute Myeloid Leukemia With Maturation; Adult Acute Myeloid Leukemia With Minimal Differentiation; Adult Acute Myeloid Leukemia With t(16;16)(p13.1;q22); CBFB-MYH11; Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); RUNX1-RUNX1T1; Adult Acute Myeloid Leukemia With t(9;11)(p22;q23); MLLT3-MLL; Adult Acute Myeloid Leukemia Without Maturation; Adult Acute Myelomonocytic Leukemia; Adult Erythroleukemia; Adult Pure Erythroid Leukemia; Alkylating Agent-Related Acute Myeloid Leukemia; Recurrent Adult Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

  12. Decitabine in Treating Patients With Myelodysplastic Syndromes or Acute Myeloid Leukemia

    ClinicalTrials.gov

    2013-09-27

    Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Atypical Chronic Myeloid Leukemia, BCR-ABL1 Negative; de Novo Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Neoplasm, Unclassifiable; Previously Treated Myelodysplastic Syndromes; Recurrent Adult Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia; Secondary Myelodysplastic Syndromes; Untreated Adult Acute Myeloid Leukemia

  13. Hepatic ischemia and reperfusion injury in the absence of myeloid cell-derived COX-2 in mice.

    PubMed

    Duarte, Sergio; Kato, Hiroyuki; Kuriyama, Naohisa; Suko, Kathryn; Ishikawa, Tomo-O; Busuttil, Ronald W; Herschman, Harvey R; Coito, Ana J

    2014-01-01

    Cyclooxygenase-2 (COX-2) is a mediator of hepatic ischemia and reperfusion injury (IRI). While both global COX-2 deletion and pharmacologic COX-2 inhibition ameliorate liver IRI, the clinical use of COX-2 inhibitors has been linked to increased risks of heart attack and stroke. Therefore, a better understanding of the role of COX-2 in different cell types may lead to improved therapeutic strategies for hepatic IRI. Macrophages of myeloid origin are currently considered to be important sources of the COX-2 in damaged livers. Here, we used a Cox-2flox conditional knockout mouse (COX-2-M/-M) to examine the function of COX-2 expression in myeloid cells during liver IRI. COX-2-M/-M mice and their WT control littermates were subjected to partial liver ischemia followed by reperfusion. COX-2-M/-M macrophages did not express COX-2 upon lipopolysaccharide stimulation and COX-2-M/-M livers showed reduced levels of COX-2 protein post-IRI. Nevertheless, selective deletion of myeloid cell-derived COX-2 failed to ameliorate liver IRI; serum transaminases and histology were comparable in both COX-2-M/-M and WT mice. COX-2-M/-M livers, like WT livers, developed extensive necrosis, vascular congestion, leukocyte infiltration and matrix metalloproteinase-9 (MMP-9) expression post-reperfusion. In addition, myeloid COX-2 deletion led to a transient increase in IL-6 levels after hepatic reperfusion, when compared to controls. Administration of celecoxib, a selective COX-2 inhibitor, resulted in significantly improved liver function and histology in both COX-2-M/-M and WT mice post-reperfusion, providing evidence that COX-2-mediated liver IRI is caused by COX-2 derived from a source(s) other than myeloid cells. In conclusion, these results support the view that myeloid COX-2, including myeloid-macrophage COX-2, is not responsible for the hepatic IRI phenotype.

  14. PHF6 mutations in adult acute myeloid leukemia.

    PubMed

    Van Vlierberghe, P; Patel, J; Abdel-Wahab, O; Lobry, C; Hedvat, C V; Balbin, M; Nicolas, C; Payer, A R; Fernandez, H F; Tallman, M S; Paietta, E; Melnick, A; Vandenberghe, P; Speleman, F; Aifantis, I; Cools, J; Levine, R; Ferrando, A

    2011-01-01

    Loss of function mutations and deletions encompassing the plant homeodomain finger 6 (PHF6) gene are present in about 20% of T-cell acute lymphoblastic leukemias (ALLs). Here, we report the identification of recurrent mutations in PHF6 in 10/353 adult acute myeloid leukemias (AMLs). Genetic lesions in PHF6 found in AMLs are frameshift and nonsense mutations distributed through the gene or point mutations involving the second plant homeodomain (PHD)-like domain of the protein. As in the case of T-ALL, where PHF6 alterations are found almost exclusively in males, mutations in PHF6 were seven times more prevalent in males than in females with AML. Overall, these results identify PHF6 as a tumor suppressor gene mutated in AML and extend the role of this X-linked tumor suppressor gene in the pathogenesis of hematologic tumors.

  15. [Chronic myeloid leukemia: "archetype" of the impact of targeted therapies].

    PubMed

    Nasr, R; Bazarbachi, A

    2012-08-01

    Chronic myeloid leukemia (CML) is a chronic blood disorder characterized by a reciprocal translocation between chromosomes 9 and 22, leading to the creation of a chimeric gene encoding the BCR-ABL fusion protein with a constitutive tyrosine kinase activity. Although long known as a disease with an inexorable progression to acute leukemia, CML history has been significantly improved by the use of imatinib, a tyrosine kinase inhibitor. Imatinib has revolutionized the treatment of CML by transforming it from an invariably fatal disease to a chronic but manageable condition. In fact, the discovery of this class of targeted therapy had an impact not only on the survival of CML patients but also on other scientific and medical fields. This review illustrates the impact of imatinib, the first example of tyrosine kinase inhibitors on the treatment of CML, on the treatment of other cancers, the impact on health systems and on the scientific research in general.

  16. ADCY7 supports development of acute myeloid leukemia

    PubMed Central

    Li, Chunling; Xie, Jingjing; Lu, Zhigang; Chen, Chen; Li, Yancun; Zhan, Renhui; Fang, Yi; Hu, Xuemei; Zhang, Cheng Cheng

    2015-01-01

    Acute myeloid leukemia (AML) is the most common adult acute leukemia. Despite treatment, the majority of the AML patients relapse within 5 years. In silico analysis of several available databases of AML patients showed that the expression of adenylate cyclase 7 (ADCY7) significantly inversely correlates with the overall survival of AML patients. To determine whether ADCY7 supports AML development, we employed an shRNA-encoding lentivirus system to inhibit adcy7 expression in human AML cells including U937, MV4-11, and THP-1 cells. The ADCY7 deficiency resulted in decreased cell growth, elvated apoptosis, and lower c-Myc expression of these leukemia cells. This indicates that G protein-coupled receptor signaling contributes to AML pathogenesis. Our study suggests that inhibition of ADCY7 may be novel strategy for treating leukemia. PMID:26220344

  17. Midostaurin and Decitabine in Treating Older Patients With Newly Diagnosed Acute Myeloid Leukemia and FLT3 Mutation

    ClinicalTrials.gov

    2016-10-10

    Acute Myeloid Leukemia With FLT3/ITD Mutation; Acute Myeloid Leukemia With Gene Mutations; FLT3 Tyrosine Kinase Domain Point Mutation; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

  18. M-CSF instructs myeloid lineage fate in single haematopoietic stem cells

    PubMed Central

    Mossadegh-Keller, Noushine; Sarrazin, Sandrine; Kandalla, Prashanth K.; Espinosa, Leon; Stanley, E. Richard; Nutt, Stephen L.; Moore, Jordan; Sieweke, Michael H.

    2013-01-01

    Under stress conditions such as infection or inflammation the body rapidly needs to generate new blood cells that are adapted to the challenge. Haematopoietic cytokines are known to increase output of specific mature cells by affecting survival, expansion and differentiation of lineage-committed progenitors1,2, but it has been debated whether long-term haematopoietic stem cells (HSCs) are susceptible to direct lineage-specifying effects of cytokines. Although genetic changes in transcription factor balance can sensitize HSCs to cytokine instruction3, the initiation of HSC commitment is generally thought to be triggered by stochastic fluctuation in cell-intrinsic regulators such as lineage-specific transcription factors4–7, leaving cytokines to ensure survival and proliferation of the progeny cells8,9. Here we show that macrophage colony-stimulating factor (M-CSF, also called CSF1), a myeloid cytokine released during infection and inflammation, can directly induce the myeloid master regulator PU.1 and instruct myeloid cell-fate change in mouse HSCs, independently of selective survival or proliferation. Video imaging and single-cell gene expression analysis revealed that stimulation of highly purified HSCs with M-CSF in culture resulted in activation of the PU.1 promoter and an increased number of PU.1+ cells with myeloid gene signature and differentiation potential. In vivo, high systemic levels of M-CSF directly stimulated M-CSF-receptor-dependent activation of endogenous PU.1 protein in single HSCs and induced a PU.1-dependent myeloid differentiation preference. Our data demonstrate that lineage-specific cytokines can act directly on HSCs in vitro and in vivo to instruct a change of cell identity. This fundamentally changes the current view of how HSCs respond to environmental challenge and implicates stress-induced cytokines as direct instructors of HSC fate. PMID:23575636

  19. Diversity of breakpoints of variant Philadelphia chromosomes in chronic myeloid leukemia in Brazilian patients

    PubMed Central

    Chauffaille, Maria de Lourdes Lopes Ferrari; Bandeira, Ana Carolina de Almeida; da Silva, Aline Schiavoni Guarnieri

    2014-01-01

    Background Chronic myeloid leukemia is a myeloproliferative disorder characterized by the Philadelphia chromosome or t(9;22)(q34.1;q11.2), resulting in the break-point cluster region-Abelson tyrosine kinase fusion gene, which encodes a constitutively active tyrosine kinase protein. The Philadelphia chromosome is detected by karyotyping in around 90% of chronic myeloid leukemia patients, but 5–10% may have variant types. Variant Philadelphia chromosomes are characterized by the involvement of another chromosome in addition to chromosome 9 or 22. It can be a simple type of variant when one other chromosome is involved, or complex, in which two or more chromosomes take part in the translocation. Few studies have reported the incidence of variant Philadelphia chromosomes or the breakpoints involved among Brazilian chronic myeloid leukemia patients. Objective The aim of this report is to describe the diversity of the variant Philadelphia chromosomes found and highlight some interesting breakpoint candidates for further studies. Methods the Cytogenetics Section Database was searched for all cases with diagnoses of chronic myeloid leukemia during a 12-year period and all the variant Philadelphia chromosomes were listed. Results Fifty (5.17%) cases out of 1071 Philadelphia-positive chronic myeloid leukemia were variants. The most frequently involved chromosome was 17, followed by chromosomes: 1, 20, 6, 11, 2, 10, 12 and 15. Conclusion Among all the breakpoints seen in this survey, six had previously been described: 11p15, 14q32, 15q11.2, 16p13.1, 17p13 and 17q21. The fact that some regions get more frequently involved in such rare rearrangements calls attention to possible predisposition that should be further studied. Nevertheless, the pathological implication of these variants remains unclear. PMID:25638762

  20. Myeloid-derived suppressor activity is mediated by monocytic lineages maintained by continuous inhibition of extrinsic and intrinsic death pathways.

    PubMed

    Haverkamp, Jessica M; Smith, Amber M; Weinlich, Ricardo; Dillon, Christopher P; Qualls, Joseph E; Neale, Geoffrey; Koss, Brian; Kim, Young; Bronte, Vincenzo; Herold, Marco J; Green, Douglas R; Opferman, Joseph T; Murray, Peter J

    2014-12-18

    Nonresolving inflammation expands a heterogeneous population of myeloid suppressor cells capable of inhibiting T cell function. This heterogeneity has confounded the functional dissection of individual myeloid subpopulations and presents an obstacle for antitumor immunity and immunotherapy. Using genetic manipulation of cell death pathways, we found the monocytic suppressor-cell subset, but not the granulocytic subset, requires continuous c-FLIP expression to prevent caspase-8-dependent, RIPK3-independent cell death. Development of the granulocyte subset requires MCL-1-mediated control of the intrinsic mitochondrial death pathway. Monocytic suppressors tolerate the absence of MCL-1 provided cytokines increase expression of the MCL-1-related protein A1. Monocytic suppressors mediate T cell suppression, whereas their granulocytic counterparts lack suppressive function. The loss of the granulocytic subset via conditional MCL-1 deletion did not alter tumor incidence implicating the monocytic compartment as the functionally immunosuppressive subset in vivo. Thus, death pathway modulation defines the development, survival, and function of myeloid suppressor cells.