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Sample records for adaptor protein appl1

  1. 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

  2. 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

  3. 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.

  4. APPL1 regulates basal NF-κB activity by stabilizing NIK

    PubMed Central

    Hupalowska, Anna; Pyrzynska, Beata; Miaczynska, Marta

    2012-01-01

    Summary APPL1 is a multifunctional adaptor protein that binds membrane receptors, signaling proteins and nuclear factors, thereby acting in endosomal trafficking and in different signaling pathways. Here, we uncover a novel role of APPL1 as a positive regulator of transcriptional activity of NF-κB under basal but not TNFα-stimulated conditions. APPL1 was found to directly interact with TRAF2, an adaptor protein known to activate canonical NF-κB signaling. APPL1 synergized with TRAF2 to induce NF-κB activation, and both proteins were necessary for this process and function upstream of the IKK complex. Although TRAF2 was not detectable on APPL endosomes, endosomal recruitment of APPL1 was required for its function in the NF-κB pathway. Importantly, in the canonical pathway, APPL1 appeared to regulate the proper spatial distribution of the p65 subunit of NF-κB in the absence of cytokine stimulation, since its overexpression enhanced and its depletion reduced the nuclear accumulation of p65. By analyzing the patterns of gene transcription upon APPL1 overproduction or depletion we found altered expression of NF-κB target genes that encode cytokines. At the molecular level, overexpressed APPL1 markedly increased the level of NIK, the key component of the noncanonical NF-κB pathway, by reducing its association with the degradative complex containing TRAF2, TRAF3 and cIAP1. In turn, high levels of NIK triggered nuclear translocation of p65. Collectively, we propose that APPL1 regulates basal NF-κB activity by modulating the stability of NIK, which affects the activation of p65. This places APPL1 as a novel link between the canonical and noncanonical machineries of NF-κB activation. PMID:22685329

  5. 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

  6. APPL1 acts as a protective factor against podocytes injury in high glucose environment

    PubMed Central

    Ji, Zhenzhong; Hu, Zhengguo; Xu, Yancheng

    2015-01-01

    APPL1, an intracellular adaptor protein, takes part in numerous metabolic reactions. Although APPL1 plays a key role in glucose metabolism via adiponectin pathway and has been proved associated with type 2 diabetes, little is known about its role in diabetic nephropathy. To explore the role of APPL1 in diabetic nephropathy, we upregulated the expression of APPL1 in cultured mouse podocytes by adenovirus infection and tested the effects of APPL1 overexpression in podocytes treated with high glucose. Here, a mouse podocyte cell line (generated from H-2Kb-tsA58 immortmouse) was cultured and divided into four groups: Group 1 (normal glucose, NG), Group 2 (high glucose, HG), Group 3 (HG and infected with control adenovirus) and Group 4 (HG and infected with Ad-APPL1). Cell vitality of Group 4 is significantly higher than Group 2, but notably lower than Group 1 (P<0.01). The apoptosis rate of Group 4 was much lower (P<0.01) than Group 2 and Group 3. A decrease in phase G0/G1 and an increase in phase S was observed in Group 4 compared with Group 2 (P<0.01). These data suggested the protective role of APPL1 overexpression in high glucose condition. Moreover, the levels of Nephrin, AMPK and p-AMPK were decreased by high-glucose treatment, but increased by APPL1 overexpression. In conclusion, in the experimental high glucose condition, APPL1 acts as a protective factor against podocytes injury through regulating AMPK signaling, and may be a new therapy target for diabetic nephropathy. PMID:26261560

  7. 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.

  8. 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

  9. The Adapter Protein APPL1 Links FSH Receptor to Inositol 1,4,5-Trisphosphate Production and Is Implicated in Intracellular Ca2+ Mobilization

    PubMed Central

    Thomas, Richard M.; Nechamen, Cheryl A.; Mazurkiewicz, Joseph E.; Ulloa-Aguirre, Alfredo

    2011-01-01

    FSH binds to its receptor (FSHR) on target cells in the ovary and testis, to regulate oogenesis and spermatogenesis, respectively. The signaling cascades activated after ligand binding are extremely complex and have been shown to include protein kinase A, mitogen-activated protein kinase, phosphatidylinositol 3-kinase/protein kinase B, and inositol 1,4,5-trisphosphate–mediated calcium signaling pathways. The adapter protein APPL1 (Adapter protein containing Pleckstrin homology domain, Phosphotyrosine binding domain and Leucine zipper motif), which has been linked to an assortment of other signaling proteins, was previously identified as an interacting protein with FSHR. Thus, alanine substitution mutations in the first intracellular loop of FSHR were generated to determine which residues are essential for FSHR-APPL1 interaction. Three amino acids were essential; when any one of them was altered, APPL1 association with FSHR mutants was abrogated. Two of the mutants (L377A and F382A) that displayed poor cell-surface expression were not studied further. Substitution of FSHR-K376A did not affect FSH binding or agonist-stimulated cAMP production in either transiently transfected human embryonic kidney cells or virally transduced human granulosa cells (KGN). In the KGN line, as well as primary cultures of rat granulosa cells transduced with wild type or mutant receptor, FSH-mediated progesterone or estradiol production was not affected by the mutation. However, in human embryonic kidney cells inositol 1,4,5-trisphosphate production was curtailed and KGN cells transduced with FSHR-K376A evidenced reduced Ca2+ mobilization from intracellular stores after FSH treatment. PMID:21285318

  10. APPL1 promotes glucose uptake in response to mechanical stretch via the PKCζ-non-muscle myosin IIa pathway in C2C12 myotubes.

    PubMed

    Saito, Tsugumichi; Okada, Shuichi; Shimoda, Yoko; Tagaya, Yuko; Osaki, Aya; Yamada, Eijiro; Shibusawa, Ryo; Nakajima, Yasuyo; Ozawa, Atsushi; Satoh, Tetsurou; Mori, Masatomo; Yamada, Masanobu

    2016-11-01

    Expression of adaptor protein, phosphotyrosine interaction, pleckstrin homology domain, and leucine zipper containing 1 (APPL1) promoted glucose transporter 4 (GLUT4) translocation and glucose uptake in adipose and muscle tissues in response to stimulation with insulin, adiponectin, or exercise. In response to mechanical stretch, knockdown of APPL1 in C2C12 myotubes suppressed glucose uptake. APPL1-induced increased glucose uptake was mediated by protein kinase C (PKC) ζ but not AKT, AMPK, or calmodulin-dependent protein kinase. In myotubes overexpressing APPL1, PKCζ was phosphorylated and translocated to the plasma membrane (PM) in response to mechanical stretch. Phosphorylated PKCζ co-immunoprecipitated with protein phosphatase 2A (PP2A) under basal conditions, but dissociated upon myotube stretching. Moreover, stretch-induced phosphorylated PKCζ co-immunoprecipitated with non-muscle myosin IIa. Blebbistatin, an inhibitor of myosin II ATPase activity, suppressed APPL1-mediated stretch-induced glucose uptake and PKCζ translocation. Taken together these data demonstrate that in response to mechanical stretch, APPL1 enhances glucose uptake by modulating the activation and localization of PKCζ, as well as its functional interaction with both PP2A and myosin IIa. These findings support a new function for non-muscle myosin IIa in differentiated myotubes. PMID:27478065

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  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. 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

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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.

  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. 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

  18. 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

  19. 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.

  20. Appl1 and Appl2 are Expendable for Mouse Development But Are Essential for HGF-Induced Akt Activation and Migration in Mouse Embryonic Fibroblasts.

    PubMed

    Tan, Yinfei; Xin, Xiaoban; Coffey, Francis J; Wiest, David L; Dong, Lily Q; Testa, Joseph R

    2016-05-01

    Although Appl1 and Appl2 have been implicated in multiple cellular activities, we and others have found that Appl1 is dispensable for mouse embryonic development, suggesting that Appl2 can substitute for Appl1 during development. To address this possibility, we generated conditionally targeted Appl2 mice. We found that ubiquitous Appl2 knockout (Appl2-/-) mice, much like Appl1-/- mice, are viable and grow normally to adulthood. Intriguingly, when Appl1-/- mice were crossed with Appl2-/- mice, we found that homozygous Appl1;Appl2 double knockout (DKO) animals are also viable and grossly normal with regard to reproductive potential and postnatal growth. Appl2-null and DKO mice were found to exhibit altered red blood cell physiology, with erythrocytes from these mice generally being larger and having a more irregular shape than erythrocytes from wild type mice. Although Appl1/2 proteins have been previously shown to have a very strong interaction with phosphatidylinositol-3 kinase (Pi3k) in thymic T cells, Pi3k-Akt signaling and cellular differentiation was unaltered in thymocytes from Appl1;Appl2 (DKO) mice. However, Appl1/2-null mouse embryonic fibroblasts exhibited defects in HGF-induced Akt activation, migration, and invasion. Taken together, these data suggest that Appl1 and Appl2 are required for robust HGF cell signaling but are dispensable for embryonic development and reproduction.

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

  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. PMID:26907692

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.'

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. Adiponectin enhances osteogenic differentiation in human adipose-derived stem cells by activating the APPL1-AMPK signaling pathway

    SciTech Connect

    Chen, Tong; Wu, Yu-wei; Lu, Hui; Guo, Yuan; Tang, Zhi-hui

    2015-05-29

    Human adipose-derived stem cells (hASCs) are multipotent progenitor cells with multi-lineage differentiation potential including osteogenesis and adipogenesis. While significant progress has been made in understanding the transcriptional control of hASC fate, little is known about how hASC differentiation is regulated by the autocrine loop. The most abundant adipocytokine secreted by adipocytes, adiponectin (APN) plays a pivotal role in glucose metabolism and energy homeostasis. Growing evidence suggests a positive association between APN and bone formation yet little is known regarding the direct effects of APN on hASC osteogenesis. Therefore, this study was designed to investigate the varied osteogenic effects and regulatory mechanisms of APN in the osteogenic commitment of hASCs. We found that APN enhanced the expression of osteoblast-related genes in hASCs, such as osteocalcin, alkaline phosphatase, and runt-related transcription factor-2 (Runx2, also known as CBFa1), in a dose- and time-dependent manner. This was further confirmed by the higher expression levels of alkaline phosphatase and increased formation of mineralization nodules, along with the absence of inhibition of cell proliferation. Importantly, APN at 1 μg/ml was the optimal concentration, resulting in maximum deposition of calcium nodules, and was significant superior to bone morphogenetic protein 2. Mechanistically, we found for the first time that APN increased nuclear translocation of the leucine zipper motif (APPL)-1 as well as AMP-activated protein kinase (AMPK) phosphorylation, which were reversed by pretreatment with APPL1 siRNA. Our results indicate that APN promotes the osteogenic differentiation of hASCs by activating APPL1-AMPK signaling, suggesting that manipulation of APN is a novel therapeutic target for controlling hASC fate. - Highlights: • Adiponectin enhances osteogenic differentiation in human adipose-derived stem cells. • The knock-down of APPL1 block the enhancement of

  9. 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.

  10. 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

  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 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

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. An APPL1-AMPK signaling axis mediates beneficial metabolic effects of adiponectin in the heart

    PubMed Central

    Fang, Xiangping; Palanivel, Rengasamy; Cresser, Justin; Schram, Kristin; Ganguly, Riya; Thong, Farah S. L.; Tuinei, Joseph; Xu, Aimin; Abel, E. Dale

    2010-01-01

    Adiponectin promotes cardioprotection by various mechanisms, and this study used primary cardiomyocytes and the isolated working perfused heart to investigate cardiometabolic effects. We show in adult cardiomyocytes that adiponectin increased CD36 translocation and fatty acid uptake as well as insulin-stimulated glucose transport and Akt phosphorylation. Coimmunoprecipitation showed that adiponectin enhanced association of AdipoR1 with APPL1, subsequent binding of APPL1 with AMPKα2, which led to phosphorylation and inhibition of ACC and increased fatty acid oxidation. Using siRNA to effectively knockdown APPL1 in neonatal cardiomyocytes, we demonstrated an essential role for APPL1 in mediating increased fatty acid uptake and oxidation by adiponectin. Importantly, enhanced fatty acid oxidation in conjunction with AMPK and ACC phosphorylation was also observed in the isolated working heart. Despite increasing fatty acid oxidation and myocardial oxygen consumption, adiponectin increased hydraulic work and maintained cardiac efficiency. In summary, the present study documents several beneficial metabolic effects mediated by adiponectin in the heart and provides novel insight into the mechanisms behind these effects, in particular the importance of APPL1. PMID:20739511

  20. 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.

  1. 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 ...

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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)

  11. 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

  12. 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,…

  13. 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...

  14. 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...

  15. 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...

  16. 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...

  17. 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...

  18. 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...

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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.

  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. 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

  1. 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...

  2. 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.

  3. 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...

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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…

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

  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. PMID:26885859

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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).

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  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. Regulatory domains of the A-Myb transcription factor and its interaction with the CBP/p300 adaptor molecules.

    PubMed Central

    Facchinetti, V; Loffarelli, L; Schreek, S; Oelgeschläger, M; Lüscher, B; Introna, M; Golay, J

    1997-01-01

    The A-Myb transcription factor belongs to the Myb family of oncoproteins and is likely to be involved in the regulation of proliferation and/or differentiation of normal B cells and Burkitt's lymphoma cells. To characterize in detail the domains of A-Myb that regulate its function, we have generated a series of deletion mutants and have investigated their trans-activation potential as well as their DNA-binding activity. Our results have allowed us to delineate the trans-activation domain as well as two separate regulatory regions. The boundaries of the trans-activation domain (amino acid residues 218-319) are centred on a sequence rich in charged amino acids (residues 259-281). A region (residues 320-482) localized immediately downstream of the trans-activation domain and containing a newly identified conserved stretch of 48 residues markedly inhibits specific DNA binding. Finally the last 110 residues of A-Myb (residues 643-752), which include a sequence conserved in all mammalian myb genes (region III), negatively regulate the maximal trans-activation potential of A-Myb. We have also investigated the functional interaction between A-Myb and the nuclear adaptor molecule CBP [cAMP response element-binding protein (CREB)-binding protein]. We demonstrate that CBP synergizes with A-Myb in a dose-dependent fashion, and that this co-operative effect can be inhibited by E1A and can also be observed with the CBP homologue p300. We show that this functional synergism requires the presence of the A-Myb charged sequence and that it involves physical interaction between A-Myb and the CREB-binding domain of CBP. PMID:9210395

  9. Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells.

    PubMed

    Reginald, Kavita; Chaoui, Karima; Roncagalli, Romain; Beau, Mathilde; Goncalves Menoita, Marisa; Monsarrat, Bernard; Burlet-Schiltz, Odile; Malissen, Marie; Gonzalez de Peredo, Anne; Malissen, Bernard

    2015-12-01

    The protein tyrosine kinase LCK plays a key role in TCR signaling, and its activity is dynamically controlled by the tyrosine kinase C-terminal Src kinase (CSK) and the tyrosine phosphatase CD45. CSK is brought in contiguity to LCK via binding to a transmembrane adaptor known as phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG). The lack of a blatant phenotype in PAG-deficient mice has impeded our understanding of the mechanisms through which PAG exerts its negative-regulatory role in TCR signaling. We used quantitative mass spectrometry and both thymocytes and CD4(+) T cells from mice in which a tag for affinity purification was knocked in the gene coding for PAG to determine the composition and dynamics of the multiprotein complexes that are found around PAG over 5 min of activation. Most of the high-confidence interactions that we observed were previously unknown. Using phosphoproteomic analysis, PAG showed low levels of tyrosine phosphorylation in resting primary mouse CD4(+) T cells; the levels of tyrosine phosphorylation increased and reached a maximum 2 min after stimulation. Analysis of the dynamics of association of the protein tyrosine phosphatase PTPN22 and lipid phosphatase SHIP-1 with PAG following T cell activation suggests that both cooperate with CSK to terminate T cell activation. Our findings provide a model of the role for PAG in mouse primary CD4(+) T cells that is consistent with recent phosphoproteomic studies of the Jurkat T cell line but difficult to reconcile with former biochemical studies indicating that PAG is constitutively phosphorylated in resting T cells and rapidly dephosphorylated once the TCR is engaged. PMID:26512138

  10. DNA as Tunable Adaptor for siRNA Polyplex Stabilization and Functionalization

    PubMed Central

    Heissig, Philipp; Klein, Philipp M.; Hadwiger, Philipp; Wagner, Ernst

    2016-01-01

    siRNA and microRNA are promising therapeutic agents, which are engaged in a natural mechanism called RNA interference that modulates gene expression posttranscriptionally. For intracellular delivery of such nucleic acid triggers, we use sequence-defined cationic polymers manufactured through solid phase chemistry. They consist of an oligoethanamino amide core for siRNA complexation and optional domains for nanoparticle shielding and cell targeting. Due to the small size of siRNA, electrostatic complexes with polycations are less stable, and consequently intracellular delivery is less efficient. Here we use DNA oligomers as adaptors to increase size and charge of cargo siRNA, resulting in increased polyplex stability, which in turn boosts transfection efficiency. Extending a single siRNA with a 181-nucleotide DNA adaptor is sufficient to provide maximum gene silencing aided by cationic polymers. Interestingly, this simple strategy was far more effective than merging defined numbers (4–10) of siRNA units into one DNA scaffolded construct. For DNA attachment, the 3′ end of the siRNA passenger strand was beneficial over the 5′ end. The impact of the attachment site however was resolved by introducing bioreducible disulfides at the connection point. We also show that DNA adaptors provide the opportunity to readily link additional functional domains to siRNA. Exemplified by the covalent conjugation of the endosomolytic influenza peptide INF-7 to siRNA via a DNA backbone strand and complexing this construct with a targeting polymer, we could form a highly functional polyethylene glycol–shielded polyplex to downregulate a luciferase gene in folate receptor–positive cells. PMID:26928236

  11. NRAGE, a p75NTR adaptor protein, is required for developmental apoptosis in vivo.

    PubMed

    Bertrand, M J M; Kenchappa, R S; Andrieu, D; Leclercq-Smekens, M; Nguyen, H N T; Carter, B D; Muscatelli, F; Barker, P A; De Backer, O

    2008-12-01

    NRAGE (also known as Maged1, Dlxin) is a member of the MAGE gene family that may play a role in the neuronal apoptosis that is regulated by the p75 neurotrophin receptor (p75NTR). To test this hypothesis in vivo, we generated NRAGE knockout mice and found that NRAGE deletion caused a defect in developmental apoptosis of sympathetic neurons of the superior cervical ganglia, similar to that observed in p75NTR knockout mice. Primary sympathetic neurons derived from NRAGE knockout mice were resistant to apoptosis induced by brain-derived neurotrophic factor (BDNF), a pro-apoptotic p75NTR ligand, and NRAGE-deficient sympathetic neurons show attenuated BDNF-dependent JNK activation. Hair follicle catagen is an apoptosis-like process that is dependent on p75NTR signaling; we show that NRAGE and p75NTR show regulated co-expression in the hair follicle and that identical defects in hair follicle catagen are present in NRAGE and p75NTR knockout mice. Interestingly, NRAGE knockout mice have severe defects in motoneuron apoptosis that are not observed in p75NTR knockout animals, raising the possibility that NRAGE may facilitate apoptosis induced by receptors other than p75NTR. Together, these studies demonstrate that NRAGE plays an important role in apoptotic-signaling in vivo. PMID:18772898

  12. NRAGE, a p75NTR adaptor protein, is required for developmental apoptosis in vivo.

    PubMed

    Bertrand, M J M; Kenchappa, R S; Andrieu, D; Leclercq-Smekens, M; Nguyen, H N T; Carter, B D; Muscatelli, F; Barker, P A; De Backer, O

    2008-12-01

    NRAGE (also known as Maged1, Dlxin) is a member of the MAGE gene family that may play a role in the neuronal apoptosis that is regulated by the p75 neurotrophin receptor (p75NTR). To test this hypothesis in vivo, we generated NRAGE knockout mice and found that NRAGE deletion caused a defect in developmental apoptosis of sympathetic neurons of the superior cervical ganglia, similar to that observed in p75NTR knockout mice. Primary sympathetic neurons derived from NRAGE knockout mice were resistant to apoptosis induced by brain-derived neurotrophic factor (BDNF), a pro-apoptotic p75NTR ligand, and NRAGE-deficient sympathetic neurons show attenuated BDNF-dependent JNK activation. Hair follicle catagen is an apoptosis-like process that is dependent on p75NTR signaling; we show that NRAGE and p75NTR show regulated co-expression in the hair follicle and that identical defects in hair follicle catagen are present in NRAGE and p75NTR knockout mice. Interestingly, NRAGE knockout mice have severe defects in motoneuron apoptosis that are not observed in p75NTR knockout animals, raising the possibility that NRAGE may facilitate apoptosis induced by receptors other than p75NTR. Together, these studies demonstrate that NRAGE plays an important role in apoptotic-signaling in vivo.

  13. Discovery of a Unique Clp Component, ClpF, in Chloroplasts: A Proposed Binary ClpF-ClpS1 Adaptor Complex Functions in Substrate Recognition and Delivery[OPEN

    PubMed Central

    Nishimura, Kenji; Apitz, Janina; Friso, Giulia; Kim, Jitae; Ponnala, Lalit; Grimm, Bernhard

    2015-01-01

    Clp proteases are found in prokaryotes, mitochondria, and plastids where they play crucial roles in maintaining protein homeostasis (proteostasis). The plant plastid Clp machinery comprises a hetero-oligomeric ClpPRT proteolytic core, ATP-dependent chaperones ClpC and ClpD, and an adaptor protein, ClpS1. ClpS1 selects substrates to the ClpPR protease-ClpC chaperone complex for degradation, but the underlying substrate recognition and delivery mechanisms are currently unclear. Here, we characterize a ClpS1-interacting protein in Arabidopsis thaliana, ClpF, which can interact with the Clp substrate glutamyl-tRNA reductase. ClpF and ClpS1 mutually stimulate their association with ClpC. ClpF, which is only found in photosynthetic eukaryotes, contains bacterial uvrB/C and YccV protein domains and a unique N-terminal domain. We propose a testable model in which ClpS1 and ClpF form a binary adaptor for selective substrate recognition and delivery to ClpC, reflecting an evolutionary adaptation of the Clp system to the plastid proteome. PMID:26419670

  14. The AP-3 adaptor complex mediates sorting of yeast and mammalian PQ-loop-family basic amino acid transporters to the vacuolar/lysosomal membrane

    PubMed Central

    Llinares, Elisa; Barry, Abdoulaye Oury; André, Bruno

    2015-01-01

    The limiting membrane of lysosomes in animal cells and that of the vacuole in yeast include a wide variety of transporters, but little is known about how these proteins reach their destination membrane. The mammalian PQLC2 protein catalyzes efflux of basic amino acids from the lysosome, and the similar Ypq1, −2, and −3 proteins of yeast perform an equivalent function at the vacuole. We here show that the Ypq proteins are delivered to the vacuolar membrane via the alkaline phosphatase (ALP) trafficking pathway, which requires the AP-3 adaptor complex. When traffic via this pathway is deficient, the Ypq proteins pass through endosomes from where Ypq1 and Ypq2 properly reach the vacuolar membrane whereas Ypq3 is missorted to the vacuolar lumen via the multivesicular body pathway. When produced in yeast, PQLC2 also reaches the vacuolar membrane via the ALP pathway, but tends to sort to the vacuolar lumen if AP-3 is defective. Finally, in HeLa cells, inhibiting the synthesis of an AP-3 subunit also impairs sorting of PQLC2 to lysosomes. Our results suggest the existence of a conserved AP-3-dependent trafficking pathway for proper delivery of basic amino acid exporters to the yeast vacuole and to lysosomes of human cells. PMID:26577948

  15. Effectiveness of Needleless Vial Adaptors and Blunt Cannulas for Drug Administration in a Microgravity Environment

    NASA Technical Reports Server (NTRS)

    Hailey, M.; Bayuse, T.

    2010-01-01

    Fluid Isolation in the medication vial: Air/ fluid isolation maneuvers were used to move the medication to the septum end of vial. This isolation may be achieved in multiple ways based on the experience of the astronaut with fluid management in microgravity. If vial adaptors/blunt cannula or syringe assembly is inserted into the to vial before fluid isolation commences, the stability of this assembly should be considered in an effort to limit the risk of "slinging off" of the vial during isolation. Alternatively, fluid isolation can be performed prior to attaching the syringe/vial adaptor assembly. Terrestrial practices for medication withdrawal from a nonvented vial require injection of an equivalent amount of air as the expected medication volume prior to withdrawing liquid. In microgravity, this action is still valid, however the injection of additional air into the vial creates a multitude of micro bubbles and increases the volume of medication mixed with air that then must be withdrawn to achieve the desired drug volume in syringe. This practice is more likely to be required when using vials >30ml in size and injection volumes >10mL. It is felt that based on the microgravity flight, the practice of air injection is more of a hindrance than help.

  16. CRM1 and its ribosome export adaptor NMD3 localize to the nucleolus and affect rRNA synthesis.

    PubMed

    Bai, Baoyan; Moore, Henna M; Laiho, Marikki

    2013-01-01

    CRM1 is an export factor that together with its adaptor NMD3 transports numerous cargo molecules from the nucleus to cytoplasm through the nuclear pore. Previous studies have suggested that CRM1 and NMD3 are detected in the nucleolus. However, their localization with subnucleolar domains or participation in the activities of the nucleolus are unclear. We demonstrate here biochemically and using imaging analyses that CRM1 and NMD3 co-localize with nucleolar marker proteins in the nucleolus. In particular, their nucleolar localization is markedly increased by inhibition of RNA polymerase I (Pol I) transcription by actinomycin D or by silencing Pol I catalytic subunit, RPA194. We show that CRM1 nucleolar localization is dependent on its activity and the expression of NMD3, whereas NMD3 nucleolar localization is independent of CRM1. This suggests that NMD3 provides nucleolar tethering of CRM1. While inhibition of CRM1 by leptomycin B inhibited processing of 28S ribosomal (r) RNA, depletion of NMD3 did not, suggesting that their effects on 28S rRNA processing are distinct. Markedly, depletion of NMD3 and inhibition of CRM1 reduced the rate of pre-47S rRNA synthesis. However, their inactivation did not lead to nucleolar disintegration, a hallmark of Pol I transcription stress, suggesting that they do not directly regulate transcription. These results indicate that CRM1 and NMD3 have complex functions in pathways that couple rRNA synthetic and processing engines and that the rRNA synthesis rate may be adjusted according to proficiency in rRNA processing and export.

  17. The late endosomal adaptor p14 is a macrophage host-defense factor against Salmonella infection.

    PubMed

    Taub, Nicole; Nairz, Manfred; Hilber, Diana; Hess, Michael W; Weiss, Günter; Huber, Lukas A

    2012-06-01

    The outcome of an infection depends on the balance between host resistance and bacterial virulence. Here, we show that the late endosomal adaptor p14 (also known as LAMTOR2) is one of the components for cellular host defense against the intracellular pathogen Salmonella enterica serovar Typhimurium. During Salmonella infection, the complex of p14 and MP1 is required for the accurately timed transport of Salmonella through the endolysosomal system. Loss of p14 opens a time window that allows Salmonella to populate a replication niche, in which early and late antimicrobial effector systems, comprising NADPH phagocytic oxidase and inducible nitric oxide synthase, respectively, are inappropriately activated. Thus, p14 supports the accurate transport of Salmonella through the endolysosomal system, thereby limiting bacterial replication in both, professional phagocytes and in non-phagocytic cells in vitro, and helps mice to successfully battle Salmonella infection in vivo.

  18. Effectiveness of Needles Vial Adaptors and Blunt Cannulas for Drug Administration in a Microgravity Environment

    NASA Technical Reports Server (NTRS)

    Hailey, Melinda; Bayuse, Tina

    2009-01-01

    The need for a new system of injectable medications aboard the International Space Station (ISS) was identified. It is desired that this system fly medications in their original manufacturer's packaging, allowing the system to comply with United States Pharmacopeia (USP) guidelines while minimizing the resupply frequency due to medication expiration. Pre-filled syringes are desired, however, the evolving nature of the healthcare marketplace requires flexibility in the redesign. If medications must be supplied in a vial, a system is required that allows for the safe withdrawal of medication from the vial into a syringe for administration in microgravity. During two reduced gravity flights, the effectiveness of two versions of a blunt cannula and needleless vial adaptors was evaluated to facilitate the withdrawal of liquid medication from a vial into a syringe for injection. Other parameters assessed included the ability to withdraw the required amount of medication and whether this is dependent on vial size, liquid, or the total volume of fluid within the vial. Injectable medications proposed for flight on ISS were used for this evaluation. Due to differing sizes of vials and the fluid properties of the medications, the needleless vial adaptors proved to be too cumbersome to recommend for use on the ISS. The blunt cannula, specifically the plastic version, proved to be more effective at removing medication from the various sizes of vials and are the recommended hardware for ISS. Fluid isolation within the vials and syringes is an important step in preparing medication for injection regardless of the hardware used. Although isolation is a challenge in the relatively short parabolas during flight, it is not an obstacle for sustained microgravity. This presentation will provide an overview of the products tested as well as the challenges identified during the microgravity flights.

  19. Nck adaptors, besides promoting N-WASP mediated actin-nucleation activity at pedestals, influence the cellular levels of enteropathogenic Escherichia coli Tir effector.

    PubMed

    Nieto-Pelegrin, Elvira; Kenny, Brendan; Martinez-Quiles, Narcisa

    2014-01-01

    Enteropathogenic Escherichia coli (EPEC) binding to human intestinal cells triggers the formation of disease-associated actin rich structures called pedestals. The latter process requires the delivery, via a Type 3 secretion system, of the translocated Intimin receptor (Tir) protein into the host plasma membrane where binding of a host kinase-modified form to the bacterial surface protein Intimin triggers pedestal formation. Tir-Intimin interaction recruits the Nck adaptor to a Tir tyrosine phosphorylated residue where it activates neural Wiskott-Aldrich syndrome protein (N-WASP); initiating the major pathway to actin polymerization mediated by the actin-related protein (Arp) 2/3 complex. Previous studies with Nck-deficient mouse embryonic fibroblasts (MEFs) identified a key role for Nck in pedestal formation, presumed to reflect a lack of N-WASP activation. Here, we show the defect relates to reduced amounts of Tir within Nck-deficient cells. Indeed, Tir delivery and, thus, pedestal formation defects were much greater for MEFs than HeLa (human epithelial) cells. Crucially, the levels of two other effectors (EspB/EspF) within Nck-deficient MEFs were not reduced unlike that of Map (Mitochondrial associated protein) which, like Tir, requires CesT chaperone function for efficient delivery. Interestingly, drugs blocking various host protein degradation pathways failed to increase Tir cellular levels unlike an inhibitor of deacetylase activity (Trichostatin A; TSA). Treatments with TSA resulted in significant recovery of Tir levels, potentiation of actin polymerization and improvement in bacterial attachment to cells. Our findings have important implications for the current model of Tir-mediated actin polymerization and opens new lines of research in this area. PMID:25482634

  20. Binding of the cSH3 domain of Grb2 adaptor to two distinct RXXK motifs within Gab1 docker employs differential mechanisms.

    PubMed

    McDonald, Caleb B; Seldeen, Kenneth L; Deegan, Brian J; Bhat, Vikas; Farooq, Amjad

    2011-01-01

    A ubiquitous component of cellular signaling machinery, Gab1 docker plays a pivotal role in routing extracellular information in the form of growth factors and cytokines to downstream targets such as transcription factors within the nucleus. Here, using isothermal titration calorimetry (ITC) in combination with macromolecular modeling (MM), we show that although Gab1 contains four distinct RXXK motifs, designated G1, G2, G3, and G4, only G1 and G2 motifs bind to the cSH3 domain of Grb2 adaptor and do so with distinct mechanisms. Thus, while the G1 motif strictly requires the PPRPPKP consensus sequence for high-affinity binding to the cSH3 domain, the G2 motif displays preference for the PXVXRXLKPXR consensus. Such sequential differences in the binding of G1 and G2 motifs arise from their ability to adopt distinct polyproline type II (PPII)- and 3(10) -helical conformations upon binding to the cSH3 domain, respectively. Collectively, our study provides detailed biophysical insights into a key protein-protein interaction involved in a diverse array of signaling cascades central to health and disease.

  1. Characterization of C-terminal adaptors, UFD-2 and UFD-3, of CDC-48 on the polyglutamine aggregation in C. elegans.

    PubMed

    Murayama, Yuki; Ogura, Teru; Yamanaka, Kunitoshi

    2015-03-27

    CDC-48 (also called VCP or p97 in mammals and Cdc48p in yeast) is a AAA (ATPases associated with diverse cellular activities) chaperone and participates in a wide range of cellular activities including modulation of protein complexes and protein aggregates. UFD-2 and UFD-3, C-terminal adaptors for CDC-48, reportedly bind to CDC-48 in a mutually exclusive manner and they may modulate the fate of substrates for CDC-48. However, their cellular functions have not yet been elucidated. In this study, we found that CDC-48 preferentially interacts with UFD-3 in Caenorhabditis elegans. We also found that the number of polyglutamine (polyQ) aggregates was reduced in the ufd-3 deletion mutant but not in the ufd-2 deletion mutant. Furthermore, the lifespan and motility of the ufd-3 deletion mutant, where polyQ40::GFP was expressed, were greatly decreased. Taken together, we propose that UFD-3 may promote the formation of polyQ aggregates to reduce the polyQ toxicity in C. elegans.

  2. B Cell Adaptor Containing Src Homology 2 Domain (Bash) Links B Cell Receptor Signaling to the Activation of Hematopoietic Progenitor Kinase 1

    PubMed Central

    Tsuji, Sachiyo; Okamoto, Mariko; Yamada, Koichi; Okamoto, Noriaki; Goitsuka, Ryo; Arnold, Rudiger; Kiefer, Friedemann; Kitamura, Daisuke

    2001-01-01

    The B cell adaptor containing src homology 2 domain (BASH; also termed BLNK or SLP-65), is crucial for B cell antigen receptor (BCR)-mediated activation, proliferation, and differentiation of B cells. BCR-mediated tyrosine-phosphorylation of BASH creates binding sites for signaling effectors such as phospholipase Cγ (PLCγ)2 and Vav, while the function of its COOH-terminal src homology 2 domain is unknown. We have now identified hematopoietic progenitor kinase (HPK)1, a STE20-related serine/threonine kinase, as a protein that inducibly interacts with the BASH SH2 domain. BCR ligation induced rapid tyrosine-phosphorylation of HPK1 mainly by Syk and Lyn, resulting in its association with BASH and catalytic activation. BCR-mediated activation of HPK1 was impaired in Syk- or BASH-deficient B cells. The functional SH2 domain of BASH and Tyr-379 within HPK1 which we identified as a Syk-phosphorylation site were both necessary for interaction of both proteins and efficient HPK1 activation after BCR stimulation. Furthermore, HPK1 augmented, whereas its kinase-dead mutant inhibited IκB kinase β (IKKβ) activation by BCR engagement. These results reveal a novel BCR signaling pathway leading to the activation of HPK1 and subsequently IKKβ, in which BASH recruits tyrosine-phosphorylated HPK1 into the BCR signaling complex. PMID:11514608

  3. Sumoylation Promotes the Stability of the DNA Sensor cGAS and the Adaptor STING to Regulate the Kinetics of Response to DNA Virus.

    PubMed

    Hu, Ming-Ming; Yang, Qing; Xie, Xue-Qin; Liao, Chen-Yang; Lin, Heng; Liu, Tian-Tian; Yin, Lei; Shu, Hong-Bing

    2016-09-20

    During viral infection, sensing of cytosolic DNA by the cyclic GMP-AMP synthase (cGAS) activates the adaptor protein STING and triggers an antiviral response. Little is known about the mechanisms that determine the kinetics of activation and deactivation of the cGAS-STING pathway, ensuring effective but controlled innate antiviral responses. Here we found that the ubiquitin ligase Trim38 targets cGas for sumoylation in uninfected cells and during the early phase of viral infection. Sumoylation of cGas prevented its polyubiquitination and degradation. Trim38 also sumoylated Sting during the early phase of viral infection, promoting both Sting activation and protein stability. In the late phase of infection, cGas and Sting were desumoylated by Senp2 and subsequently degraded via proteasomal and chaperone-mediated autophagy pathways, respectively. Our findings reveal an essential role for Trim38 in the innate immune response to DNA virus and provide insight into the mechanisms that ensure optimal activation and deactivation of the cGAS-STING pathway. PMID:27637147

  4. iRhom2 is essential for innate immunity to DNA viruses by mediating trafficking and stability of the adaptor STING.

    PubMed

    Luo, Wei-Wei; Li, Shu; Li, Chen; Lian, Huan; Yang, Qing; Zhong, Bo; Shu, Hong-Bing

    2016-09-01

    STING is a central adaptor in the innate immune response to DNA viruses. However, the manner in which STING activity is regulated remains unclear. We identified iRhom2 ('inactive rhomboid protein 2') as a positive regulator of DNA-virus-triggered induction of type I interferons. iRhom2 deficiency markedly impaired DNA-virus- and intracellular-DNA-induced signaling in cells, and iRhom2-deficient mice were more susceptible to lethal herpes simplex virus type 1 (HSV-1) infection. iRhom2 was constitutively associated with STING and acted in two distinct processes to regulate STING activity. iRhom2 recruited the translocon-associated protein TRAPβ to the STING complex to facilitate trafficking of STING from the endoplasmic reticulum to perinuclear microsomes. iRhom2 also recruited the deubiquitination enzyme EIF3S5 to maintain the stability of STING through removal of its K48-linked polyubiquitin chains. These results suggest that iRhom2 is essential for STING activity, as it regulates TRAPβ-mediated translocation and EIF3S5-mediated deubiquitination of STING. PMID:27428826

  5. A hematopoietic cell-driven mechanism involving SLAMF6 receptor, SAP adaptors and SHP-1 phosphatase regulates NK cell education.

    PubMed

    Wu, Ning; Zhong, Ming-Chao; Roncagalli, Romain; Pérez-Quintero, Luis-Alberto; Guo, Huaijian; Zhang, Zhanguang; Lenoir, Christelle; Dong, Zhongjun; Latour, Sylvain; Veillette, André

    2016-04-01

    Activation of natural killer (NK) cells by hematopoietic target cells is controlled by the SLAM family of receptors and by the associated SAP family of adaptors. Here we found that SLAM receptors also enhanced NK cell activation by nonhematopoietic target cells, which lack ligands for SLAM receptors. This function was mediated by SLAMF6, a homotypic SLAM receptor found on NK cells and other hematopoietic cells, and was regulated by SAP adaptors, which uncoupled SLAM receptors from phosphatase SHP-1 and diminished the effect of SLAMF6 on NK cell responsiveness toward nonhematopoietic cells. Thus, in addition to their role in NK cell activation by hematopoietic cells, the SLAM-SAP pathways influence responsiveness toward nonhematopoietic targets by a process akin to NK cell 'education'.

  6. HIV-1 Vpr Induces the Degradation of ZIP and sZIP, Adaptors of the NuRD Chromatin Remodeling Complex, by Hijacking DCAF1/VprBP

    PubMed Central

    Maudet, Claire; Sourisce, Adèle; Dragin, Loïc; Lahouassa, Hichem; Rain, Jean-Christophe; Bouaziz, Serge; Ramirez, Bertha Cécilia; Margottin-Goguet, Florence

    2013-01-01

    The Vpr protein from type 1 and type 2 Human Immunodeficiency Viruses (HIV-1 and HIV-2) is thought to inactivate several host proteins through the hijacking of the DCAF1 adaptor of the Cul4A ubiquitin ligase. Here, we identified two transcriptional regulators, ZIP and sZIP, as Vpr-binding proteins degraded in the presence of Vpr. ZIP and sZIP have been shown to act through the recruitment of the NuRD chromatin remodeling complex. Strikingly, chromatin is the only cellular fraction where Vpr is present together with Cul4A ubiquitin ligase subunits. Components of the NuRD complex and exogenous ZIP and sZIP were also associated with this fraction. Several lines of evidence indicate that Vpr induces ZIP and sZIP degradation by hijacking DCAF1: (i) Vpr induced a drastic decrease of exogenously expressed ZIP and sZIP in a dose-dependent manner, (ii) this decrease relied on the proteasome activity, (iii) ZIP or sZIP degradation was impaired in the presence of a DCAF1-binding deficient Vpr mutant or when DCAF1 expression was silenced. Vpr-mediated ZIP and sZIP degradation did not correlate with the growth-related Vpr activities, namely G2 arrest and G2 arrest-independent cytotoxicity. Nonetheless, infection with HIV-1 viruses expressing Vpr led to the degradation of the two proteins. Altogether our results highlight the existence of two host transcription factors inactivated by Vpr. The role of Vpr-mediated ZIP and sZIP degradation in the HIV-1 replication cycle remains to be deciphered. PMID:24116224

  7. The Molecular Basis for the Endocytosis of Small R-SNAREs by the Clathrin Adaptor CALM

    PubMed Central

    Miller, Sharon E.; Sahlender, Daniela A.; Graham, Stephen C.; Höning, Stefan; Robinson, Margaret S.; Peden, Andrew A.; Owen, David J.

    2011-01-01

    Summary SNAREs provide a large part of the specificity and energy needed for membrane fusion and, to do so, must be localized to their correct membranes. Here, we show that the R-SNAREs VAMP8, VAMP3, and VAMP2, which cycle between the plasma membrane and endosomes, bind directly to the ubiquitously expressed, PtdIns4,5P2-binding, endocytic clathrin adaptor CALM/PICALM. X-ray crystallography shows that the N-terminal halves of their SNARE motifs bind the CALMANTH domain as helices in a manner that mimics SNARE complex formation. Mutation of residues in the CALM:SNARE interface inhibits binding in vitro and prevents R-SNARE endocytosis in vivo. Thus, CALM:R-SNARE interactions ensure that R-SNAREs, required for the fusion of endocytic clathrin-coated vesicles with endosomes and also for subsequent postendosomal trafficking, are sorted into endocytic vesicles. CALM's role in directing the endocytosis of small R-SNAREs may provide insight into the association of CALM/PICALM mutations with growth retardation, cognitive defects, and Alzheimer's disease. PMID:22118466

  8. A conserved serine residue regulates the stability of Drosophila Salvador and human WW domain-containing adaptor 45 through proteasomal degradation

    SciTech Connect

    Wu, Di Wu, Shian

    2013-04-19

    Highlights: •Ser-17 is key for the stability of Drosophila Sav. •Ala mutation of Ser-17 promotes the proteasomal degradation of Sav. •Ser-17 residue is not the main target of Hpo-induced Sav stabilization. •Hpo-dependent and -independent mechanisms regulate Sav stability. •This mechanism is conserved in the homologue of Sav, human WW45. -- Abstract: The Hippo (Hpo) pathway is a conserved tumor suppressor pathway that controls organ size through the coordinated regulation of apoptosis and proliferation. Drosophila Salvador (Sav), which limits organ size, is a core component of the Hpo pathway. In this study, Ser-17 was shown to be important for the stability of Sav. Alanine mutation of Ser-17 promoted the proteasomal degradation of Sav. Destabilization and stabilization of the Sav protein mediated by alanine mutation of Ser-17 and by Hpo, respectively, were independent of each other. This implies that the stability of Sav is controlled by two mechanisms, one that is Ser-17-dependent and Hpo-independent, and another that is Ser-17-independent and Hpo-dependent. These dual mechanisms also regulated the human counterpart of Drosophila Sav, WW domain-containing adaptor 45 (WW45). The conservation of this regulation adds to its significance in normal physiology and tumorigenesis.

  9. Hydrological Modeling Reproducibility Through Data Management and Adaptors for Model Interoperability

    NASA Astrophysics Data System (ADS)

    Turner, M. A.

    2015-12-01

    Because of a lack of centralized planning and no widely-adopted standards among hydrological modeling research groups, research communities, and the data management teams meant to support research, there is chaos when it comes to data formats, spatio-temporal resolutions, ontologies, and data availability. All this makes true scientific reproducibility and collaborative integrated modeling impossible without some glue to piece it all together. Our Virtual Watershed Integrated Modeling System provides the tools and modeling framework hydrologists need to accelerate and fortify new scientific investigations by tracking provenance and providing adaptors for integrated, collaborative hydrologic modeling and data management. Under global warming trends where water resources are under increasing stress, reproducible hydrological modeling will be increasingly important to improve transparency and understanding of the scientific facts revealed through modeling. The Virtual Watershed Data Engine is capable of ingesting a wide variety of heterogeneous model inputs, outputs, model configurations, and metadata. We will demonstrate one example, starting from real-time raw weather station data packaged with station metadata. Our integrated modeling system will then create gridded input data via geostatistical methods along with error and uncertainty estimates. These gridded data are then used as input to hydrological models, all of which are available as web services wherever feasible. Models may be integrated in a data-centric way where the outputs too are tracked and used as inputs to "downstream" models. This work is part of an ongoing collaborative Tri-state (New Mexico, Nevada, Idaho) NSF EPSCoR Project, WC-WAVE, comprised of researchers from multiple universities in each of the three states. The tools produced and presented here have been developed collaboratively alongside watershed scientists to address specific modeling problems with an eye on the bigger picture of

  10. Role of β-arrestins and arrestin domain-containing proteins in G protein-coupled receptor trafficking.

    PubMed

    Kang, Dong Soo; Tian, Xufan; Benovic, Jeffrey L

    2014-04-01

    The arrestin clan can now be broadly divided into three structurally similar subgroups: the originally identified arrestins (visual and β-arrestins), the α-arrestins and a group of Vps26-related proteins. The visual and β-arrestins selectively bind to agonist-occupied phosphorylated G protein-coupled receptors (GPCRs) and inhibit GPCR coupling to heterotrimeric G proteins while the β-arrestins also function as adaptor proteins to regulate GPCR trafficking and G protein-independent signaling. The α-arrestins have also recently been implicated in regulating GPCR trafficking while Vps26 regulates retrograde trafficking. In this review, we provide an overview of the α-arrestins and β-arrestins with a focus on our current understanding of how these adaptor proteins regulate GPCR trafficking.

  11. Functional analyses of Src-like adaptor (SLA), a glucocorticoid-regulated gene in acute lymphoblastic leukemia.

    PubMed

    Mansha, Muhammad; Carlet, Michela; Ploner, Christian; Gruber, Georg; Wasim, Muhammad; Wiegers, Gerrit Jan; Rainer, Johannes; Geley, Stephan; Kofler, Reinhard

    2010-04-01

    Glucocorticoids (GCs) cause apoptosis and cell cycle arrest in lymphoid cells and are used in the therapy of lymphoid malignancies. SLA (Src-like-adaptor), an inhibitor of T- and B-cell receptor signaling, is a promising candidate derived from expression profiling analyses in children with acute lymphoblastic leukemia (ALL). Over-expression and knock-down experiments in ALL in vitro model revealed that transgenic SLA alone had no effect on survival or cell cycle progression, nor did it affect sensitivity to, or kinetics of, GC-induced apoptosis. Although SLA is a prominent GC response gene, it does not seem to contribute to the anti-leukemic effects of GC.

  12. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  13. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  14. Adaptor identity modulates adaptation effects in familiar face identification and their neural correlates.

    PubMed

    Walther, Christian; Schweinberger, Stefan R; Kovács, Gyula

    2013-01-01

    Adaptation-related aftereffects (AEs) show how face perception can be altered by recent perceptual experiences. Along with contrastive behavioural biases, modulations of the early event-related potentials (ERPs) were typically reported on categorical levels. Nevertheless, the role of the adaptor stimulus per se for face identity-specific AEs is not completely understood and was therefore investigated in the present study. Participants were adapted to faces (S1s) varying systematically on a morphing continuum between pairs of famous identities (identities A and B), or to Fourier phase-randomized faces, and had to match the subsequently presented ambiguous faces (S2s; 50/50% identity A/B) to one of the respective original faces. We found that S1s identical with or near to the original identities led to strong contrastive biases with more identity B responses following A adaptation and vice versa. In addition, the closer S1s were to the 50/50% S2 on the morphing continuum, the smaller the magnitude of the AE was. The relation between S1s and AE was, however, not linear. Additionally, stronger AEs were accompanied by faster reaction times. Analyses of the simultaneously recorded ERPs revealed categorical adaptation effects starting at 100 ms post-stimulus onset, that were most pronounced at around 125-240 ms for occipito-temporal sites over both hemispheres. S1-specific amplitude modulations were found at around 300-400 ms. Response-specific analyses of ERPs showed reduced voltages starting at around 125 ms when the S1 biased perception in a contrastive way as compared to when it did not. Our results suggest that face identity AEs do not only depend on physical differences between S1 and S2, but also on perceptual factors, such as the ambiguity of S1. Furthermore, short-term plasticity of face identity processing might work in parallel to object-category processing, and is reflected in the first 400 ms of the ERP.

  15. Adaptor Identity Modulates Adaptation Effects in Familiar Face Identification and Their Neural Correlates

    PubMed Central

    Walther, Christian; Schweinberger, Stefan R.; Kovács, Gyula

    2013-01-01

    Adaptation-related aftereffects (AEs) show how face perception can be altered by recent perceptual experiences. Along with contrastive behavioural biases, modulations of the early event-related potentials (ERPs) were typically reported on categorical levels. Nevertheless, the role of the adaptor stimulus per se for face identity-specific AEs is not completely understood and was therefore investigated in the present study. Participants were adapted to faces (S1s) varying systematically on a morphing continuum between pairs of famous identities (identities A and B), or to Fourier phase-randomized faces, and had to match the subsequently presented ambiguous faces (S2s; 50/50% identity A/B) to one of the respective original faces. We found that S1s identical with or near to the original identities led to strong contrastive biases with more identity B responses following A adaptation and vice versa. In addition, the closer S1s were to the 50/50% S2 on the morphing continuum, the smaller the magnitude of the AE was. The relation between S1s and AE was, however, not linear. Additionally, stronger AEs were accompanied by faster reaction times. Analyses of the simultaneously recorded ERPs revealed categorical adaptation effects starting at 100 ms post-stimulus onset, that were most pronounced at around 125–240 ms for occipito-temporal sites over both hemispheres. S1-specific amplitude modulations were found at around 300–400 ms. Response-specific analyses of ERPs showed reduced voltages starting at around 125 ms when the S1 biased perception in a contrastive way as compared to when it did not. Our results suggest that face identity AEs do not only depend on physical differences between S1 and S2, but also on perceptual factors, such as the ambiguity of S1. Furthermore, short-term plasticity of face identity processing might work in parallel to object-category processing, and is reflected in the first 400 ms of the ERP. PMID:23990908

  16. MyD88 Adaptor-Dependent Microbial Sensing by Regulatory T cells Promotes Mucosal Tolerance and Enforces Commensalism

    PubMed Central

    Wang, Sen; Charbonnier, Louis-Marie; Rivas, Magali Noval; Georgiev, Peter; Li, Ning; Gerber, Georg; Bry, Lynn; Chatila, Talal A

    2015-01-01

    SUMMARY Commensal microbiota promote mucosal tolerance in part by engaging regulatory T (Treg) cells via Toll like receptors (TLR). We report that Treg cell-specific deletion of the TLR adaptor MyD88 resulted in deficiency of intestinal Treg cells, a reciprocal increase in T helper-17 (Th17) cells and heightened interleukin-17 (IL-17)-dependent inflammation in experimental colitis. It also precipitated dysbiosis with overgrowth of segmented filamentous bacteria (SFB) and increased microbial loads in deep tissues. The Th17 cell dysregulation and bacterial dysbiosis were linked to impaired anti-microbial intestinal IgA responses, related to defective MyD88 adaptor- and Stat3 transcription factor-dependent T follicular regulatory and helper cell differentiation in the Peyer’s patches. These findings establish an essential role for MyD88-dependent microbial sensing by Treg cells in enforcing mucosal tolerance and maintaining commensalism by promoting intestinal Treg cell formation and anti-commensal IgA-responses. PMID:26231118

  17. MyD88 Adaptor-Dependent Microbial Sensing by Regulatory T Cells Promotes Mucosal Tolerance and Enforces Commensalism.

    PubMed

    Wang, Sen; Charbonnier, Louis-Marie; Noval Rivas, Magali; Georgiev, Peter; Li, Ning; Gerber, Georg; Bry, Lynn; Chatila, Talal A

    2015-08-18

    Commensal microbiota promote mucosal tolerance in part by engaging regulatory T (Treg) cells via Toll-like receptors (TLRs). We report that Treg-cell-specific deletion of the TLR adaptor MyD88 resulted in deficiency of intestinal Treg cells, a reciprocal increase in T helper 17 (Th17) cells and heightened interleukin-17 (IL-17)-dependent inflammation in experimental colitis. It also precipitated dysbiosis with overgrowth of segmented filamentous bacteria (SFB) and increased microbial loads in deep tissues. The Th17 cell dysregulation and bacterial dysbiosis were linked to impaired anti-microbial intestinal IgA responses, related to defective MyD88 adaptor- and Stat3 transcription factor-dependent T follicular regulatory and helper cell differentiation in the Peyer's patches. These findings establish an essential role for MyD88-dependent microbial sensing by Treg cells in enforcing mucosal tolerance and maintaining commensalism by promoting intestinal Treg cell formation and anti-commensal IgA responses.

  18. Interferon-γ and granulocyte/monocyte colony-stimulating factor production by natural killer cells involves different signaling pathways and the adaptor stimulator of interferon genes (STING).

    PubMed

    Souza-Fonseca-Guimaraes, Fernando; Parlato, Marianna; de Oliveira, Rosane B; Golenbock, Douglas; Fitzgerald, Katherine; Shalova, Irina N; Biswas, Subhra K; Cavaillon, Jean-Marc; Adib-Conquy, Minou

    2013-04-12

    Natural killer (NK) cells are important for innate immunity in particular through the production of IFN-γ and GM-CSF. Both cytokines are important in restoration of immune function of tolerized leukocytes under inflammatory events. The expression of TLRs in NK cells has been widely studied by analyzing the mRNA of these receptors, rarely seeking their protein expression. We previously showed that murine spleen NK cells express TLR9 intracellularly and respond to CpG oligodeoxynucleotide (CpG-ODN) by producing IFN-γ and GM-CSF. However, to get such production the presence of accessory cytokines (such as IL-15 and IL-18) was required, whereas CpG-ODN or accessory cytokines alone did not induce IFN-γ or GM-CSF. We show here that TLR9 overlaps with the Golgi apparatus in NK cells. Furthermore, CpG-ODN stimulation in the presence of accessory cytokines induces the phosphorylation of c-Jun, STAT3, and IκBα. IFN-γ and GM-CSF production requires NF-κB and STAT3 activation as well as Erk-dependent mechanisms for IFN-γ and p38 signaling for GM-CSF. Using knock-out-mice, we show that UNC93b1 and IL-12 (produced by NK cells themselves) are also necessary for IFN-γ and GM-CSF production. IFN-γ production was found to be MyD88- and TLR9-dependent, whereas GM-CSF was TLR9-independent but dependent on STING (stimulator of interferon genes), a cytosolic adaptor recently described for DNA sensing. Our study thereby allows us to gain insight into the mechanisms of synergy between accessory cytokines and CpG-ODN in NK cells. It also identifies a new and alternative signaling pathway for CpG-ODN in murine NK cells.

  19. Proteins

    NASA Astrophysics Data System (ADS)

    Regnier, Fred E.; Gooding, Karen M.

    Because of the complexity of cellular material and body fluids, it is seldom possible to analyze a natural product directly. Qualitative and quantitative analyses must often be preceded by some purification step that separates the molecular species being examined from interfering materials. In the case of proteins, column liquid chromatography has been used extensively for these fractionations. With the advent of gel permeation, cation exchange, anion exchange, hydrophobic, and affinity chromatography, it became possible to resolve proteins through their fundamental properties of size, charge, hydrophobicity, and biological affinity. The chromatographic separations used in the early isolation and characterization of many proteins later became analytical tools in their routine analysis. Unfortunately, these inherently simple and versatile column chromatographic techniques introduced in the 50s and 60s have a severe limitation in routine analysis-separation time. It is common to encounter 1-24 h separation times with the classical gel-type supports.

  20. p97 Disease Mutations Modulate Nucleotide-Induced Conformation to Alter Protein-Protein Interactions.

    PubMed

    Bulfer, Stacie L; Chou, Tsui-Fen; Arkin, Michelle R

    2016-08-19

    The AAA+ ATPase p97/VCP adopts at least three conformations that depend on the binding of ADP and ATP and alter the orientation of the N-terminal protein-protein interaction (PPI) domain into "up" and "down" conformations. Point mutations that cause multisystem proteinopathy 1 (MSP1) are found at the interface of the N domain and D1-ATPase domain and potentially alter the conformational preferences of p97. Additionally, binding of "adaptor" proteins to the N-domain regulates p97's catalytic activity. We propose that p97/adaptor PPIs are coupled to p97 conformational states. We evaluated the binding of nucleotides and the adaptor proteins p37 and p47 to wild-type p97 and MSP1 mutants. Notably, p47 and p37 bind 8-fold more weakly to the ADP-bound conformation of wild-type p97 compared to the ATP-bound conformation. However, MSP1 mutants lose this nucleotide-induced conformational coupling because they destabilize the ADP-bound, "down" conformation of the N-domain. Loss in conformation coupling to PPIs could contribute to the mechanism of MSP1. PMID:27267671

  1. Structural analysis of the STING adaptor protein reveals a hydrophobic dimer interface and mode of cyclic di-GMP binding.

    PubMed

    Ouyang, Songying; Song, Xianqiang; Wang, Yaya; Ru, Heng; Shaw, Neil; Jiang, Yan; Niu, Fengfeng; Zhu, Yanping; Qiu, Weicheng; Parvatiyar, Kislay; Li, Yang; Zhang, Rongguang; Cheng, Genhong; Liu, Zhi-Jie

    2012-06-29

    STING is an essential signaling molecule for DNA and cyclic di-GMP (c-di-GMP)-mediated type I interferon (IFN) production via TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) pathway. It contains an N-terminal transmembrane region and a cytosolic C-terminal domain (CTD). Here, we describe crystal structures of STING CTD alone and complexed with c-di-GMP in a unique binding mode. The strictly conserved aa 153-173 region was shown to be cytosolic and participated in dimerization via hydrophobic interactions. The STING CTD functions as a dimer and the dimerization was independent of posttranslational modifications. Binding of c-di-GMP enhanced interaction of a shorter construct of STING CTD (residues 139-344) with TBK1. This suggests an extra TBK1 binding site, other than serine 358. This study provides a glimpse into the unique architecture of STING and sheds light on the mechanism of c-di-GMP-mediated TBK1 signaling.

  2. Stability of HIB-Cul3 E3 ligase adaptor HIB Is Regulated by Self-degradation and Availability of Its Substrates

    PubMed Central

    Zhou, Zizhang; Xu, Congyu; Chen, Ping; Liu, Chen; Pang, Shu; Yao, Xia; Zhang, Qing

    2015-01-01

    The HIB-Cul3 complex E3 ligase regulates physiological homeostasis through regulating its substrate stability and its activity can be modulated by changing HIB abundance. However, regulation of HIB remains elusive. Here we provide evidence that HIB is degraded through the proteasome by Cul3-mediated polyubiquitination in K48 manner in Drosophila. Strikingly, HIB is targeted for degradation by itself. We further identify that three degrons (52LKSS56T, 76LDEE80S and 117MESQ121R) and K185 and K198 of HIB are essential for its auto-degradation. Finally, we demonstrate that HIB-Cul3 substrates, Ci and Puc, can effectively protect HIB from HIB-Cul3-mediated degradation. Taken together, our study indicates that there is an exquisite equilibrium between the adaptor and targets to achieve the tight control of the HIB, which is essential for maintaining suitable Hh and JNK signaling. And the mechanism of adaptor self-degradation and reciprocal control of the abundance between adaptor and its substrates is also applied to BTB-Cul3 E3 ligase adaptor dKeap1, dDiablo and dKLHL18. PMID:26263855

  3. Sorting of the Alzheimer's Disease Amyloid Precursor Protein Mediated by the AP-4 Complex

    SciTech Connect

    Burgos, Patricia V.; Mardones, Gonzalo A.; Rojas, Adriana L.; daSilva, Luis L.P.; Prabhu, Yogikala; Hurley, James H.; Bonifacino, Juan S.

    2010-08-12

    Adaptor protein 4 (AP-4) is the most recently discovered and least well-characterized member of the family of heterotetrameric adaptor protein (AP) complexes that mediate sorting of transmembrane cargo in post-Golgi compartments. Herein, we report the interaction of an YKFFE sequence from the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) with the {micro}4 subunit of AP-4. Biochemical and X-ray crystallographic analyses reveal that the properties of the APP sequence and the location of the binding site on 4 are distinct from those of other signal-adaptor interactions. Disruption of the APP-AP-4 interaction decreases localization of APP to endosomes and enhances {gamma}-secretase-catalyzed cleavage of APP to the pathogenic amyloid-{beta} peptide. These findings demonstrate that APP and AP-4 engage in a distinct type of signal-adaptor interaction that mediates transport of APP from the trans-Golgi network (TGN) to endosomes, thereby reducing amyloidogenic processing of the protein.

  4. A Patient-Controlled Analgesia Adaptor to Mitigate Postsurgical Pain for Combat Casualties With Multiple Limb Amputation: A Case Series.

    PubMed

    Pasquina, Paul F; Isaacson, Brad M; Johnson, Elizabeth; Rhoades, Daniel S; Lindholm, Mark P; Grindle, Garrett G; Cooper, Rory A

    2016-08-01

    The use of explosive armaments during Operation Iraqi Freedom, Operation Enduring Freedom, and Operation New Dawn has resulted in a significant number of injured U.S. service members. These weapons often generate substantial extremity trauma requiring multiple surgical procedures to preserve life, limb, and restore function. For those individuals who require multiple surgeries, the use of patient-controlled analgesia (PCA) devices can be an effective way to achieve adequate pain management and promote successful rehabilitation and recovery during inpatient treatment. A subpopulation of patients are unable to independently control a PCA device because of severe multiple limb dysfunction and/or loss. In response to the needs of these patients, our team designed and developed a custom adaptor to assist service members who would otherwise not be able to use a PCA. Patient feedback of the device indicated a positive response, improved independence, and overall satisfaction during inpatient hospitalization. PMID:27483540

  5. Ascent Heating Thermal Analysis on the Spacecraft Adaptor (SA) Fairings and the Interface with the Crew Launch Vehicle (CLV)

    NASA Technical Reports Server (NTRS)

    Wang, Xiao-Yen; Yuko, James; Motil, Brian

    2009-01-01

    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 aero heating case. Heating rates from Thermal Environment (TE) 3 aero heating analysis computed by engineers at Marshall Space Flight Center (MSFC) are used in the thermal analysis. Both MSC Patran 2007r1b/Pthermal and C&R Thermal Desktop 5.1/Sinda models are built to validate each other. The numerical results are also compared with those reported by Lockheed Martin (LM) and show a reasonably good agreement.

  6. Monoamine receptor agonists, acting preferentially at presynaptic autoreceptors and heteroreceptors, downregulate the cell fate adaptor FADD in rat brain cortex.

    PubMed

    García-Fuster, M Julia; García-Sevilla, Jesús A

    2015-02-01

    FADD is a crucial adaptor of death receptors that can engage apoptosis or survival actions (e.g. neuroplasticity) through its phosphorylated form (p-FADD). Although FADD was shown to participate in receptor mechanisms related to drugs of abuse, little is known on its role in the signaling of classic neurotransmitters (dopamine, noradrenaline, and serotonin) in brain. This study assessed the modulation of FADD (and p-FADD/FADD ratio, as an index of neuroplasticity) and FLIP-L (a neuroprotective FADD interacting partner), as well as the role of MEK-ERK signaling, after activation of monoamine auto/heteroreceptors by selective agonists in rat cortex. Acute depletion of monoamines with reserpine, but not with AMPT or PCPA, reduced FADD (28%) and increased p-FADD/FADD ratio (1.34-fold). Activation of presynaptic α2A-adrenoceptors (UK-14304 and clonidine), 5-HT1A receptors (8-OH-DPAT), and D2 dopamine receptor (bromocriptine) dose-dependently decreased FADD (up to 54%) and increased p-FADD (up to 29%) and p-FADD/FADD ratios (up to 2.93-fold), through specific receptor mechanisms. Activation of rat 5-HT1B autoreceptor in axon terminals by CP-94253 did not modulate FADD forms. Activation of postsynaptic D1 dopamine receptor by SKF-81297 also reduced FADD (25%) and increased p-FADD (32%). Disruption of MEK-ERK activation with SL327 did not modify clonidine (α2A-adrenoceptor)-induced FADD inhibition, indicating that agonist effect was not dependent on ERK signaling. The various monoamine receptor agonists and antagonists did not alter FLIP-L content, or the activation of executioner caspase-3 and PARP-1 cleavage, indicating that the agonists attenuated apoptotic signals and promoted neuroplasticity through FADD regulation. These novel results indicate that inhibition of pro-apoptotic FADD adaptor could function as a common signaling step in the initial activation of monoamine receptors in the brain.

  7. Monoamine receptor agonists, acting preferentially at presynaptic autoreceptors and heteroreceptors, downregulate the cell fate adaptor FADD in rat brain cortex.

    PubMed

    García-Fuster, M Julia; García-Sevilla, Jesús A

    2015-02-01

    FADD is a crucial adaptor of death receptors that can engage apoptosis or survival actions (e.g. neuroplasticity) through its phosphorylated form (p-FADD). Although FADD was shown to participate in receptor mechanisms related to drugs of abuse, little is known on its role in the signaling of classic neurotransmitters (dopamine, noradrenaline, and serotonin) in brain. This study assessed the modulation of FADD (and p-FADD/FADD ratio, as an index of neuroplasticity) and FLIP-L (a neuroprotective FADD interacting partner), as well as the role of MEK-ERK signaling, after activation of monoamine auto/heteroreceptors by selective agonists in rat cortex. Acute depletion of monoamines with reserpine, but not with AMPT or PCPA, reduced FADD (28%) and increased p-FADD/FADD ratio (1.34-fold). Activation of presynaptic α2A-adrenoceptors (UK-14304 and clonidine), 5-HT1A receptors (8-OH-DPAT), and D2 dopamine receptor (bromocriptine) dose-dependently decreased FADD (up to 54%) and increased p-FADD (up to 29%) and p-FADD/FADD ratios (up to 2.93-fold), through specific receptor mechanisms. Activation of rat 5-HT1B autoreceptor in axon terminals by CP-94253 did not modulate FADD forms. Activation of postsynaptic D1 dopamine receptor by SKF-81297 also reduced FADD (25%) and increased p-FADD (32%). Disruption of MEK-ERK activation with SL327 did not modify clonidine (α2A-adrenoceptor)-induced FADD inhibition, indicating that agonist effect was not dependent on ERK signaling. The various monoamine receptor agonists and antagonists did not alter FLIP-L content, or the activation of executioner caspase-3 and PARP-1 cleavage, indicating that the agonists attenuated apoptotic signals and promoted neuroplasticity through FADD regulation. These novel results indicate that inhibition of pro-apoptotic FADD adaptor could function as a common signaling step in the initial activation of monoamine receptors in the brain. PMID:25286119

  8. AP-1 and AP-3 mediate sorting of melanosomal and lysosomal membrane proteins into distinct post-Golgi trafficking pathways.

    PubMed

    Chapuy, Björn; Tikkanen, Ritva; Mühlhausen, Chris; Wenzel, Dirk; von Figura, Kurt; Höning, Stefan

    2008-07-01

    The adaptor complexes AP-1 and AP-3 are localized to endosomes and/or the trans Golgi network (TGN). Because of limitations in analysing intracellular adaptor function directly, their site of function is a matter of ongoing uncertainty. To overcome this problem and to analyse adaptor sorting at the TGN, we reconstituted vesicle formation from Golgi/TGN-enriched membranes in a novel in vitro budding assay. Melanocytes were metabolically labelled followed by a 19 degrees C temperature block to accumulate newly synthesized proteins in Golgi membranes, which were then enriched by subcellular fractionation and used as donor membranes for vesicle formation in vitro. The incorporation of the melanosomal proteins tyrosinase and tyrosinase-related protein 1 (TRP-1) as well as Lamp-1 and 46 kDa mannose-6-phosphate receptor (MPR46) into Golgi/TGN-derived vesicles was temperature, nucleotide, cytosol, ADP ribosylation factor 1 and adaptor dependent. We show that sorting of TRP-1 and MPR46 was AP-1 dependent, while budding of tyrosinase and Lamp-1 required AP-3. Depletion of clathrin inhibited sorting of all four cargo proteins, suggesting that AP-1 and AP-3 are involved in the formation of distinct types of clathrin-coated vesicles, each of which is characterized by the incorporation of specific cargo membrane proteins.

  9. Small-Molecule Targeting of E3 Ligase Adaptor SPOP in Kidney Cancer.

    PubMed

    Guo, Zhong-Qiang; Zheng, Tong; Chen, Baoen; Luo, Cheng; Ouyang, Sisheng; Gong, Shouzhe; Li, Jiafei; Mao, Liu-Liang; Lian, Fulin; Yang, Yong; Huang, Yue; Li, Li; Lu, Jing; Zhang, Bidong; Zhou, Luming; Ding, Hong; Gao, Zhiwei; Zhou, Liqun; Li, Guoqiang; Zhou, Ran; Chen, Ke; Liu, Jingqiu; Wen, Yi; Gong, Likun; Ke, Yuwen; Yang, Shang-Dong; Qiu, Xiao-Bo; Zhang, Naixia; Ren, Jin; Zhong, Dafang; Yang, Cai-Guang; Liu, Jiang; Jiang, Hualiang

    2016-09-12

    In the cytoplasm of virtually all clear-cell renal cell carcinoma (ccRCC), speckle-type POZ protein (SPOP) is overexpressed and misallocated, which may induce proliferation and promote kidney tumorigenesis. In normal cells, however, SPOP is located in the nucleus and induces apoptosis. Here we show that a structure-based design and subsequent hit optimization yield small molecules that can inhibit the SPOP-substrate protein interaction and can suppress oncogenic SPOP-signaling pathways. These inhibitors kill human ccRCC cells that are dependent on oncogenic cytoplasmic SPOP. Notably, these inhibitors minimally affect the viability of other cells in which SPOP is not accumulated in the cytoplasm. Our findings validate the SPOP-substrate protein interaction as an attractive target specific to ccRCC that may yield novel drug discovery efforts. PMID:27622336

  10. VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex.

    PubMed

    Bondage, Devanand D; Lin, Jer-Sheng; Ma, Lay-Sun; Kuo, Chih-Horng; Lai, Erh-Min

    2016-07-01

    Type VI secretion system (T6SS) is a macromolecular machine used by many Gram-negative bacteria to inject effectors/toxins into eukaryotic hosts or prokaryotic competitors for survival and fitness. To date, our knowledge of the molecular determinants and mechanisms underlying the transport of these effectors remains limited. Here, we report that two T6SS encoded valine-glycine repeat protein G (VgrG) paralogs in Agrobacterium tumefaciens C58 specifically control the secretion and interbacterial competition activity of the type VI DNase toxins Tde1 and Tde2. Deletion and domain-swapping analysis identified that the C-terminal extension of VgrG1 specifically confers Tde1 secretion and Tde1-dependent interbacterial competition activity in planta, and the C-terminal variable region of VgrG2 governs this specificity for Tde2. Functional studies of VgrG1 and VgrG2 variants with stepwise deletion of the C terminus revealed that the C-terminal 31 aa (C31) of VgrG1 and 8 aa (C8) of VgrG2 are the molecular determinants specifically required for delivery of each cognate Tde toxin. Further in-depth studies on Tde toxin delivery mechanisms revealed that VgrG1 interacts with the adaptor/chaperone-effector complex (Tap-1-Tde1) in the absence of proline-alanine-alanine-arginine (PAAR) and the VgrG1-PAAR complex forms independent of Tap-1 and Tde1. Importantly, we identified the regions involved in these interactions. Although the entire C31 segment is required for binding with the Tap-1-Tde1 complex, only the first 15 aa of this region are necessary for PAAR binding. These results suggest that the VgrG1 C terminus interacts sequentially or simultaneously with the Tap-1-Tde1 complex and PAAR to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity. PMID:27313214

  11. FADD adaptor and PEA-15/ERK1/2 partners in major depression and schizophrenia postmortem brains: basal contents and effects of psychotropic treatments.

    PubMed

    García-Fuster, M J; Díez-Alarcia, R; Ferrer-Alcón, M; La Harpe, R; Meana, J J; García-Sevilla, J A

    2014-09-26

    Enhanced brain apoptosis (neurons and glia) may be involved in major depression (MD) and schizophrenia (SZ), mainly through the activation of the intrinsic (mitochondrial) apoptotic pathway. In the extrinsic death pathway, pro-apoptotic Fas-associated death domain (FADD) adaptor and its non-apoptotic p-Ser194 FADD form have critical roles interacting with other death regulators such as phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) and extracellular signal-regulated kinase (ERK). The basal status of FADD (protein and messenger RNA (mRNA)) and the effects of psychotropic drugs (detected in blood/urine samples) were first assessed in postmortem prefrontal cortex of MD and SZ subjects (including a non-MD/SZ suicide group). In MD, p-FADD, but not total FADD (and mRNA), was increased (26%, n=24; all MD subjects) as well as p-FADD/FADD ratio (a pro-survival marker) in antidepressant-free MD subjects (50%, n=10). In contrast, cortical FADD (and mRNA), p-FADD, and p-FADD/FADD were not altered in SZ brains (n=21) regardless of antipsychotic medications (except enhanced mRNA in treated subjects). Similar negative results were quantified in the non-MD/SZ suicide group. In MD, the regulation of multifunctional PEA-15 (i.e., p-Ser116 PEA-15 blocks pro-apoptotic FADD and PEA-15 prevents pro-survival ERK action) and the modulation of p-ERK1/2 were also investigated. Cortical p-PEA-15 was not changed whereas PEA-15 was increased mainly in antidepressant-treated subjects (16-20%). Interestingly, cortical p-ERK1/2/ERK1/2 ratio was reduced (33%) in antidepressant-free when compared to antidepressant-treated MD subjects. The neurochemical adaptations of brain FADD (increased p-FADD and pro-survival p-FADD/FADD ratio), as well as its interaction with PEA-15, could play a major role to counteract the known activation of the mitochondrial apoptotic pathway in MD.

  12. FADD adaptor and PEA-15/ERK1/2 partners in major depression and schizophrenia postmortem brains: basal contents and effects of psychotropic treatments.

    PubMed

    García-Fuster, M J; Díez-Alarcia, R; Ferrer-Alcón, M; La Harpe, R; Meana, J J; García-Sevilla, J A

    2014-09-26

    Enhanced brain apoptosis (neurons and glia) may be involved in major depression (MD) and schizophrenia (SZ), mainly through the activation of the intrinsic (mitochondrial) apoptotic pathway. In the extrinsic death pathway, pro-apoptotic Fas-associated death domain (FADD) adaptor and its non-apoptotic p-Ser194 FADD form have critical roles interacting with other death regulators such as phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) and extracellular signal-regulated kinase (ERK). The basal status of FADD (protein and messenger RNA (mRNA)) and the effects of psychotropic drugs (detected in blood/urine samples) were first assessed in postmortem prefrontal cortex of MD and SZ subjects (including a non-MD/SZ suicide group). In MD, p-FADD, but not total FADD (and mRNA), was increased (26%, n=24; all MD subjects) as well as p-FADD/FADD ratio (a pro-survival marker) in antidepressant-free MD subjects (50%, n=10). In contrast, cortical FADD (and mRNA), p-FADD, and p-FADD/FADD were not altered in SZ brains (n=21) regardless of antipsychotic medications (except enhanced mRNA in treated subjects). Similar negative results were quantified in the non-MD/SZ suicide group. In MD, the regulation of multifunctional PEA-15 (i.e., p-Ser116 PEA-15 blocks pro-apoptotic FADD and PEA-15 prevents pro-survival ERK action) and the modulation of p-ERK1/2 were also investigated. Cortical p-PEA-15 was not changed whereas PEA-15 was increased mainly in antidepressant-treated subjects (16-20%). Interestingly, cortical p-ERK1/2/ERK1/2 ratio was reduced (33%) in antidepressant-free when compared to antidepressant-treated MD subjects. The neurochemical adaptations of brain FADD (increased p-FADD and pro-survival p-FADD/FADD ratio), as well as its interaction with PEA-15, could play a major role to counteract the known activation of the mitochondrial apoptotic pathway in MD. PMID:25075716

  13. Molecular cloning and functional analysis of the duck TIR domain-containing adaptor inducing IFN-β (TRIF) gene.

    PubMed

    Wei, Xiaoqin; Qian, Wei; Sizhu, Suolang; Shi, Lijuan; Jin, Meilin; Zhou, Hongbo

    2016-12-01

    Toll-like receptors (TLRs) trigger the innate immune response by responding to specific components of microorganisms. The TIR domain-containing adaptor inducing IFN-β (TRIF) plays an essential role in mammalian TLR-mediated signaling. The role of TRIF in ducks (duTRIF) remains poorly understood. In this study, we cloned and characterized the full-length coding sequence of duTRIF from duck embryo fibroblasts (DEFs). In healthy ducks, duTRIF transcripts were broadly expressed in different tissues, with higher expression levels in the spleen and liver. Using quantitative real-time PCR (qRT-PCR), we demonstrated the upregulation of duTRIF in DEFs infected with AIV or DTMUV, and DEFs treated with Poly I:C or LPS. Overexpression of duTRIF was able to induce the NF-κB and IFN-β expression. Furthermore, the IFN induction function of duTRIF was impaired when Ala517 was mutated to Pro or His. Taken together, these results suggested that duTRIF regulated duck innate immune responses. PMID:27539203

  14. Selection of the Most Potent Specific On/Off Adaptor-Hepatitis Delta Virus Ribozymes for Use in Gene Targeting

    PubMed Central

    Lévesque, Michel V.; Rouleau, Samuel G.; Perreault, Jean-Pierre

    2016-01-01

    The Hepatitis Delta Virus (HDV) ribozyme, which is well adapted to the environment of the human cell, is an excellent candidate for the future development of gene-inactivation systems. On top of this, a new generation of HDV ribozymes now exists that benefits from the addition of a specific on/off adaptor (specifically the SOFA-HDV ribozymes) which greatly increases both the ribozyme’s specificity and its cleavage activity. Unlike RNAi and hammerhead ribozymes, the designing of SOFA-HDV ribozymes to cleave, in trans, given RNA species has never been the object of a systematic optimization study, even with their recent use for the gene knockdown of various targets. This report aims at both improving and clarifying the design process of SOFA-HDV ribozymes. Both the ribozyme and the targeted RNA substrate were analyzed in order to provide new criteria that are useful in the selection of the most potent SOFA-HDV ribozymes. The crucial features present in both the ribozyme’s biosensor and blocker, as well as at the target site, were identified and characterized. Simple rules were derived and tested using hepatitis C virus NS5B RNA as a model target. Overall, this method should promote the use of the SOFA-HDV ribozymes in a plethora of applications in both functional genomics and gene therapy. PMID:21793786

  15. Usefulness of the paralens fluorescent microscope adaptor for the identification of mycobacteria in both field and laboratory settings.

    PubMed

    Kuhn, Walter; Armstrong, Derek; Atteberry, Suzanne; Dewbrey, Euline; Smith, Diane; Hooper, Nancy

    2010-04-30

    The presence of acid-fast bacilli (AFB) in laboratories has traditionally been demonstrated using the fluorochrome method, which requires a fluorescent microscope or the Ziehl-Neelsen (ZN) method employing light microscopy. Low sensitivity of the ZN method and high costs of fluoroscopy make the need for a more effective means of diagnosis a top priority, especially in developing countries where the burden of tuberculosis is high. The QBC ParaLens attachment (QBC Diagnostic Inc., Port Matilda, PA) is a substitute for conventional fluoroscopy in the identification of AFB. To evaluate the efficacy of the ParaLens LED (light-emitting diode) system, the authors performed a two-part study, looking at usefulness, functionality and durability in urban/rural health clinics around the world, as well as in a controlled state public health laboratory setting. In the field, the ParaLens was durable and functioned well with various power sources and lighting conditions. Results from the state laboratory indicated agreement between standard fluorescent microscopy and fluorescent microscopy using the ParaLens. This adaptor is a welcome addition to laboratories in resource-limited settings as a useful alternative to conventional fluoroscopy for detection of mycobacterial species.

  16. Basolateral sorting of chloride channel 2 is mediated by interactions between a dileucine motif and the clathrin adaptor AP-1

    PubMed Central

    de la Fuente-Ortega, Erwin; Gravotta, Diego; Bay, Andres Perez; Benedicto, Ignacio; Carvajal-Gonzalez, Jose Maria; Lehmann, Guillermo L.; Lagos, Carlos F.; Rodríguez-Boulan, Enrique

    2015-01-01

    In spite of the many key cellular functions of chloride channels, the mechanisms that mediate their subcellular localization are largely unknown. ClC-2 is a ubiquitous chloride channel usually localized to the basolateral domain of epithelia that regulates cell volume, ion transport, and acid–base balance; mice knocked out for ClC-2 are blind and sterile. Previous work suggested that CLC-2 is sorted basolaterally by TIFS812LL, a dileucine motif in CLC-2's C-terminal domain. However, our in silico modeling of ClC-2 suggested that this motif was buried within the channel's dimerization interface and identified two cytoplasmically exposed dileucine motifs, ESMI623LL and QVVA635LL, as candidate sorting signals. Alanine mutagenesis and trafficking assays support a scenario in which ESMI623LL acts as the authentic basolateral signal of ClC-2. Silencing experiments and yeast three-hybrid assays demonstrated that both ubiquitous (AP-1A) and epithelium-specific (AP-1B) forms of the tetrameric clathrin adaptor AP-1 are capable of carrying out basolateral sorting of ClC-2 through interactions of ESMI623LL with a highly conserved pocket in their γ1-σ1A hemicomplex. PMID:25739457

  17. 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

  18. Structure of a myosin•adaptor complex and pairing by cargo

    PubMed Central

    Shi, Hang; Singh, Nimisha; Esselborn, Filipp; Blobel, Günter

    2014-01-01

    Myosin 4 protein (Myo4p), one of five distinct myosins of yeast, is dedicated to cytoplasmic transport of two types of cargos, zipcoded messenger ribonucleoprotein particles (mRNPs) and tubular endoplasmic reticulum (tER). Neither cargo binds directly to Myo4p. Instead, swi5p-dependent HO expression 3 protein (She3p) serves as an “adaptor” that contains three binding modules, one for Myo4p and one each for zipcoded mRNP and tER. The assembly of a transport-competent motor complex is poorly understood. Here, we report that Myo4p•She3p forms a stable 1:2 heterotrimer in solution. In the Myo4p•She3p crystal structure, Myo4p’s C-terminal domain (CTD) assumes a lobster claw-shaped form, the minor prong of which adheres to a pseudocoiled-coil region of She3p. The extensive Myo4p•She3p interactome buries 3,812 Å2 surface area and is primarily hydrophobic. Because the Myo4p•She3p heterotrimer contains only one myosin molecule, it is not transport-competent. By stepwise reconstitution, we found a single molecule of synthetic oligonucleotide (representing the mRNA zipcode element) bound to a single tetramer of zipcode binding protein She2p to be sufficient for Myo4p•She3p dimerization. Therefore, cargo initiates cross-linking of two Myo4p•She3p heterotrimers to an ensemble that contains two myosin molecules obligatory for movement. An additional crystal structure comprising an overlapping upstream portion of She3p showed continuation of the pseudocoiled-coil structure and revealed another highly conserved surface region. We suggest this region as a candidate binding site for a yet unidentified tER ligand. We propose a model whereby zipcoded mRNP and/or tER ligands couple two Myo4p•She3p heterotrimers and thereby generate a transport-competent motor complex either for separate transport or cotransport of these two cargos. PMID:24522109

  19. A Molecular Toolkit to Visualize Native Protein Assemblies in the Context of Human Disease.

    PubMed

    Gilmore, Brian L; Winton, Carly E; Demmert, Andrew C; Tanner, Justin R; Bowman, Sam; Karageorge, Vasilea; Patel, Kaya; Sheng, Zhi; Kelly, Deborah F

    2015-01-01

    We present a new molecular toolkit to investigate protein assemblies natively formed in the context of human disease. The system employs tunable microchips that can be decorated with switchable adaptor molecules to select for target proteins of interest and analyze them using molecular microscopy. Implementing our new streamlined microchip approach, we could directly visualize BRCA1 gene regulatory complexes from patient-derived cancer cells for the first time. PMID:26395823

  20. Regulation of Cell Migration and β1 Integrin Trafficking by the Endosomal Adaptor GGA3.

    PubMed

    Ratcliffe, Colin D H; Sahgal, Pranshu; Parachoniak, Christine A; Ivaska, Johanna; Park, Morag

    2016-06-01

    The integrin family of cell adhesion receptors link extracellular matrices to intracellular signaling pathways and the actin cytoskeleton; and regulate cell migration, proliferation and survival in normal and diseased tissues. The subcellular location of integrin receptors is critical for their function and deregulated trafficking is implicated in various human diseases. Here we identify a role for Golgi-localized gamma-ear containing Arf-binding protein 3 (GGA3), in regulating trafficking of β1 integrin. GGA3 knockdown reduces cell surface and total levels of α2, α5 and β1 integrin subunits, inhibits cell spreading, reduces focal adhesion number, as well as cell migration. In the absence of GGA3, integrins are increasingly retained inside the cell, traffic toward the perinuclear lysosomal compartment and their degradation is enhanced. Integrin traffic and maintenance of integrin levels are dependent on the integrity of the Arf binding site of GGA3. Furthermore, sorting nexin 17 (SNX17), a critical regulator of integrin recycling, becomes mislocalized to enlarged late endosomes upon GGA3 depletion. These data support a model whereby GGA3, through its ability to regulate SNX17 endosomal localization and through interaction with Arf6 diverts integrins from the degradative pathway supporting cell migration. PMID:26935970

  1. pH responsive Janus-like supramolecular fusion proteins for functional protein delivery.

    PubMed

    Kuan, Seah Ling; Ng, David Y W; Wu, Yuzhou; Förtsch, Christina; Barth, Holger; Doroshenko, Mikheil; Koynov, Kaloian; Meier, Christoph; Weil, Tanja

    2013-11-20

    A facile, noncovalent solid-phase immobilization platform is described to assemble Janus-like supramolecular fusion proteins that are responsive to external stimuli. A chemically postmodified transporter protein, DHSA, is fused with (imino)biotinylated cargo proteins via an avidin adaptor with a high degree of spatial control. Notably, the derived heterofusion proteins are able to cross cellular membranes, dissociate at acidic pH due to the iminobiotin linker and preserve the enzymatic activity of the cargo proteins β-galactosidase and the enzymatic subunit of Clostridium botulinum C2 toxin. The mix-and-match strategy described herein opens unique opportunities to access macromolecular architectures of high structural definition and biological activity, thus complementing protein ligation and recombinant protein expression techniques. PMID:24156787

  2. The Adaptor CARD9 Is Required for Adaptive but Not Innate Immunity to Oral Mucosal Candida albicans Infections

    PubMed Central

    Bishu, Shrinivas; Hernández-Santos, Nydiaris; Simpson-Abelson, Michelle R.; Huppler, Anna R.; Conti, Heather R.; Ghilardi, Nico; Mamo, Anna J.

    2014-01-01

    Oropharyngeal candidiasis (OPC [thrush]) is an opportunistic infection caused by the commensal fungus Candida albicans. OPC is common in individuals with HIV/AIDS, infants, patients on chemotherapy, and individuals with congenital immune defects. Immunity to OPC is strongly dependent on the interleukin-23 (IL-23)/IL-17R axis, as mice and humans with defects in IL-17R signaling (IL17F, ACT1, IL-17RA) or in genes that direct Th17 differentiation (STAT3, STAT1, CARD9) are prone to mucocutaneous candidiasis. Conventional Th17 cells are induced in response to C. albicans infection via signals from C-type lectin receptors, which signal through the adaptor CARD9, leading to production of Th17-inducing cytokines such as IL-6, IL-1β, and IL-23. Recent data indicate that IL-17 can also be made by numerous innate cell subsets. These innate “type 17” cells resemble conventional Th17 cells, but they can be activated without need for prior antigen exposure. Because C. albicans is not a commensal organism in rodents and mice are thus naive to this fungus, we had the opportunity to assess the role of CARD9 in innate versus adaptive responses using an OPC infection model. As expected, CARD9−/− mice failed to mount an adaptive Th17 response following oral Candida infection. Surprisingly, however, CARD9−/− mice had preserved innate IL-17-dependent responses to Candida and were almost fully resistant to OPC. Thus, CARD9 is important primarily for adaptive immunity to C. albicans, whereas alternate recognition systems appear to be needed for effective innate responses. PMID:24379290

  3. AP-3 mediated trafficking of TLR2 ligands controls specificity of inflammatory responses but not adaptor complex assembly

    PubMed Central

    Petnicki-Ocwieja, Tanja; Kern, Aurelie; Killpack, Tess L.; Bunnell, Stephen C.; Hu, Linden T.

    2015-01-01

    Innate immune engagement results in the activation of host defenses that produce microbe-specific inflammatory responses. A long-standing interest in the field of innate immunity is to understand how varied host responses are generated through the signaling of just a limited number of receptors. Recently, intracellular trafficking and compartmental partitioning have been identified as mechanism that provide signaling specificity for receptors by regulating signaling platform assembly. We show that cytokine activation as a result of Toll-like receptor 2 (TLR2) stimulation occurs at different intracellular locations and is mediated by the phagosomal trafficking molecule AP-3. AP-3 is required for trafficking TLR2 purified ligands or the Lyme disease causing bacterium, Borrelia burgdorferi, to LAMP-1 lysosomal compartments. The presence of AP-3 is necessary for the activation of cytokines such as IL-6 but not TNF-α or type I interferons, suggesting induction of these cytokines occurs from a different compartment. Lack of AP-3 does not interfere with the recruitment of TLR signaling adaptors TRAM and MyD88 to the phagosome, indicating that the TLR-MyD88 signaling complex is assembled at a pre-lysosomal stage and that IL-6 activation depends on proper localization of signaling molecules downstream of MyD88. Finally, infection of AP-3 deficient mice with B. burgdorferi resulted in altered joint inflammation during murine Lyme arthritis. Our studies further elucidate the effects of phagosomal trafficking on tailoring immune responses in vitro and in vivo. PMID:26423153

  4. Reciprocal influence of connexins and apical junction proteins on their expressions and functions

    PubMed Central

    Derangeon, Mickaël; Spray, David C.; Bourmeyster, Nicolas; Sarrouilhe, Denis; Hervé, Jean-Claude

    2009-01-01

    Membranes of adjacent cells form intercellular junctional complexes to mechanically anchor neighbour cells (anchoring junctions), to seal the paracellular space and to prevent diffusion of integral proteins within the plasma membrane (tight junctions) and to allow cell-to-cell diffusion of small ions and molecules (gap junctions). These different types of specialised plasma membrane microdomains, sharing common adaptor molecules, particularly zonula occludens proteins, frequently present intermingled relationships where the different proteins co-assemble into macromolecular complexes and their expressions are co-ordinately regulated. Proteins forming gap junction channels (connexins, particularly) and proteins fulfilling cell attachment or forming tight junction strands mutually influence expression and functions of one another. PMID:19046940

  5. MdCOP1 Ubiquitin E3 Ligases Interact with MdMYB1 to Regulate Light-Induced Anthocyanin Biosynthesis and Red Fruit Coloration in Apple1[W][OA

    PubMed Central

    Li, Yuan-Yuan; Mao, Ke; Zhao, Cheng; Zhao, Xian-Yan; Zhang, Hua-Lei; Shu, Huai-Rui; Hao, Yu-Jin

    2012-01-01

    MdMYB1 is a crucial regulator of light-induced anthocyanin biosynthesis and fruit coloration in apple (Malus domestica). In this study, it was found that MdMYB1 protein accumulated in the light but degraded via a ubiquitin-dependent pathway in the dark. Subsequently, the MdCOP1-1 and MdCOP1-2 genes were isolated from apple fruit peel and were functionally characterized in the Arabidopsis (Arabidopsis thaliana) cop1-4 mutant. Yeast (Saccharomyces cerevisiae) two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays showed that MdMYB1 interacts with the MdCOP1 proteins. Furthermore, in vitro and in vivo experiments indicated that MdCOP1s are necessary for the ubiquitination and degradation of MdMYB1 protein in the dark and are therefore involved in the light-controlled stability of the MdMYB1 protein. Finally, a viral vector-based transformation approach demonstrated that MdCOP1s negatively regulate the peel coloration of apple fruits by modulating the degradation of the MdMYB1 protein. Our findings provide new insight into the mechanism by which light controls anthocyanin accumulation and red fruit coloration in apple and even other plant species. PMID:22855936

  6. Intrinsically disordered proteins drive membrane curvature

    NASA Astrophysics Data System (ADS)

    Busch, David J.; Houser, Justin R.; Hayden, Carl C.; Sherman, Michael B.; Lafer, Eileen M.; Stachowiak, Jeanne C.

    2015-07-01

    Assembly of highly curved membrane structures is essential to cellular physiology. The prevailing view has been that proteins with curvature-promoting structural motifs, such as wedge-like amphipathic helices and crescent-shaped BAR domains, are required for bending membranes. Here we report that intrinsically disordered domains of the endocytic adaptor proteins, Epsin1 and AP180 are highly potent drivers of membrane curvature. This result is unexpected since intrinsically disordered domains lack a well-defined three-dimensional structure. However, in vitro measurements of membrane curvature and protein diffusivity demonstrate that the large hydrodynamic radii of these domains generate steric pressure that drives membrane bending. When disordered adaptor domains are expressed as transmembrane cargo in mammalian cells, they are excluded from clathrin-coated pits. We propose that a balance of steric pressure on the two surfaces of the membrane drives this exclusion. These results provide quantitative evidence for the influence of steric pressure on the content and assembly of curved cellular membrane structures.

  7. Cloning and subcellular location of an Arabidopsis receptor-like protein that shares common features with protein-sorting receptors of eukaryotic cells.

    PubMed Central

    Ahmed, S U; Bar-Peled, M; Raikhel, N V

    1997-01-01

    Many receptors involved in clathrin-mediated protein transport through the endocytic and secretory pathways of yeast and animal cells share common features. They are all type I integral membrane proteins containing cysteine-rich lumenal domains and cytoplasmic tails with tyrosine-containing sorting signals. The cysteine-rich domains are thought to be involved in ligand binding, whereas the cytoplasmic tyrosine motifs interact with clathrin-associated adaptor proteins during protein sorting along these pathways. In addition, tyrosine-containing signals are required for the retention and recycling of some of these membrane proteins to the trans-Golgi network. Here we report the characterization of an approximately 80-kD epidermal growth factor receptor-like type I integral membrane protein containing all of these functional motifs from Arabidopsis thaliana (called AtELP for A. thaliana Epidermal growth factor receptor-Like Protein). Biochemical analysis indicates that AtELP is a membrane protein found at high levels in the roots of both monocots and dicots. Subcellular fractionation studies indicate that the AtELP protein is present in two membrane fractions corresponding to a novel, undefined compartment and a fraction enriched in vesicles containing clathrin and its associated adaptor proteins. AtELP may therefore serve as a marker for compartments involved in intracellular protein trafficking in the plant cell. PMID:9159954

  8. Rapid whole genome sequencing of Miyazaki-Bali/2007 Pteropine orthoreovirus by modified rolling circular amplification with adaptor ligation – next generation sequencing

    PubMed Central

    Singh, Harpal; Yoshikawa, Tomoki; Kobayashi, Takeshi; Fukushi, Shuetsu; Tani, Hideki; Taniguchi, Satoshi; Fukuma, Aiko; Yang, Ming; Sugamata, Masami; Shimojima, Masayuki; Saijo, Masayuki

    2015-01-01

    The emergence of orthoreoviruses as the causative agent of human respiratory illness over the past few years has led to a demand to determine their viral genome sequences. The whole genome sequencing of such RNA viruses using traditional methods, such as Sanger dideoxy sequencing following rapid amplification of cDNA ends presents a laborious challenge due to the numerous preparatory steps required before sequencing can commence. We developed a practical, time-efficient novel combination method capable of reducing the total time required from months to less than a week in the determination of whole genome sequence of Pteropine orthoreoviruses (PRV); through a combination of viral RNA purification and enrichment, adaptor ligation, reverse transcription, cDNA circularization and amplification, and next generation sequencing. We propose to call the method “modified rolling circular amplification with adaptor ligation – next generation sequencing (mRCA-NGS)”. Here, we describe the technological focus and advantage of mRCA-NGS and its expansive application, exemplified through the phylogenetic understanding of the Miyazaki-Bali/2007 PRV. PMID:26558341

  9. Rapid whole genome sequencing of Miyazaki-Bali/2007 Pteropine orthoreovirus by modified rolling circular amplification with adaptor ligation - next generation sequencing.

    PubMed

    Singh, Harpal; Yoshikawa, Tomoki; Kobayashi, Takeshi; Fukushi, Shuetsu; Tani, Hideki; Taniguchi, Satoshi; Fukuma, Aiko; Yang, Ming; Sugamata, Masami; Shimojima, Masayuki; Saijo, Masayuki

    2015-01-01

    The emergence of orthoreoviruses as the causative agent of human respiratory illness over the past few years has led to a demand to determine their viral genome sequences. The whole genome sequencing of such RNA viruses using traditional methods, such as Sanger dideoxy sequencing following rapid amplification of cDNA ends presents a laborious challenge due to the numerous preparatory steps required before sequencing can commence. We developed a practical, time-efficient novel combination method capable of reducing the total time required from months to less than a week in the determination of whole genome sequence of Pteropine orthoreoviruses (PRV); through a combination of viral RNA purification and enrichment, adaptor ligation, reverse transcription, cDNA circularization and amplification, and next generation sequencing. We propose to call the method "modified rolling circular amplification with adaptor ligation - next generation sequencing (mRCA-NGS)". Here, we describe the technological focus and advantage of mRCA-NGS and its expansive application, exemplified through the phylogenetic understanding of the Miyazaki-Bali/2007 PRV. PMID:26558341

  10. The origin of polynucleotide-directed protein synthesis

    NASA Technical Reports Server (NTRS)

    Orgel, Leslie E.

    1989-01-01

    If protein synthesis evolved in an RNA world it was probably preceded by simpler processes by means of which interaction with amino acids conferred selective advantage on replicating RNA molecules. It is suggested that at first the simple attachment of amino acids to the 2'(3') termini of RNA templates favored initiation of replication at the end of the template rather than at internal positions. The second stage in the evolution of protein synthesis would probably have been the association of pairs of charged RNA adaptors in such a way as to favor noncoded formation of peptides. Only after this process had become efficient could coded synthesis have begun.

  11. NMR structure and binding studies confirm that PA4608 from Pseudomonas aeruginosa is a PilZ domain and a c-di-GMP binding protein

    SciTech Connect

    Ramelot, Theresa A; Yee, Adelinda; Cort, John R; Semesi, Anthony; Arrowsmith, Cheryl H; Kennedy, Michael A

    2007-02-01

    PA4608 is a 125 residue protein with a proposed identification as a PilZ domain and c-di-GMP adaptor protein that plays a role in bacterial second-messenger regulated processes. The NMR structure of PA4608 has been determined and c-di-GMP binding has been confirmed by NMR titration studies. The monomeric structure of PA4608 contains a six-stranded anti-parallel β barrel flanked by three helices. Conserved surface residues among PA4608 homologs suggest the the c-di-GMP binding site is at one end of the barrel and includes residues in the helices as well as in the unstructured N-terminus. Chemical shift changes upon binding to c-di-GMP confirm that PA4608 binds to c-di-GMP. This evidence supports the hypothesis that proteins containing PilZ domains are the long-sought c-di-GMP adaptor proteins.

  12. How anchoring proteins shape pain.

    PubMed

    Fischer, Michael J M; McNaughton, Peter A

    2014-09-01

    Cellular responsiveness to external stimuli can be altered by extracellular mediators which activate membrane receptors, in turn signalling to the intracellular space via calcium, cyclic nucleotides, membrane lipids or enzyme activity. These signalling events trigger a cascade leading to an effector which can be a channel, an enzyme or a transcription factor. The effectiveness of these intracellular events is enhanced when they are maintained in close proximity by anchoring proteins, which assemble complexes of signalling molecules such as kinases together with their targets, and in this way enhance both the speed and the precision of intracellular signalling. The A kinase anchoring protein (AKAP) family are adaptor proteins originally named for their ability to associate Protein Kinase A and its targets, but several other enzymes bound by AKAPs have now been found and a wide variety of target structures has been described. This review provides an overview of anchoring proteins involved in pain signalling. The key anchoring proteins and their ion channel targets in primary sensory neurons responding to painful stimuli (nociceptors) are discussed.

  13. Protein maturation and proteolysis in plant plastids, mitochondria, and peroxisomes.

    PubMed

    van Wijk, Klaas J

    2015-01-01

    Plastids, mitochondria, and peroxisomes are key organelles with dynamic proteomes in photosynthetic eukaryotes. Their biogenesis and activity must be coordinated and require intraorganellar protein maturation, degradation, and recycling. The three organelles together are predicted to contain ∼200 presequence peptidases, proteases, aminopeptidases, and specific protease chaperones/adaptors, but the substrates and substrate selection mechanisms are poorly understood. Similarly, lifetime determinants of organellar proteins, such as N-end degrons and tagging systems, have not been identified, but the substrate recognition mechanisms likely share similarities between organelles. Novel degradomics tools for systematic analysis of protein lifetime and proteolysis could define such protease-substrate relationships, degrons, and protein lifetime. Intraorganellar proteolysis is complemented by autophagy of whole organelles or selected organellar content, as well as by cytosolic protein ubiquitination and degradation by the proteasome. This review summarizes (putative) plant organellar protease functions and substrate-protease relationships. Examples illustrate key proteolytic events.

  14. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway

    PubMed Central

    Shi, Tujin; Niepel, Mario; McDermott, Jason E.; Gao, Yuqian; Nicora, Carrie D.; Chrisler, William B.; Markillie, Lye M.; Petyuk, Vladislav A.; Smith, Richard D.; Rodland, Karin D.; Sorger, Peter K.; Qian, Wei-Jun; Wiley, H. Steven

    2016-01-01

    Various genetic mutations associated with cancer are known to alter cell signaling, but it is not clear whether they dysregulate signaling pathways by altering the abundance of pathway proteins. Using a combination of RNA sequencing and ultrasensitive targeted proteomics, we defined the primary components—16 core proteins and 10 feedback regulators—of the epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) pathway in normal human mammary epithelial cells and then quantified their absolute abundance across a panel of normal and breast cancer cell lines as well as fibroblasts. We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species. In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations. The absolute abundance of most core proteins was between 50,000 and 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower amounts (2000 to 5000 copies per cell). MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable amounts of feedback regulators and receptors. The low abundance of adaptors relative to EGFR could be responsible for previous observations that only a fraction of total cell surface EGFR is capable of rapid endocytosis, high-affinity binding, and mitogenic signaling. PMID:27405981

  15. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway.

    PubMed

    Shi, Tujin; Niepel, Mario; McDermott, Jason E; Gao, Yuqian; Nicora, Carrie D; Chrisler, William B; Markillie, Lye M; Petyuk, Vladislav A; Smith, Richard D; Rodland, Karin D; Sorger, Peter K; Qian, Wei-Jun; Wiley, H Steven

    2016-01-01

    Various genetic mutations associated with cancer are known to alter cell signaling, but it is not clear whether they dysregulate signaling pathways by altering the abundance of pathway proteins. Using a combination of RNA sequencing and ultrasensitive targeted proteomics, we defined the primary components-16 core proteins and 10 feedback regulators-of the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway in normal human mammary epithelial cells and then quantified their absolute abundance across a panel of normal and breast cancer cell lines as well as fibroblasts. We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species. In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations. The absolute abundance of most core proteins was between 50,000 and 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower amounts (2000 to 5000 copies per cell). MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable amounts of feedback regulators and receptors. The low abundance of adaptors relative to EGFR could be responsible for previous observations that only a fraction of total cell surface EGFR is capable of rapid endocytosis, high-affinity binding, and mitogenic signaling. PMID:27405981

  16. AP-2 Is the Crucial Clathrin Adaptor Protein for CD4 Downmodulation by HIV-1 Nef in Infected Primary CD4+ T Cells.

    PubMed

    Gondim, Marcos Vinicius; Wiltzer-Bach, Linda; Maurer, Brigitte; Banning, Carina; Arganaraz, Enrique; Schindler, Michael

    2015-12-01

    HIV-1 Nef-mediated CD4 downmodulation involves various host factors. We investigated the importance of AP-1, AP-2, AP-3, V1H-ATPase, β-COP, and ACOT8 for CD4 downmodulation in HIV-1-infected short hairpin RNA (shRNA)-expressing CD4(+) T cells and characterized direct interaction with Nef by Förster resonance energy transfer (FRET). Binding of lentiviral Nefs to CD4 and AP-2 was conserved, and only AP-2 knockdown impaired Nef-mediated CD4 downmodulation from primary T cells. Altogether, among the factors tested, AP-2 is the most important player for Nef-mediated CD4 downmodulation.

  17. TLR signaling adaptor protein MyD88 in primary sensory neurons contributes to persistent inflammatory and neuropathic pain and neuroinflammation

    PubMed Central

    Liu, Xing-Jun; Liu, Tong; Chen, Gang; Wang, Bing; Yu, Xiao-Lu; Yin, Cui; Ji, Ru-Rong

    2016-01-01

    Increasing evidence suggests that neuro-immune and neuro-glial interactions are critically involved in chronic pain sensitization. It is well studied how immune/glial mediators sensitize pain, but how sensory neurons control neuroinflammation remains unclear. We employed Myd88 conditional knockout (CKO) mice, in which Myd88 was deleted in sodium channel subunit Nav1.8-expressing primary sensory neurons, to examine the unique role of neuronal MyD88 in regulating acute and chronic pain, and possible underlying mechanisms. We found that baseline pain and the formalin induced acute inflammatory pain were intact in CKO mice. However, the late phase inflammatory pain following complete Freund’s adjuvant injection and the late phase neuropathic pain following chronic constriction injury (CCI), were reduced in CKO mice. CCI induced up-regulation of MyD88 and chemokine C-C motif ligand 2 expression in DRG neurons and macrophage infiltration into DRGs, and microglia activation in spinal dorsal horns in wild-type mice, but all these changes were compromised in CKO mice. Finally, the pain hypersensitivity induced by intraplantar IL-1β was reduced in CKO mice. Our findings suggest that MyD88 in primary sensory neurons plays an active role in regulating IL-1β signaling and neuroinflammation in the peripheral and the central nervous systems, and contributes to the maintenance of persistent pain. PMID:27312666

  18. Ubiquitination and degradation of CFTR by the E3 ubiquitin ligase MARCH2 through its association with adaptor proteins CAL and STX6.

    PubMed

    Cheng, Jie; Guggino, William

    2013-01-01

    Golgi-localized cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) and syntaxin 6 (STX6) regulate the abundance of mature, post-ER CFTR by forming a CAL/STX6/CFTR complex (CAL complex) that promotes CFTR degradation in lysosomes. However, the molecular mechanism underlying this degradation is unknown. Here we investigated the interaction of a Golgi-localized, membrane-associated RING-CH E3 ubiquitin ligase, MARCH2, with the CAL complex and the consequent binding, ubiquitination, and degradation of mature CFTR. We found that MARCH2 not only co-immunoprecipitated and co-localized with CAL and STX6, but its binding to CAL was also enhanced by STX6, suggesting a synergistic interaction. In vivo ubiquitination assays demonstrated the ubiquitination of CFTR by MARCH2, and overexpression of MARCH2, like that of CAL and STX6, led to a dose-dependent degradation of mature CFTR that was blocked by bafilomycin A1 treatment. A catalytically dead MARCH2 RING mutant was unable to promote CFTR degradation. In addition, MARCH2 had no effect on a CFTR mutant lacking the PDZ motif, suggesting that binding to the PDZ domain of CAL is required for MARCH2-mediated degradation of CFTR. Indeed, silencing of endogenous CAL ablated the effect of MARCH2 on CFTR. Consistent with its Golgi localization, MARCH2 had no effect on ER-localized ΔF508-CFTR. Finally, siRNA-mediated silencing of endogenous MARCH2 in the CF epithelial cell line CFBE-CFTR increased the abundance of mature CFTR. Taken together, these data suggest that the recruitment of the E3 ubiquitin ligase MARCH2 to the CAL complex and subsequent ubiquitination of CFTR are responsible for the CAL-mediated lysosomal degradation of mature CFTR.

  19. Absence of the Autophagy Adaptor SQSTM1/p62 Causes Childhood-Onset Neurodegeneration with Ataxia, Dystonia, and Gaze Palsy.

    PubMed

    Haack, Tobias B; Ignatius, Erika; Calvo-Garrido, Javier; Iuso, Arcangela; Isohanni, Pirjo; Maffezzini, Camilla; Lönnqvist, Tuula; Suomalainen, Anu; Gorza, Matteo; Kremer, Laura S; Graf, Elisabeth; Hartig, Monika; Berutti, Riccardo; Paucar, Martin; Svenningsson, Per; Stranneheim, Henrik; Brandberg, Göran; Wedell, Anna; Kurian, Manju A; Hayflick, Susan A; Venco, Paola; Tiranti, Valeria; Strom, Tim M; Dichgans, Martin; Horvath, Rita; Holinski-Feder, Elke; Freyer, Christoph; Meitinger, Thomas; Prokisch, Holger; Senderek, Jan; Wredenberg, Anna; Carroll, Christopher J; Klopstock, Thomas

    2016-09-01

    SQSTM1 (sequestosome 1; also known as p62) encodes a multidomain scaffolding protein involved in various key cellular processes, including the removal of damaged mitochondria by its function as a selective autophagy receptor. Heterozygous variants in SQSTM1 have been associated with Paget disease of the bone and might contribute to neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Using exome sequencing, we identified three different biallelic loss-of-function variants in SQSTM1 in nine affected individuals from four families with a childhood- or adolescence-onset neurodegenerative disorder characterized by gait abnormalities, ataxia, dysarthria, dystonia, vertical gaze palsy, and cognitive decline. We confirmed absence of the SQSTM1/p62 protein in affected individuals' fibroblasts and found evidence of a defect in the early response to mitochondrial depolarization and autophagosome formation. Our findings expand the SQSTM1-associated phenotypic spectrum and lend further support to the concept of disturbed selective autophagy pathways in neurodegenerative diseases. PMID:27545679

  20. Regulation of Latent Membrane Protein 1 Signaling through Interaction with Cytoskeletal Proteins

    PubMed Central

    Holthusen, Kirsten; Talaty, Pooja

    2015-01-01

    ABSTRACT Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) induces constitutive signaling in EBV-infected cells to ensure the survival of the latently infected cells. LMP1 is localized to lipid raft domains to induce signaling. In the present study, a genome-wide screen based on bimolecular fluorescence complementation (BiFC) was performed to identify LMP1-binding proteins. Several actin cytoskeleton-associated proteins were identified in the screen. Overexpression of these proteins affected LMP1-induced signaling. BiFC between the identified proteins and LMP1 was localized to lipid raft domains and was dependent on LMP1-induced signaling. Proximity biotinylation assays with LMP1 induced biotinylation of the actin-associated proteins, which were shifted in molecular mass. Together, the findings of this study suggest that the association of LMP1 with lipid rafts is mediated at least in part through interactions with the actin cytoskeleton. IMPORTANCE LMP1 signaling requires oligomerization, lipid raft partitioning, and binding to cellular adaptors. The current study utilized a genome-wide screen to identify several actin-associated proteins as candidate LMP1-binding proteins. The interaction between LMP1 and these proteins was localized to lipid rafts and dependent on LMP1 signaling. This suggests that the association of LMP1 with lipid rafts is mediated through interactions with actin-associated proteins. PMID:25948738

  1. Coexistence of Phases in a Protein Heterodimer

    PubMed Central

    Krokhotin, Andrey; Liwo, Adam; Niemi, Antti J.; Scheraga, Harold A.

    2012-01-01

    A heterodimer consisting of two or more different kinds of proteins can display an enormous number of distinct molecular architectures. The conformational entropy is an essential ingredient in the Helmholtz free energy and, consequently, these heterodimers can have a very complex phase structure. Here, it is proposed that there is a state of proteins, in which the different components of a heterodimer exist in different phases. For this purpose, the structures in the protein data bank (PDB) have been analyzed, with radius of gyration as the order parameter. Two major classes of heterodimers with their protein components coexisting in different phases have been identified. An example is the PDB structure 3DXC. This is a transcriptionally active dimer. One of the components is an isoform of the intra-cellular domain of the Alzheimer-disease related amyloid precursor protein (AICD), and the other is a nuclear multidomain adaptor protein in the Fe65 family. It is concluded from the radius of gyration that neither of the two components in this dimer is in its own collapsed phase, corresponding to a biologically active protein. The UNRES energy function has been utilized to confirm that, if the two components are separated from each other, each of them collapses. The results presented in this work show that heterodimers whose protein components coexist in different phases, can have intriguing physical properties with potentially important biological consequences. PMID:22830730

  2. The inositol 5-phosphatase SHIP-1 and adaptors Dok-1 and 2 play central roles in CD4-mediated inhibitory signaling

    PubMed Central

    Waterman, Paul M.; Marschner, Susanne; Brandl, Erin; Cambier, John C.

    2012-01-01

    CD4 functions to enhance the sensitivity of T cells to antigenic peptide/MHC class II. However, if aggregated in isolation, e.g. in the absence of T cell receptor (TCR), CD4 can transduce yet undefined signals that lead to T cell unresponsiveness to antigen and apoptosis. In Human Immunodeficiency Virus-1 (HIV-1) disease, CD4+ T cell loss can result from gp120-induced CD4 signaling in uninfected cells. We show here that CD4 aggregation leads to Lck-dependent phosphorylation of the RasGAP adaptors Downstream of kinase-1/2 (Dok-1/2) and the inositol 5-phosphatase-1 (SHIP-1) and association of the two molecules. Studies using SHIP-1 shRNA, knockout mice and decoy inhibitors further indicate that CD4-mediated inhibition of TCR-mediated T cell activation is SHIP-1 and Dok-1/2 dependent, and involves SHIP-1 hydrolysis of Phosphatidylinositol 3,4,5-trisphosophate (PI(3,4,5)P3) needed for TCR signaling. Our studies provide evidence for a novel mechanism by which ill-timed CD4-mediated signals activated by ligands such as HIV-1 gp120 lead to disarmament of the immune system. PMID:22370159

  3. Clnk, a Novel Slp-76–Related Adaptor Molecule Expressed in Cytokine-Stimulated Hemopoietic Cells

    PubMed Central

    Cao, Ming Yu; Davidson, Dominique; Yu, Jie; Latour, Sylvain; Veillette, André

    1999-01-01

    We have identified a novel Src homology 2 domain–containing leukocyte protein of 76 kD (SLP-76)–related molecule which we have termed Clnk (for cytokine-dependent hemopoietic cell linker). Unlike its relatives SLP-76 and B cell linker protein (Blnk), Clnk is not expressed uniformly within a given hemopoietic cell lineage. Even though it can be detected in several cell types, including T cells, natural killer cells, and mast cells, its expression seems to be strictly dependent on sustained exposure to cytokines such as interleukin (IL)-2 and IL-3. Strong support for the notion that Clnk is involved in immunoreceptor signaling was provided by the observation that it inducibly associated with at least one tyrosine-phosphorylated polypeptide (p92) in response to immunoreceptor stimulation. Moreover, transient expression of Clnk caused an increase in immunoreceptor-mediated signaling events in a T cell line. Taken together, these results show that Clnk is a novel member of the SLP-76 family selectively expressed in cytokine-stimulated hemopoietic cells. Furthermore, they suggest that Clnk may be involved in a cross-talk mechanism between cytokine receptor and immunoreceptor signaling. PMID:10562326

  4. Analytical reduction of combinatorial complexity arising from multiple protein modification sites

    PubMed Central

    Birtwistle, Marc R.

    2015-01-01

    Combinatorial complexity is a major obstacle to ordinary differential equation (ODE) modelling of biochemical networks. For example, a protein with 10 sites that can each be unphosphorylated, phosphorylated or bound to adaptor protein requires 310 ODEs. This problem is often dealt with by making ad hoc assumptions which have unclear validity and disallow modelling of site-specific dynamics. Such site-specific dynamics, however, are important in many biological systems. We show here that for a common biological situation where adaptors bind modified sites, binding is slow relative to modification/demodification, and binding to one modified site hinders binding to other sites, for a protein with n modification sites and m adaptor proteins the number of ODEs needed to simulate the site-specific dynamics of biologically relevant, lumped bound adaptor states is independent of the number of modification sites and equal to m + 1, giving a significant reduction in system size. These considerations can be relaxed considerably while retaining reasonably accurate descriptions of the true system dynamics. We apply the theory to model, using only 11 ODEs, the dynamics of ligand-induced phosphorylation of nine tyrosines on epidermal growth factor receptor (EGFR) and primary recruitment of six signalling proteins (Grb2, PI3K, PLCγ1, SHP2, RasA1 and Shc1). The model quantitatively accounts for experimentally determined site-specific phosphorylation and dephosphorylation rates, differential affinities of binding proteins for the phosphorylated sites and binding protein expression levels. Analysis suggests that local concentration of site-specific phosphatases such as SHP2 in membrane subdomains by a factor of approximately 107 is critical for effective site-specific regulation. We further show how our framework can be extended with minimal effort to consider binding cooperativity between Grb2 and c-Cbl, which is important for receptor trafficking. Our theory has potentially broad

  5. Yeast Irc6p is a novel type of conserved clathrin coat accessory factor related to small G proteins.

    PubMed

    Gorynia, Sabine; Lorenz, Todd C; Costaguta, Giancarlo; Daboussi, Lydia; Cascio, Duilio; Payne, Gregory S

    2012-11-01

    Clathrin coat accessory proteins play key roles in transport mediated by clathrin-coated vesicles. Yeast Irc6p and the related mammalian p34 are putative clathrin accessory proteins that interact with clathrin adaptor complexes. We present evidence that Irc6p functions in clathrin-mediated traffic between the trans-Golgi network and endosomes, linking clathrin adaptor complex AP-1 and the Rab GTPase Ypt31p. The crystal structure of the Irc6p N-terminal domain revealed a G-protein fold most related to small G proteins of the Rab and Arf families. However, Irc6p lacks G-protein signature motifs and high-affinity GTP binding. Also, mutant Irc6p lacking candidate GTP-binding residues retained function. Mammalian p34 rescued growth defects in irc6 cells, indicating functional conservation, and modeling predicted a similar N-terminal fold in p34. Irc6p and p34 also contain functionally conserved C-terminal regions. Irc6p/p34-related proteins with the same two-part architecture are encoded in genomes of species as diverse as plants and humans. Together these results define Irc6p/p34 as a novel type of conserved clathrin accessory protein and founding members of a new G protein-like family.

  6. ShcC proteins: brain aging and beyond.

    PubMed

    Sagi, Orli; Budovsky, Arie; Wolfson, Marina; Fraifeld, Vadim E

    2015-01-01

    To date, most studies of Shc family of signaling adaptor proteins have been focused on the near-ubiquitously expressed ShcA, indicating its relevance to age-related diseases and longevity. Although the role of the neuronal ShcC protein is much less investigated, accumulated evidence suggests its importance for neuroprotection against such aging-associated conditions as brain ischemia and oxidative stress. Here, we summarize more than decade of studies on the ShcC expression and function in normal brain, age-related brain pathologies and immune disorders with a focus on the interactions of ShcC with signaling proteins/pathways, and the possible implications of these interactions for changes associated with aging.

  7. Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2.

    PubMed

    Höning, Stefan; Ricotta, Doris; Krauss, Michael; Späte, Kira; Spolaore, Barbara; Motley, Alison; Robinson, Margaret; Robinson, Carol; Haucke, Volker; Owen, David J

    2005-05-27

    The alpha,beta2,mu2,sigma2 heterotetrameric AP2 complex is recruited exclusively to the phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2))-rich plasma membrane where, amongst other roles, it selects motif-containing cargo proteins for incorporation into clathrin-coated vesicles. Unphosphorylated and mu2Thr156-monophosphorylated AP2 mutated in their alphaPtdIns4,5P(2), mu2PtdIns4,5P(2), and mu2Yxxvarphi binding sites were produced, and their interactions with membranes of different phospholipid and cargo composition were measured by surface plasmon resonance. We demonstrate that recognition of Yxxvarphi and acidic dileucine motifs is dependent on corecognition with PtdIns4,5P(2), explaining the selective recruitment of AP2 to the plasma membrane. The interaction of AP2 with PtdIns4,5P(2)/Yxxvarphi-containing membranes is two step: initial recruitment via the alphaPtdIns4,5P(2) site and then stabilization through the binding of mu2Yxxvarphi and mu2PtdIns4,5P(2) sites to their ligands. The second step is facilitated by a conformational change favored by mu2Thr156 phosphorylation. The binding of AP2 to acidic-dileucine motifs occurs at a different site from Yxxvarphi binding and is not enhanced by mu2Thr156 phosphorylation.

  8. Kelch Domain of Gigaxonin Interacts with Intermediate Filament Proteins Affected in Giant Axonal Neuropathy

    PubMed Central

    Johnson-Kerner, Bethany L.; Garcia Diaz, Alejandro; Ekins, Sean; Wichterle, Hynek

    2015-01-01

    Patients with giant axonal neuropathy (GAN) show progressive loss of motor and sensory function starting in childhood and typically live for less than 30 years. GAN is caused by autosomal recessive mutations leading to low levels of gigaxonin (GIG), a ubiquitously-expressed BTB/Kelch cytoplasmic protein believed to be an E3 ligase substrate adaptor. GAN pathology is characterized by aggregates of intermediate filaments (IFs) in multiple tissues. To delineate the molecular pathway between GIG deficiency and IF pathology, we undertook a proteomic screen to identify the normal binding partners of GIG. Prominent among them were several classes of IFs, including the neurofilament subunits whose accumulation leads to the axonal swellings for which GAN is named. We showed these interactions were dependent on the Kelch domain of GIG. Furthermore, we identified the E3 ligase MYCBP2 and the heat shock proteins HSP90AA1/AB1 as interactors with the BTB domain that may result in the ubiquitination and subsequent degradation of intermediate filaments. Our open-ended proteomic screen provides support to GIG’s role as an adaptor protein, linking IF proteins through its Kelch domain to the ubiquitin pathway proteins via its BTB domain, and points to future approaches for reversing the phenotype in human patients. PMID:26460568

  9. Loss of TGF-β Adaptor β2SP Activates Notch Signaling and SOX9 Expression in Esophageal Adenocarcinoma

    PubMed Central

    Song, Shumei; Maru, Dipen M.; Ajani, Jaffer A.; Chan, Chia-Hsin; Honjo, Soichiro; Lin, Hui-Kuan; Correa, Arlene; Hofstetter, Wayne L.; Davila, Marta; Stroehlein, John; Mishra, Lopa

    2013-01-01

    TGF-β and Notch signaling pathways play important roles in regulating self-renewal of stem cells and gastrointestinal carcinogenesis. Loss of TGF-β signaling components activates Notch signaling in esophageal adenocarcinoma, but the basis for this effect has been unclear. Here we report that loss of TGF-β adapter β2SP (SPNB2) activates Notch signaling and its target SOX9 in primary fibroblasts or esophageal adenocarcinoma cells. Expression of the stem cell marker SOX9 was markedly higher in esophageal adenocarcinoma tumor tissues than normal tissues, and its higher nuclear staining in tumors correlated with poorer survival and lymph node invasion in esophageal adenocarcinoma patients. Downregulation of β2SP by lentivirus short hairpin RNA increased SOX9 transcription and expression, enhancing nuclear localization for both active Notch1 (intracellular Notch1, ICN1) and SOX9. In contrast, reintroduction into esophageal adenocarcinoma cells of β2SP and a dominant-negative mutant of the Notch coactivator mastermind-like (dnMAN) decreased SOX9 promoter activity. Tumor sphere formation and invasive capacity in vitro and tumor growth in vivo were increased in β2SP-silenced esophageal adenocarcinoma cells. Conversely, SOX9 silencing rescued the phenotype of esophageal adenocarcinoma cells with loss of β2SP. Interaction between Smad3 and ICN1 via Smad3 MH1 domain was also observed, with loss of β2SP increasing the binding between these proteins, inducing expression of Notch targets SOX9 and C-MYC, and decreasing expression of TGF-β targets p21(CDKN1A), p27 (CDKN1B), and E-cadherin. Taken together, our findings suggest that loss of β2SP switches TGF-β signaling from tumor suppression to tumor promotion by engaging Notch signaling and activating SOX9. PMID:23536563

  10. Fit-to-Flow (F2F) interconnects: universal reversible adhesive-free microfluidic adaptors for lab-on-a-chip systems.

    PubMed

    Chen, Arnold; Pan, Tingrui

    2011-02-21

    World-to-chip (macro-to-micro) interface continues to be one of the most complicated, ineffective, and unreliable components in the development of emerging lab-on-a-chip systems involving integrated microfluidic operations. A number of irreversible (e.g., adhesive gluing) and reversible techniques (e.g., press fitting) have attempted to provide dedicated fluidic passage from standard tubing to miniature on-chip devices, none of which completely addresses the above concerns. In this paper, we present standardized adhesive-free microfluidic adaptors, referred to as Fit-to-Flow (F2F) Interconnects, to achieve reliable hermetic seal, high-density tube packing, self-aligned plug-in, reworkable connectivity, straightforward scalability and expandability, and applicability to broad lab-on-a-chip platforms; analogous to the modular plug-and-play USB architecture employed in modern electronics. Specifically, two distinct physical packaging mechanisms are applied, with one utilizing induced tensile stress in elastomeric socket to establish reversible seal and the other using negative pressure to provide on demand vacuum shield, both of which can be adapted to a variety of experimental configurations. The non-leaking performance (up to 336 kPa) along with high tube-packing density (of 1 tube/mm(2)) and accurate self-guided alignment (of 10 μm) have been characterized. In addition, a 3D microfluidic mixer and a 6-level chemical gradient generator paired with the corresponding F2F Interconnects have been devised to illustrate the applicability of the universal fluidic connections to classic lab-on-a-chip operations.

  11. Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders.

    PubMed

    Zheng, S J; Henken, B; Sofiari, E; Jacobsen, E; Krens, F A; Kik, C

    2001-06-01

    Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA 105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66 bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration.

  12. Rab4b controls an early endosome sorting event by interacting with the γ-subunit of the clathrin adaptor complex 1.

    PubMed

    Perrin, Laura; Laura, Perrin; Lacas-Gervais, Sandra; Sandra, Lacas-Gervais; Gilleron, Jérôme; Jérôme, Gilleron; Ceppo, Franck; Franck, Ceppo; Prodon, François; François, Prodon; Benmerah, Alexandre; Alexandre, Benmerah; Tanti, Jean-François; Jean-François, Tanti; Cormont, Mireille; Mireille, Cormont

    2013-11-01

    The endocytic pathway is essential for cell homeostasis and numerous small Rab GTPases are involved in its control. The endocytic trafficking step controlled by Rab4b has not been elucidated, although recent data suggested it could be important for glucose homeostasis, synaptic homeostasis or adaptive immunity. Here, we show that Rab4b is required for early endosome sorting of transferrin receptors (TfRs) to the recycling endosomes, and we identified the AP1γ subunit of the clathrin adaptor AP-1 as a Rab4b effector and key component of the machinery of early endosome sorting. We show that internalised transferrin (Tf) does not reach Vamp3/Rab11 recycling endosomes in the absence of Rab4b, whereas it is rapidly recycled back to the plasma membrane. By contrast, overexpression of Rab4b leads to the accumulation of internalised Tf within AP-1- and clathrin-coated vesicles. These vesicles are poor in early and recycling endocytic markers except for TfR and require AP1γ for their formation. Furthermore, the targeted overexpression of the Rab4b-binding domain of AP1γ to early endosome upon its fusion with FYVE domains inhibited the interaction between Rab4b and endogenous AP1γ, and perturbed Tf traffic. We thus proposed that the interaction between early endocytic Rab4b and AP1γ could allow the budding of clathrin-coated vesicles for subsequent traffic to recycling endosomes. The data also uncover a novel type of endosomes, characterised by low abundance of either early or recycling endocytic markers, which could potentially be generated in cell types that naturally express high level of Rab4b.

  13. Improved cytotoxic T-lymphocyte immune responses to a tumor antigen by vaccines co-expressing the SLAM-associated adaptor EAT-2.

    PubMed

    Aldhamen, Y A; Seregin, S S; Kousa, Y A; Rastall, D P W; Appledorn, D M; Godbehere, S; Schutte, B C; Amalfitano, A

    2013-10-01

    The signaling lymphocytic activation molecule-associated adaptor Ewing's sarcoma's-activated transcript 2 (EAT-2) is primarily expressed in dendritic cells, macrophages and natural killer cells. Including EAT-2 in a vaccination regimen enhanced innate and adaptive immune responses toward pathogen-derived antigens, even in the face of pre-existing vaccine immunity. Herein, we investigate whether co-vaccinations with two recombinant Ad5 (rAd5) vectors, one expressing the carcinoembryonic antigen (CEA) and one expressing EAT-2, can induce more potent CEA-specific cytotoxic T lymphocyte (CTL) and antitumor activity in the therapeutic CEA-expressing MC-38 tumor model. Our results suggest that inclusion of EAT-2 significantly alters the kinetics of Th1-biasing proinflammatory cytokine and chemokine responses, and enhances anti-CEA-specific CTL responses. As a result, rAd5-EAT2-augmented rAd5-CEA vaccinations are more efficient in eliminating CEA-expressing target cells as measured by an in vivo CTL assay. Administration of rAd5-EAT2 vaccines also reduced the rate of growth of MC-38 tumor growth in vivo. Also, an increase in MC-38 tumor cell apoptosis (as measured by hematoxylin and eosin staining, active caspase-3 and granzyme B levels within the tumors) was observed. These data provide evidence that more efficient, CEA-specific effector T cells are generated by rAd5 vaccines expressing CEA, when augmented by rAd5 vaccines expressing EAT-2, and this regimen may be a promising approach for cancer immunotherapy in general.

  14. Ratcheting up protein translocation with anthrax toxin

    PubMed Central

    Feld, Geoffrey K; Brown, Michael J; Krantz, Bryan A

    2012-01-01

    Energy-consuming nanomachines catalyze the directed movement of biopolymers in the cell. They are found both dissolved in the aqueous cytosol as well as embedded in lipid bilayers. Inquiries into the molecular mechanism of nanomachine-catalyzed biopolymer transport have revealed that these machines are equipped with molecular parts, including adjustable clamps, levers, and adaptors, which interact favorably with substrate polypeptides. Biological nanomachines that catalyze protein transport, known as translocases, often require that their substrate proteins unfold before translocation. An unstructured protein chain is likely entropically challenging to bind, push, or pull in a directional manner, especially in a way that produces an unfolding force. A number of ingenious solutions to this problem are now evident in the anthrax toxin system, a model used to study protein translocation. Here we highlight molecular ratchets and current research on anthrax toxin translocation. A picture is emerging of proton-gradient-driven anthrax toxin translocation, and its associated ratchet mechanism likely applies broadly to other systems. We suggest a cyclical thermodynamic order-to-disorder mechanism (akin to a heat-engine cycle) is central to underlying protein translocation: peptide substrates nonspecifically bind to molecular clamps, which possess adjustable affinities; polypeptide substrates compress into helical structures; these clamps undergo proton-gated switching; and the substrate subsequently expands regaining its unfolded state conformational entropy upon translocation. PMID:22374876

  15. Evidence for the association of the human regulatory protein Ki-1/57 with the translational machinery.

    PubMed

    Gonçalves, Kaliandra de Almeida; Bressan, Gustavo Costa; Saito, Angela; Morello, Luis Gustavo; Zanchin, Nilson Ivo T; Kobarg, Jörg

    2011-08-19

    Ki-1/57 is a cytoplasmic and nuclear protein of 57 kDa first identified in malignant cells from Hodgkin's lymphoma. Based on yeast-two hybrid protein interaction we found out that Ki-1/57 interacts with adaptor protein RACK1 (receptor of activated kinase 1), CIRP (cold-inducible RNA-binding protein), RPL38 (ribosomal protein L38) and FXR1 (fragile X mental retardation-related protein 1). Since these proteins are involved in the regulation of translation we suspected that Ki-1/57 may have a role in it. We show by immunoprecipitation the association of Ki-1/57 with FMRP. Confocal microscopy revealed that Ki-1/57 colocalizes with FMRP/FXR1/2 to stress granules. Furthermore Ki-1/57 cosediments with free ribosomal particles and enhances translation, when tethered to a reporter mRNA, suggesting that Ki-1/57 may be involved in translational regulation.

  16. Mouse disabled (mDab1): a Src binding protein implicated in neuronal development.

    PubMed Central

    Howell, B W; Gertler, F B; Cooper, J A

    1997-01-01

    Here, we identify a mouse homolog of the Drosophila Disabled (Dab) protein, mDab1, and show it is an adaptor molecule functioning in neural development. We find that mDab1 is expressed in certain neuronal and hematopoietic cell lines, and is localized to the growing nerves of embryonic mice. During mouse embryogenesis, mDab1 is tyrosine phosphorylated when the nervous system is undergoing dramatic expansion. However, when nerve tracts are established, mDab1 lacks detectable phosphotyrosine. Tyrosine-phosphorylated mDab1 associates with the SH2 domains of Src, Fyn and Abl. An interaction between mDab1 and Src is observed when P19 embryonal carcinoma (EC) cells undergo differentiation into neuronal cell types. mDab1 can also form complexes with cellular phosphotyrosyl proteins through a domain that is related to the phosphotyrosine binding (PTB) domains of the Shc family of adaptor proteins. The mDab1 PTB domain binds to phosphotyrosine-containing proteins of 200, 120 and 40 kDa from extracts of embryonic mouse heads. The properties of mDab1 and genetic analysis of Dab in Drosophila suggest that these molecules function in key signal transduction pathways involved in the formation of neural networks. PMID:9009273

  17. 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.

  18. THE RNAissance Family: SR proteins as multifaceted regulators of gene expression

    PubMed Central

    Howard, Jonathan M.; Sanford, Jeremy R.

    2014-01-01

    Serine and Arginine-rich (SR) proteins play multiple roles in the eukaryotic gene expression pathway. Initially described as constitutive and alternative splicing factors, it is now clear that SR proteins are key determinants of exon identity and function as molecular adaptors, linking the pre-mRNA to the splicing machinery. In addition, SR proteins are now implicated in many aspects of mRNA and ncRNA processing well beyond splicing. These unexpected roles, including RNA transcription, export, translation and decay may prove to be the rule rather than the exception. To simply define this family of RNA binding proteins as splicing factors belies the broader roles of SR proteins in post-transcriptional gene expression. PMID:25155147

  19. Essential roles for the FE65 amyloid precursor protein-interacting proteins in brain development

    PubMed Central

    Guénette, Suzanne; Chang, Yang; Hiesberger, Thomas; Richardson, James A; Eckman, Christopher B; Eckman, Elizabeth A; Hammer, Robert E; Herz, Joachim

    2006-01-01

    Targeted deletion of two members of the FE65 family of adaptor proteins, FE65 and FE65L1, results in cortical dysplasia. Heterotopias resembling those found in cobblestone lissencephalies in which neuroepithelial cells migrate into superficial layers of the developing cortex, aberrant cortical projections and loss of infrapyramidal mossy fibers arise in FE65/FE65L1 compound null animals, but not in single gene knockouts. The disruption of pial basal membranes underlying the heterotopias and poor organization of fibrillar laminin by isolated meningeal fibroblasts from double knockouts suggests that FE65 proteins are involved in basement membrane assembly. A similar phenotype is observed in triple mutant mice lacking the APP family members APP, APLP1 and APLP2, all of which interact with FE65 proteins, suggesting that this phenotype may be caused by decreased transmission of an APP-dependent signal through the FE65 proteins. The defects observed in the double knockout may also involve the family of Ena/Vasp proteins, which participate in actin cytoskeleton remodeling and interact with the WW domains of FE65 proteins. PMID:16407979

  20. The B Cell Adaptor Molecule Bam32 Is Critically Important for Optimal Antibody Response and Resistance to Trypanosoma congolense Infection in Mice

    PubMed Central

    Onyilagha, Chukwunonso; Jia, Ping; Jayachandran, Nipun; Hou, Sen; Okwor, Ifeoma; Kuriakose, Shiby; Marshall, Aaron; Uzonna, Jude E.

    2015-01-01

    Background Bam32, a 32 kDa adaptor molecule, plays important role in B cell receptor signalling, T cell receptor signalling and antibody affinity maturation in germinal centres. Since antibodies against trypanosome variant surface glycoproteins (VSG) are critically important for control of parasitemia, we hypothesized that Bam32 deficient (Bam32-/-) mice would be susceptible to T. congolense infection. Methodology/Principal Findings We found that T. congolense-infected Bam32-/- mice successfully control the first wave of parasitemia but then fail to control subsequent waves and ultimately succumb to their infection unlike wild type (WT) C57BL6 mice which are relatively resistant. Although infected Bam32-/- mice had significantly higher hepatomegaly and splenomegaly, their serum AST and ALT levels were not different, suggesting that increased liver pathology may not be responsible for the increased susceptibility of Bam32-/- mice to T. congolense. Using direct ex vivo flow cytometry and ELISA, we show that CD4+ T cells from infected Bam32-/- mice produced significantly increased amounts of disease-exacerbating proinflammatory cytokines (including IFN-γ, TNF-α and IL-6). However, the percentages of regulatory T cells and IL-10-producing CD4+ cells were similar in infected WT and Bam32-/- mice. While serum levels of parasite-specific IgM antibodies were normal, the levels of parasite-specific IgG, (particularly IgG1 and IgG2a) were significantly lower in Bam32-/- mice throughout infection. This was associated with impaired germinal centre response in Bam32-/- mice despite increased numbers of T follicular helper (Tfh) cells. Adoptive transfer studies indicate that intrinsic B cell defect was responsible for the enhanced susceptibility of Bam32-/- mice to T. congolense infection. Conclusions/Significance Collectively, our data show that Bam32 is important for optimal anti-trypanosome IgG antibody response and suppression of disease-promoting proinflammatory cytokines

  1. A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones

    PubMed Central

    Siebert, Matthias; Böhme, Mathias A; Driller, Jan H; Babikir, Husam; Mampell, Malou M; Rey, Ulises; Ramesh, Niraja; Matkovic, Tanja; Holton, Nicole; Reddy-Alla, Suneel; Göttfert, Fabian; Kamin, Dirk; Quentin, Christine; Klinedinst, Susan; Andlauer, Till FM; Hell, Stefan W; Collins, Catherine A; Wahl, Markus C; Loll, Bernhard; Sigrist, Stephan J

    2015-01-01

    Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). We here demonstrate axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. We show in atomic detail that RBP C-terminal SH3 domains bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Pointmutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes. DOI: http://dx.doi.org/10.7554/eLife.06935.001 PMID:26274777

  2. A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones.

    PubMed

    Siebert, Matthias; Böhme, Mathias A; Driller, Jan H; Babikir, Husam; Mampell, Malou M; Rey, Ulises; Ramesh, Niraja; Matkovic, Tanja; Holton, Nicole; Reddy-Alla, Suneel; Göttfert, Fabian; Kamin, Dirk; Quentin, Christine; Klinedinst, Susan; Andlauer, Till Fm; Hell, Stefan W; Collins, Catherine A; Wahl, Markus C; Loll, Bernhard; Sigrist, Stephan J

    2015-08-14

    Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). We here demonstrate axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. We show in atomic detail that RBP C-terminal SH3 domains bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Pointmutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes.

  3. Programmable DNA scaffolds for spatially-ordered protein assembly

    NASA Astrophysics Data System (ADS)

    Chandrasekaran, Arun Richard

    2016-02-01

    Ever since the notion of using DNA as a material was realized, it has been employed in the construction of complex structures that facilitate the assembly of nanoparticles or macromolecules with nanometer-scale precision. Specifically, tiles fashioned from DNA strands and DNA origami sheets have been shown to be suitable as scaffolds for immobilizing proteins with excellent control over their spatial positioning. Supramolecular assembly of proteins into periodic arrays in one or more dimensions is one of the most challenging aspects in the design of scaffolds for biomolecular investigations and macromolecular crystallization. This review provides a brief overview of how various biomolecular interactions with high degree of specificity such as streptavidin-biotin, antigen-antibody, and aptamer-protein interactions have been used to fabricate linear and multidimensional assemblies of structurally intact and functional proteins. The use of DNA-binding proteins as adaptors, polyamide recognition on DNA scaffolds and oligonucleotide linkers for protein assembly are also discussed.Ever since the notion of using DNA as a material was realized, it has been employed in the construction of complex structures that facilitate the assembly of nanoparticles or macromolecules with nanometer-scale precision. Specifically, tiles fashioned from DNA strands and DNA origami sheets have been shown to be suitable as scaffolds for immobilizing proteins with excellent control over their spatial positioning. Supramolecular assembly of proteins into periodic arrays in one or more dimensions is one of the most challenging aspects in the design of scaffolds for biomolecular investigations and macromolecular crystallization. This review provides a brief overview of how various biomolecular interactions with high degree of specificity such as streptavidin-biotin, antigen-antibody, and aptamer-protein interactions have been used to fabricate linear and multidimensional assemblies of structurally

  4. ENTH and ANTH domain proteins participate in AP2-independent clathrin-mediated endocytosis.

    PubMed

    Manna, Paul T; Gadelha, Catarina; Puttick, Amy E; Field, Mark C

    2015-06-01

    Clathrin-mediated endocytosis (CME) is a major route of entry into eukaryotic cells. A core of evolutionarily ancient genes encodes many components of this system but much of our mechanistic understanding of CME is derived from a phylogenetically narrow sampling of a few model organisms. In the parasite Trypanosoma brucei, which is distantly related to the better characterised animals and fungi, exceptionally fast endocytic turnover aids its evasion of the host immune system. Although clathrin is absolutely essential for this process, the adaptor protein complex 2 (AP2) has been secondarily lost, suggesting mechanistic divergence. Here, we characterise two phosphoinositide-binding monomeric clathrin adaptors, T. brucei (Tb)EpsinR and TbCALM, which in trypanosomes are represented by single genes, unlike the expansions present in animals and fungi. Depletion of these gene products reveals essential, but partially redundant, activities in CME. Ultrastructural analysis of TbCALM and TbEpsinR double-knockdown cells demonstrated severe defects to clathrin-coated pit formation and morphology associated with a dramatic inhibition of endocytosis. Depletion of TbCALM alone, however, produced a distinct lysosomal segregation phenotype, indicating an additional non-redundant role for this protein. Therefore, TbEpsinR and TbCALM represent ancient phosphoinositide-binding proteins with distinct and vital roles in AP2-independent endocytosis.

  5. Mitochondrial trafficking in neurons and the role of the Miro family of GTPase proteins.

    PubMed

    Birsa, Nicol; Norkett, Rosalind; Higgs, Nathalie; Lopez-Domenech, Guillermo; Kittler, Josef T

    2013-12-01

    Correct mitochondrial dynamics are essential to neuronal function. These dynamics include mitochondrial trafficking and quality-control systems that maintain a precisely distributed and healthy mitochondrial network, so that local energy demands or Ca2+-buffering requirements within the intricate architecture of the neuron can be met. Mitochondria make use of molecular machinery that couples these organelles to microtubule-based transport via kinesin and dynein motors, facilitating the required long-range movements. These motors in turn are associated with a variety of adaptor proteins allowing additional regulation of the complex dynamics demonstrated by these organelles. Over recent years, a number of new motor and adaptor proteins have been added to a growing list of components implicated in mitochondrial trafficking and distribution. Yet, there are major questions that remain to be addressed about the regulation of mitochondrial transport complexes. One of the core components of this machinery, the mitochondrial Rho GTPases Miro1 (mitochondrial Rho 1) and Miro2 have received special attention due to their Ca2+-sensing and GTPase abilities, marking Miro an exceptional candidate for co-ordinating mitochondrial dynamics and intracellular signalling pathways. In the present paper, we discuss the wealth of literature regarding Miro-mediated mitochondrial transport in neurons and recently highlighted involvement of Miro proteins in mitochondrial turnover, emerging as a key process affected in neurodegeneration. PMID:24256248

  6. ENTH and ANTH domain proteins participate in AP2-independent clathrin-mediated endocytosis

    PubMed Central

    Manna, Paul T.; Gadelha, Catarina; Puttick, Amy E.; Field, Mark C.

    2015-01-01

    ABSTRACT Clathrin-mediated endocytosis (CME) is a major route of entry into eukaryotic cells. A core of evolutionarily ancient genes encodes many components of this system but much of our mechanistic understanding of CME is derived from a phylogenetically narrow sampling of a few model organisms. In the parasite Trypanosoma brucei, which is distantly related to the better characterised animals and fungi, exceptionally fast endocytic turnover aids its evasion of the host immune system. Although clathrin is absolutely essential for this process, the adaptor protein complex 2 (AP2) has been secondarily lost, suggesting mechanistic divergence. Here, we characterise two phosphoinositide-binding monomeric clathrin adaptors, T. brucei (Tb)EpsinR and TbCALM, which in trypanosomes are represented by single genes, unlike the expansions present in animals and fungi. Depletion of these gene products reveals essential, but partially redundant, activities in CME. Ultrastructural analysis of TbCALM and TbEpsinR double-knockdown cells demonstrated severe defects to clathrin-coated pit formation and morphology associated with a dramatic inhibition of endocytosis. Depletion of TbCALM alone, however, produced a distinct lysosomal segregation phenotype, indicating an additional non-redundant role for this protein. Therefore, TbEpsinR and TbCALM represent ancient phosphoinositide-binding proteins with distinct and vital roles in AP2-independent endocytosis. PMID:25908855

  7. Identification of AOSC-binding proteins in neurons

    NASA Astrophysics Data System (ADS)

    Liu, Ming; Nie, Qin; Xin, Xianliang; Geng, Meiyu

    2008-11-01

    Acidic oligosaccharide sugar chain (AOSC), a D-mannuronic acid oligosaccharide, derived from brown algae polysaccharide, has been completed Phase I clinical trial in China as an anti-Alzheimer’s Disease (AD) drug candidate. The identification of AOSC-binding protein(s) in neurons is very important for understanding its action mechanism. To determine the binding protein(s) of AOSC in neurons mediating its anti-AD activities, confocal microscopy, affinity chromatography, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were used. Confocal microscopy analysis shows that AOSC binds to SH-SY5Y cells in concentration-, time-, and temperature-dependent fashions. The AOSC binding proteins were purified by affinity chromatography and identified by LC-MS/MS analysis. The results showed that there are 349 proteins binding AOSC, including clathrin, adaptor protein-2 (AP-2) and amyloid precursor protein (APP). These results suggest that the binding/entrance of AOSC to neurons is probably responsible for anti-AD activities.

  8. Identification of novel CBP interacting proteins in embryonic orofacial tissue

    SciTech Connect

    Yin Xiaolong; Warner, Dennis R.; Roberts, Emily A.; Pisano, M. Michele; Greene, Robert M. . E-mail: greene@louisville.edu

    2005-04-15

    cAMP response element-binding protein (CREB)-binding protein (CBP) plays an important role as a general co-integrator of multiple signaling pathways and interacts with a large number of transcription factors and co-factors, through its numerous protein-binding domains. To identify nuclear factors associated with CBP in developing orofacial tissue, a yeast two-hybrid screen of a cDNA library derived from orofacial tissue from gestational day 11 to 13 mouse embryos was conducted. Using the carboxy terminus (amino acid residues 1676-2441) of CBP as bait, several novel proteins that bind CBP were identified, including an Msx-interacting-zinc finger protein, CDC42 interaction protein 4/thyroid hormone receptor interactor 10, SH3-domain GRB2-like 1, CCR4-NOT transcription complex subunit 3, adaptor protein complex AP-1 {beta}1 subunit, eukaryotic translation initiation factor 2B subunit 1 ({alpha}), and cyclin G-associated kinase. Results of the yeast two-hybrid screen were confirmed by glutathione S-transferase pull-down assays. The identification of these proteins as novel CBP-binding partners allows exploration of new mechanisms by which CBP regulates and integrates diverse cell signaling pathways.

  9. The tight junction protein ZO-2 blocks cell cycle progression and inhibits cyclin D1 expression.

    PubMed

    Gonzalez-Mariscal, Lorenza; Tapia, Rocio; Huerta, Miriam; Lopez-Bayghen, Esther

    2009-05-01

    ZO-2 is an adaptor protein of the tight junction that belongs to the MAGUK protein family. ZO-2 is a dual localization protein that in sparse cultures is present at the cell borders and the nuclei, whereas in confluent cultures it is concentrated at the cell boundaries. Here we have studied whether ZO-2 is able to regulate the expression of cyclin D1 (CD1) and cell proliferation. We have demonstrated that ZO-2 negatively regulates CD1 transcription by interacting with c-Myc at an E box present in CD1 promoter. We have further found that ZO-2 transfection into epithelial MDCK cells triggers a diminished expression of CD1 protein and decreases the rate of cell proliferation in a wound-healing assay.

  10. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments.

    PubMed

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.

  11. The Functions of the HIV1 protein Vpr and its action through the DCAF1•DDB1•Cullin4 ubiquitin ligase

    PubMed Central

    Casey, Laurieann; Wen, Xiaoyun; de Noronha, Carlos M.C.

    2010-01-01

    Among the proteins encoded by human and simian immunodeficiency viruses (HIV and SIV) at least three, Vif, Vpu and Vpr, subvert cellular ubiquitin ligases to block the action of anti-viral defenses. This review focuses on Vpr and its HIV2/SIV counterparts, Vpx and Vpr, which all engage the DDB1•Cullin4 ubiquitin ligase complex through the DCAF1 adaptor protein. Here, we discuss the multiple functions that have been linked to Vpr expression and summarize the current knowledge on the role of the ubiquitin ligase complex in carrying out a subset of these activities. PMID:20347598

  12. Structural Basis for Cul3 Protein Assembly with the BTB-Kelch Family of E3 Ubiquitin Ligases*

    PubMed Central

    Canning, Peter; Cooper, Christopher D. O.; Krojer, Tobias; Murray, James W.; Pike, Ashley C. W.; Chaikuad, Apirat; Keates, Tracy; Thangaratnarajah, Chancievan; Hojzan, Viktorija; Marsden, Brian D.; Gileadi, Opher; Knapp, Stefan; von Delft, Frank; Bullock, Alex N.

    2013-01-01

    Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein “3-box” motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design. PMID:23349464

  13. Bimolecular complementation affinity purification (BiCAP) reveals dimer-specific protein interactions for ERBB2 dimers.

    PubMed

    Croucher, David R; Iconomou, Mary; Hastings, Jordan F; Kennedy, Sean P; Han, Jeremy Z R; Shearer, Robert F; McKenna, Jessie; Wan, Adrian; Lau, Joseph; Aparicio, Samuel; Saunders, Darren N

    2016-01-01

    The dynamic assembly of multiprotein complexes is a central mechanism of many cell signaling pathways. This process is key to maintaining the spatiotemporal specificity required for an accurate, yet adaptive, response to rapidly changing cellular conditions. We describe a technique for the specific isolation and downstream proteomic characterization of any two interacting proteins, to the exclusion of their individual moieties and competing binding partners. We termed the approach bimolecular complementation affinity purification (BiCAP) because it combines the use of conformation-specific nanobodies with a protein-fragment complementation assay with affinity purification. Using BiCAP, we characterized the specific interactome of the epidermal growth factor receptor (EGFR) family member ERBB2 when in the form of a homodimer or when in the form of a heterodimer with either EGFR or ERBB3. We identified dimer-specific interaction patterns for key adaptor proteins and identified a number of previously unknown interacting partners. Functional analysis for one of these newly identified partners revealed a noncanonical mechanism of extracellular signal-regulated kinase (ERK) activation that is specific to the ERBB2:ERBB3 heterodimer and acts through the adaptor protein FAM59A in breast cancer cells. PMID:27405979

  14. Hrr25 triggers selective autophagy–related pathways by phosphorylating receptor proteins

    PubMed Central

    Tanaka, Chikara; Tan, Li-Jing; Mochida, Keisuke; Kirisako, Hiromi; Koizumi, Michiko; Asai, Eri; Sakoh-Nakatogawa, Machiko; Ohsumi, Yoshinori

    2014-01-01

    In selective autophagy, degradation targets are specifically recognized, sequestered by the autophagosome, and transported into the lysosome or vacuole. Previous studies delineated the molecular basis by which the autophagy machinery recognizes those targets, but the regulation of this process is still poorly understood. In this paper, we find that the highly conserved multifunctional kinase Hrr25 regulates two distinct selective autophagy–related pathways in Saccharomyces cerevisiae. Hrr25 is responsible for the phosphorylation of two receptor proteins: Atg19, which recognizes the assembly of vacuolar enzymes in the cytoplasm-to-vacuole targeting pathway, and Atg36, which recognizes superfluous peroxisomes in pexophagy. Hrr25-mediated phosphorylation enhances the interactions of these receptors with the common adaptor Atg11, which recruits the core autophagy-related proteins that mediate the formation of the autophagosomal membrane. Thus, this study introduces regulation of selective autophagy as a new role of Hrr25 and, together with other recent studies, reveals that different selective autophagy–related pathways are regulated by a uniform mechanism: phosphoregulation of the receptor–adaptor interaction. PMID:25287303

  15. Cherry Valley Ducks Mitochondrial Antiviral-Signaling Protein-Mediated Signaling Pathway and Antiviral Activity Research

    PubMed Central

    Li, Ning; Hong, Tianqi; Li, Rong; Wang, Yao; Guo, Mengjiao; Cao, Zongxi; Cai, Yumei; Liu, Sidang; Chai, Tongjie; Wei, Liangmeng

    2016-01-01

    Mitochondrial antiviral-signaling protein (MAVS), an adaptor protein of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs)-mediated signal pathway, is involved in innate immunity. In this study, Cherry Valley duck MAVS (duMAVS) was cloned from the spleen and analyzed. duMAVS was determined to have a caspase activation and recruitment domain at N-terminal, followed by a proline-rich domain and a transmembrane domain at C-terminal. Quantitative real-time PCR indicated that duMAVS was expressed in all tissues tested across a broad expression spectrum. The expression of duMAVS was significantly upregulated after infection with duck Tembusu virus (DTMUV). Overexpression of duMAVS could drive the activation of interferon (IFN)-β, nuclear factor-κB, interferon regulatory factor 7, and many downstream factors (such as Mx, PKR, OAS, and IL-8) in duck embryo fibroblast cells. What is more, RNA interference further confirmed that duMAVS was an important adaptor for IFN-β activation. The antiviral assay showed that duMAVS could suppress the various viral replications (DTMUV, novel reovirus, and duck plague virus) at early stages of infection. Overall, these results showed that the main signal pathway mediated by duMAVS and it had a broad-spectrum antiviral ability. This research will be helpful to better understanding the innate immune system of ducks. PMID:27708647

  16. Regulation of cargo-selective endocytosis by dynamin 2 GTPase-activating protein girdin.

    PubMed

    Weng, Liang; Enomoto, Atsushi; Miyoshi, Hiroshi; Takahashi, Kiyofumi; Asai, Naoya; Morone, Nobuhiro; Jiang, Ping; An, Jian; Kato, Takuya; Kuroda, Keisuke; Watanabe, Takashi; Asai, Masato; Ishida-Takagishi, Maki; Murakumo, Yoshiki; Nakashima, Hideki; Kaibuchi, Kozo; Takahashi, Masahide

    2014-09-17

    In clathrin-mediated endocytosis (CME), specificity and selectivity for cargoes are thought to be tightly regulated by cargo-specific adaptors for distinct cellular functions. Here, we show that the actin-binding protein girdin is a regulator of cargo-selective CME. Girdin interacts with dynamin 2, a GTPase that excises endocytic vesicles from the plasma membrane, and functions as its GTPase-activating protein. Interestingly, girdin depletion leads to the defect in clathrin-coated pit formation in the center of cells. Also, we find that girdin differentially interacts with some cargoes, which competitively prevents girdin from interacting with dynamin 2 and confers the cargo selectivity for CME. Therefore, girdin regulates transferrin and E-cadherin endocytosis in the center of cells and their subsequent polarized intracellular localization, but has no effect on integrin and epidermal growth factor receptor endocytosis that occurs at the cell periphery. Our results reveal that girdin regulates selective CME via a mechanism involving dynamin 2, but not by operating as a cargo-specific adaptor.

  17. Negative effects of desiccation on the protein sorting and post-translational modification.

    PubMed

    Wang, Xiaoqin; He, Yikun

    2009-05-01

    Bryophytes as the first land plants are believed to have colonized the land from a fresh water origin, requiring adaptive mechanisms that survival of dehydration. Physcomitrella patens is such a non-vascular bryophyte and shows rare desiccation tolerance in its vegetative tissues. Previous studies showed that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling and microtubule depolymerization. And proteomic alteration supported the cellular structural changes in respond to desiccation stress. In this addendum, we report that Golgi bodies are absent and adaptor protein complex AP-1 large subunit is downregulated during the course of dehydration. Those phenomena may be adverse in protein posttranslational modification, protein sorting and cell walls synthesis under the desiccation condition. PMID:19816114

  18. Overproduction, purification, crystallization and preliminary X-ray analysis of human Fe65-PTB2 in complex with the amyloid precursor protein intracellular domain

    SciTech Connect

    Radzimanowski, Jens; Beyreuther, Konrad; Sinning, Irmgard; Wild, Klemens

    2008-05-01

    Alzheimer’s disease is characterized by proteolytic processing of the amyloid precursor protein (APP), which releases the aggregation-prone amyloid-β (Aβ) peptide and liberates the intracellular domain (AICD) that interacts with various adaptor proteins. The crystallized AICD–Fe65-PTB2 complex is of central importance for APP translocation, nuclear signalling, processing and Aβ generation. Alzheimer’s disease is associated with typical brain deposits (senile plaques) that mainly contain the neurotoxic amyloid β peptide. This peptide results from proteolytic processing of the type I transmembrane protein amyloid precursor protein (APP). During this proteolytic pathway the APP intracellular domain (AICD) is released into the cytosol, where it associates with various adaptor proteins. The interaction of the AICD with the C-terminal phosphotyrosine-binding domain of Fe65 (Fe65-PTB2) regulates APP translocation, signalling and processing. Human AICD and Fe65-PTB2 have been cloned, overproduced and purified in large amounts in Escherichia coli. A complex of Fe65-PTB2 with the C-terminal 32 amino acids of the AICD gave well diffracting hexagonal crystals and data have been collected to 2.1 Å resolution. Initial phases obtained by the molecular-replacement method are of good quality and revealed well defined electron density for the substrate peptide.

  19. DAXX is a new AIRE-interacting protein.

    PubMed

    Meloni, Alessandra; Meloni, Allesandra; Fiorillo, Edoardo; Corda, Denise; Incani, Federica; Serra, Maria Luisa; Contini, Antonella; Cao, Antonio; Rosatelli, Maria Cristina

    2010-04-23

    The AIRE protein plays a remarkable role as a regulator of central tolerance by controlling the promiscuous expression of tissue-specific antigens in thymic medullary epithelial cells. Defects in the AIRE gene cause the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, a rare disease frequent in Iranian Jews, Finns, and Sardinian population. To this day, the precise function of the AIRE protein in regulating transcription and its interacting proteins has yet to be entirely clarified. The knowledge of novel AIRE interactors and their precise role will improve our knowledge of its biological activity and address some of the foremost autoimmunity-related questions. In this study, we have used a yeast two-hybrid system to identify AIRE-interacting proteins. This approach led us to the discovery of a new AIRE-interacting protein called DAXX. The protein is known to be a multifunctional adaptor with functions both in apoptosis and in transcription regulation pathways. The interaction between AIRE and DAXX has been validated by in vivo coimmunoprecipitation analysis and colocalization study in mammalian cells. The interaction has been further confirmed by showing in transactivation assays that DAXX exerts a strong repressive role on the transcriptional activity of AIRE.

  20. SR proteins SRp20 and 9G8 contribute to efficient export of herpes simplex virus 1 mRNAs.

    PubMed

    Escudero-Paunetto, Laurimar; Li, Ling; Hernandez, Felicia P; Sandri-Goldin, Rozanne M

    2010-06-01

    Herpes simplex virus 1 (HSV-1) mRNAs are exported to the cytoplasm through the export receptor TAP/NFX1. HSV-1 multifunctional protein ICP27 interacts with TAP/NXF1, binds viral RNAs, and is required for efficient viral RNA export. In ICP27 mutant infections, viral RNA export is reduced but not ablated, indicating that other export adaptors can aid in viral RNA export. Export adaptor protein Aly/REF is recruited to viral replication compartments, however, Aly/REF knockdown has little effect on viral RNA export. SR proteins SRp20 and 9G8 interact with TAP/NXF1 and mediate export of some cellular RNAs. We report that siRNA knockdown of SRp20 or 9G8 resulted in about a 10 fold decrease in virus yields and in nuclear accumulation of polyA+ RNA. In infected cells depleted of SRp20, newly transcribed Bromouridine-labeled RNA also accumulated in the nucleus. We conclude that SRp20 and 9G8 contribute to HSV-1 RNA export. PMID:20227104

  1. SR proteins SRp20 and 9G8 contribute to efficient export of herpes simplex virus 1 mRNAs

    SciTech Connect

    Escudero-Paunetto, Laurimar; Li Ling; Hernandez, Felicia P.; Sandri-Goldin, Rozanne M.

    2010-06-05

    Herpes simplex virus 1 (HSV-1) mRNAs are exported to the cytoplasm through the export receptor TAP/NFX1. HSV-1 multifunctional protein ICP27 interacts with TAP/NXF1, binds viral RNAs, and is required for efficient viral RNA export. In ICP27 mutant infections, viral RNA export is reduced but not ablated, indicating that other export adaptors can aid in viral RNA export. Export adaptor protein Aly/REF is recruited to viral replication compartments, however, Aly/REF knockdown has little effect on viral RNA export. SR proteins SRp20 and 9G8 interact with TAP/NXF1 and mediate export of some cellular RNAs. We report that siRNA knockdown of SRp20 or 9G8 resulted in about a 10 fold decrease in virus yields and in nuclear accumulation of poly(A+) RNA. In infected cells depleted of SRp20, newly transcribed Bromouridine-labeled RNA also accumulated in the nucleus. We conclude that SRp20 and 9G8 contribute to HSV-1 RNA export.

  2. Expression and localization of X11 family proteins in neurons.

    PubMed

    Motodate, Rika; Saito, Yuhki; Hata, Saori; Suzuki, Toshiharu

    2016-09-01

    The X11/Mint family of proteins comprises X11/X11α/Mint1, X11L/X11β/Mint2, and X11L2/X11γ/Mint3. Each of these molecules is an adaptor protein that contains a phosphotyrosine interaction/binding (PI/PTB) and two PDZ domains in its carboxy-terminal region. X11/Mint family members associate with a broad spectrum of membrane proteins, including Alzheimer's β-amyloid precursor protein (APP), alcadeins, and low density lipoprotein receptor proteins, as well as various cytoplasmic proteins including Arf, kalirin-7, and Munc18. In particular, X11 and X11L are thought to play various roles in the regulation of neural functions in brain. Nevertheless, the protein levels and respective localization of individual family members remain controversial. We analyzed the protein levels of X11 and X11L in the corresponding single- and double-knockout mice. X11 and X11L did not exhibit obvious changes of their protein levels when the other was absent, especially in cerebrum in which they were widely co-expressed. In cerebellum, X11 and X11L localized in characteristic patterns in various types of neurons, and X11 protein level increased without an obvious ectopic localization in X11L-knockout mice. Interestingly, only X11L protein existed specifically in brain, whereas, contrary to the accepted view, X11 protein was detected at the highest levels in brain but was also strongly detected in pancreas, testis, and paranephros. Together, our results indicate that both X11 and X11L exert largely in brain neurons, but X11 may also function in peripheral tissues. PMID:27268412

  3. T Cell Receptor (TCR) Interacting Molecule (TRIM), A Novel Disulfide-linked Dimer Associated with the TCR–CD3–ζ Complex, Recruits Intracellular Signaling Proteins to the Plasma Membrane

    PubMed Central

    Bruyns, Eddy; Marie-Cardine, Anne; Kirchgessner, Henning; Sagolla, Karin; Shevchenko, Andrej; Mann, Matthias; Autschbach, Frank; Bensussan, Armand; Meuer, Stefan; Schraven, Burkhart

    1998-01-01

    The molecular mechanisms regulating recruitment of intracellular signaling proteins like growth factor receptor–bound protein 2 (Grb2), phospholipase Cγ1, or phosphatidylinositol 3-kinase (PI3-kinase) to the plasma membrane after stimulation of the T cell receptor (TCR)– CD3–ζ complex are not very well understood. We describe here purification, tandem mass spectrometry sequencing, molecular cloning, and biochemical characterization of a novel transmembrane adaptor protein which associates and comodulates with the TCR–CD3–ζ complex in human T lymphocytes and T cell lines. This protein was termed T cell receptor interacting molecule (TRIM). TRIM is a disulfide-linked homodimer which is comprised of a short extracellular domain of 8 amino acids, a 19–amino acid transmembrane region, and a 159–amino acid cytoplasmic tail. In its intracellular domain, TRIM contains several tyrosine-based signaling motifs that could be involved in SH2 domain–mediated protein–protein interactions. Indeed, after T cell activation, TRIM becomes rapidly phosphorylated on tyrosine residues and then associates with the 85-kD regulatory subunit of PI3-kinase via an YxxM motif. Thus, TRIM represents a TCR-associated transmembrane adaptor protein which is likely involved in targeting of intracellular signaling proteins to the plasma membrane after triggering of the TCR. PMID:9687533

  4. Quantitative comparison of CrkL-SH3 binding proteins from embryonic murine brain and liver: Implications for developmental signaling and the quantification of protein species variants in bottom-up proteomics.

    PubMed

    Cheerathodi, Mujeeburahim; Vincent, James J; Ballif, Bryan A

    2015-07-01

    A major aim of proteomics is to comprehensively identify and quantify all protein species variants from a given biological source. However, in spite of its tremendous utility, bottom-up proteomic strategies can do little to provide true quantification of distinct whole protein species variants given its reliance on proteolysis. This is particularly true when molecular size information is lost as in gel-free proteomics. Crk and CrkL comprise a family of adaptor proteins that couple upstream phosphotyrosine signals to downstream effectors by virtue of their SH2 and SH3 domains respectively. Here we compare the identification and quantification of CrkL-SH3 binding partners between embryonic murine brain and liver. We also uncover and quantify tissue-specific variants in CrkL-SH3 binding proteins.

  5. mRNA and protein dataset of autophagy markers (LC3 and p62) in several cell lines

    PubMed Central

    Gómez-Sánchez, Rubén; Yakhine-Diop, Sokhna M.S.; Rodríguez-Arribas, Mario; Bravo-San Pedro, José M.; Martínez-Chacón, Guadalupe; Uribe-Carretero, Elisabet; Pinheiro de Castro, Diana C.J.; Pizarro-Estrella, Elisa; Fuentes, José M.; González-Polo, Rosa A.

    2016-01-01

    We characterized the dynamics of autophagy in vitro using four different cell systems and analyzing markers widely used in this field, i.e. LC3 (microtubule-associated protein 1 light chain 3; protein recruited from the cytosol (LC3-I) to the autophagosomal membrane where it is lipidated (LC3-II)) and p62/SQSTM1 (adaptor protein that serves as a link between LC3 and ubiquitinated substrates), (Klionsky et al., 2016) [1]. Data provided include analyses of protein levels of LC3 and p62 by Western-blotting and endogenous immunofluorescence experiments, but also p62 mRNA levels obtained by quantitative PCR (qPCR). To monitor the turnover of these autophagy markers and, thus, measure the flux of this pathway, cells were under starvation conditions and/or treated with bafilomycin A1 (Baf. A1) to block fusion of autophagosomes with lysosomes. PMID:27054171

  6. Novel activation domain derived from Che-1 cofactor coupled with the artificial protein Jazz drives utrophin upregulation.

    PubMed

    Desantis, Agata; Onori, Annalisa; Di Certo, Maria Grazia; Mattei, Elisabetta; Fanciulli, Maurizio; Passananti, Claudio; Corbi, Nicoletta

    2009-02-01

    Our aim is to upregulate the expression level of the dystrophin related gene utrophin in Duchenne muscular dystrophy, thus complementing the lack of dystrophin functions. To this end, we have engineered synthetic zinc finger based transcription factors. We have previously shown that the artificial three-zinc finger protein named Jazz fused with the Vp16 activation domain, is able to bind utrophin promoter A and to increase the endogenous level of utrophin in transgenic mice. Here, we report on an innovative artificial protein, named CJ7, that consists of Jazz DNA binding domain fused to a novel activation domain derived from the regulatory multivalent adaptor protein Che-1/AATF. This transcriptional activation domain is 100 amino acids in size and it is very powerful as compared to the Vp16 activation domain. We show that CJ7 protein efficiently promotes transcription and accumulation of the acetylated form of histone H3 on the genomic utrophin promoter locus.

  7. A TIR Domain Protein from E. faecalis Attenuates MyD88-Mediated Signaling and NF-κB Activation

    PubMed Central

    Zou, Jun; Baghdayan, Arto S.; Payne, Sarah J.; Shankar, Nathan

    2014-01-01

    Toll-like receptor signaling, mediated by functional Toll/interleukin-1 receptor (TIR) domains, plays a critical role in activating the innate immune response responsible for controlling and clearing infection. Bacterial protein mimics of components of this signaling pathway have been identified and function through inhibition of interactions between Toll-like receptors (TLRs) and their adaptor proteins, mediated by TIR domains. A previously uncharacterized gene, which we have named tcpF (for TIR domain-containing protein in E. faecalis) was identified in the genome of Enterococcus faecalis V583, and predicted to encode a protein resembling mammalian and bacterial TIR proteins. We overexpressed and purified TcpF from E. coli and found that the recombinant protein could bind to phosphatidylinositol phosphates in vitro, suggesting a mechanism by which TcpF may be anchored to the plasma membrane in close proximity to TIR domains of TLRs and adaptor proteins. Purified TcpF was also found to interact specifically with the TIR adaptor protein MyD88, and this interaction was dependent on the BB loop domain in the Box 2 region of TcpF. Despite no evidence of TcpF being a secreted protein, recombinant TcpF was effectively able to enter RAW264.7 cells in vitro although the mechanism by which this occurs remains to be determined. Overexpression of TcpF in mammalian cells suppressed the NF-κB activation induced by bacterial lipoteichoic acid. A mutant lacking the tcpF gene was attenuated for survival in macrophages, with increased ability to activate NF-κB compared to the wild type strain. Complementation in trans restored growth, and inhibition of NF-κB, to that of wild type levels. No appreciable difference in bacterial persistence, dissemination or pathogenesis was observed between the wild type and mutant in a mouse peritonitis model however, which suggested either a subtle role for TcpF or functional overlap with other redundant factor(s) in this virulence model. PMID

  8. Sphingolipid transfer proteins defined by the GLTP-fold

    PubMed Central

    Malinina, Lucy; Simanshu, Dhirendra K.; Zhai, Xiuhong; Samygina, Valeria R.; Kamlekar, RaviKanth; Kenoth, Roopa; Ochoa-Lizarralde, Borja; Malakhova, Margarita L.; Molotkovsky, Julian G.; Patel, Dinshaw J.; Brown, Rhoderick E.

    2015-01-01

    Glycolipid transfer proteins (GLTPs) originally were identified as small (~24 kDa), soluble, amphitropic proteins that specifically accelerate the intermembrane transfer of glycolipids. GLTPs and related homologs now are known to adopt a unique, helically dominated, two-layer ‘sandwich’ architecture defined as the GLTP-fold that provides the structural underpinning for the eukaryotic GLTP superfamily. Recent advances now provide exquisite insights into structural features responsible for lipid headgroup selectivity as well as the adaptability of the hydrophobic compartment for accommodating hydrocarbon chains of differing length and unsaturation. A new understanding of the structural versatility and evolutionary premium placed on the GLTP motif has emerged. Human GLTP-motifs have evolved to function not only as glucosylceramide binding/transferring domains for phosphoinositol 4-phosphate adaptor protein-2 during glycosphingolipid biosynthesis but also as selective binding/transfer proteins for ceramide-1-phosphate. The latter, known as ceramide-l-phosphate transfer protein, recently has been shown to form GLTP-fold while critically regulating Group-IV cytoplasmic phospholipase A2 activity and pro-inflammatory eicosanoid production. PMID:25797198

  9. DAZ Family Proteins, Key Players for Germ Cell Development

    PubMed Central

    Fu, Xia-Fei; Cheng, Shun-Feng; Wang, Lin-Qing; Yin, Shen; De Felici, Massimo; Shen, Wei

    2015-01-01

    DAZ family proteins are found almost exclusively in germ cells in distant animal species. Deletion or mutations of their encoding genes usually severely impair either oogenesis or spermatogenesis or both. The family includes Boule (or Boll), Dazl (or Dazla) and DAZ genes. Boule and Dazl are situated on autosomes while DAZ, exclusive of higher primates, is located on the Y chromosome. Deletion of DAZ gene is the most common causes of infertility in humans. These genes, encoding for RNA binding proteins, contain a highly conserved RNA recognition motif and at least one DAZ repeat encoding for a 24 amino acids sequence able to bind other mRNA binding proteins. Basically, Daz family proteins function as adaptors for target mRNA transport and activators of their translation. In some invertebrate species, BOULE protein play a pivotal role in germline specification and a conserved regulatory role in meiosis. Depending on the species, DAZL is expressed in primordial germ cells (PGCs) and/or pre-meiotic and meiotic germ cells of both sexes. Daz is found in fetal gonocytes, spermatogonia and spermatocytes of adult testes. Here we discuss DAZ family genes in a phylogenic perspective, focusing on the common and distinct features of these genes, and their pivotal roles during gametogenesis evolved during evolution. PMID:26327816

  10. 14-3-3 proteins play a role in the cell cycle by shielding cdt2 from ubiquitin-mediated degradation.

    PubMed

    Dar, Ashraf; Wu, David; Lee, Nicholas; Shibata, Etsuko; Dutta, Anindya

    2014-11-01

    Cdt2 is the substrate recognition adaptor of CRL4(Cdt2) E3 ubiquitin ligase complex and plays a pivotal role in the cell cycle by mediating the proteasomal degradation of Cdt1 (DNA replication licensing factor), p21 (cyclin-dependent kinase [CDK] inhibitor), and Set8 (histone methyltransferase) in S phase. Cdt2 itself is attenuated by SCF(FbxO11)-mediated proteasomal degradation. Here, we report that 14-3-3 adaptor proteins interact with Cdt2 phosphorylated at threonine 464 (T464) and shield it from polyubiquitination and consequent proteasomal degradation. Depletion of 14-3-3 proteins promotes the interaction of FbxO11 with Cdt2. Overexpressing 14-3-3 proteins shields Cdt2 that has a phospho-mimicking mutation (T464D [change of T to D at position 464]) but not Cdt2(T464A) from ubiquitination. Furthermore, the delay of the cell cycle in the G2/M phase and decrease in cell proliferation seen upon depletion of 14-3-3γ is partly due to the accumulation of the CRL4(Cdt2) substrate, Set8 methyltransferase. Therefore, the stabilization of Cdt2 is an important function of 14-3-3 proteins in cell cycle progression.

  11. 14-3-3 Proteins Play a Role in the Cell Cycle by Shielding Cdt2 from Ubiquitin-Mediated Degradation

    PubMed Central

    Dar, Ashraf; Wu, David; Lee, Nicholas; Shibata, Etsuko

    2014-01-01

    Cdt2 is the substrate recognition adaptor of CRL4Cdt2 E3 ubiquitin ligase complex and plays a pivotal role in the cell cycle by mediating the proteasomal degradation of Cdt1 (DNA replication licensing factor), p21 (cyclin-dependent kinase [CDK] inhibitor), and Set8 (histone methyltransferase) in S phase. Cdt2 itself is attenuated by SCFFbxO11-mediated proteasomal degradation. Here, we report that 14-3-3 adaptor proteins interact with Cdt2 phosphorylated at threonine 464 (T464) and shield it from polyubiquitination and consequent proteasomal degradation. Depletion of 14-3-3 proteins promotes the interaction of FbxO11 with Cdt2. Overexpressing 14-3-3 proteins shields Cdt2 that has a phospho-mimicking mutation (T464D [change of T to D at position 464]) but not Cdt2(T464A) from ubiquitination. Furthermore, the delay of the cell cycle in the G2/M phase and decrease in cell proliferation seen upon depletion of 14-3-3γ is partly due to the accumulation of the CRL4Cdt2 substrate, Set8 methyltransferase. Therefore, the stabilization of Cdt2 is an important function of 14-3-3 proteins in cell cycle progression. PMID:25154416

  12. The SWIRM domain: a conserved module found in chromosomal proteins points to novel chromatin-modifying activities

    PubMed Central

    Aravind, L; Iyer, Lakshminarayan M

    2002-01-01

    Background Eukaryotic chromosomal components, especially histones, are subject to a wide array of covalent modifications and catalytic reorganization. These modifications have an important role in the regulation of chromatin structure and are mediated by large multisubunit complexes that contain modular proteins with several conserved catalytic and noncatalytic adaptor domains. Results Using computational sequence-profile analysis methods, we identified a previously uncharacterized, predicted α-helical domain of about 85 residues in chromosomal proteins such as Swi3p, Rsc8p, Moira and several other uncharacterized proteins. This module, termed the SWIRM domain, is predicted to mediate specific protein-protein interactions in the assembly of chromatin-protein complexes. In one group of proteins, which are highly conserved throughout the crown-group eukaryotes, the SWIRM domain is linked to a catalytic domain related to the monoamine and polyamine oxidases. Another human protein has the SWIRM domain linked to a JAB domain that is involved in protein degradation through the ubiquitin pathway. Conclusions Identification of the SWIRM domain could help in directed experimental analysis of specific interactions in chromosomal proteins. We predict that the proteins in which it is combined with an amino-oxidase domain define a novel class of chromatin-modifying enzymes, which are likely to oxidize either the amino group of basic residues in histones and other chromosomal proteins or the polyamines in chromatin, and thereby alter the charge distribution. Other forms, such as KIAA1915, may link chromatin modification to ubiquitin-dependent protein degradation. PMID:12186646

  13. Evidence for the interaction of the regulatory protein Ki-1/57 with p53 and its interacting proteins

    SciTech Connect

    Nery, Flavia C.; Rui, Edmilson; Kuniyoshi, Tais M.; Kobarg, Joerg . E-mail: jkobarg@lnls.br

    2006-03-17

    Ki-1/57 is a cytoplasmic and nuclear phospho-protein of 57 kDa and interacts with the adaptor protein RACK1, the transcription factor MEF2C, and the chromatin remodeling factor CHD3, suggesting that it might be involved in the regulation of transcription. Here, we describe yeast two-hybrid studies that identified a total of 11 proteins interacting with Ki-1/57, all of which interact or are functionally associated with p53 or other members of the p53 family of proteins. We further found that Ki-1/57 is able to interact with p53 itself in the yeast two-hybrid system when the interaction was tested directly. This interaction could be confirmed by pull down assays with purified proteins in vitro and by reciprocal co-immunoprecipitation assays from the human Hodgkin analogous lymphoma cell line L540. Furthermore, we found that the phosphorylation of p53 by PKC abolishes its interaction with Ki-1/57 in vitro.

  14. Identification of contractile vacuole proteins in Trypanosoma cruzi.

    PubMed

    Ulrich, Paul N; Jimenez, Veronica; Park, Miyoung; Martins, Vicente P; Atwood, James; Moles, Kristen; Collins, Dalis; Rohloff, Peter; Tarleton, Rick; Moreno, Silvia N J; Orlando, Ron; Docampo, Roberto

    2011-03-18

    and provided information on the potential participation of adaptor protein complexes in their biogenesis.

  15. MicroRNAs regulate tight junction proteins and modulate epithelial/endothelial barrier functions.

    PubMed

    Cichon, Christoph; Sabharwal, Harshana; Rüter, Christian; Schmidt, M Alexander

    2014-01-01

    Tightly controlled epithelial and endothelial barriers are a prerequisite for life as these barriers separate multicellular organisms from their environment and serve as first lines of defense. Barriers between neighboring epithelial cells are formed by multiple intercellular junctions including the 'apical junctional complex-AJC' with tight junctions (TJ), adherens junctions (AJ), and desmosomes. TJ consist of tetraspan transmembrane proteins like occludin, various claudins that directly control paracellular permeability, and the 'Junctional Adhesion Molecules' (JAMs). For establishing tight barriers TJ are essential but at the same time have to allow also selective permeability. For this, TJ need to be tightly regulated and controlled. This is organized by a variety of adaptor molecules, i.e., protein kinases, phosphatases and GTPases, which in turn are regulated and fine-tuned involving microRNAs (miRNAs). In this review we summarize available data on the role and targeting of miRNAs in the maintenance of epithelial and/or endothelial barriers.

  16. A presynaptic role for the cytomatrix protein GIT in synaptic vesicle recycling.

    PubMed

    Podufall, Jasmin; Tian, Rui; Knoche, Elena; Puchkov, Dmytro; Walter, Alexander M; Rosa, Stefanie; Quentin, Christine; Vukoja, Anela; Jung, Nadja; Lampe, Andre; Wichmann, Carolin; Böhme, Mathias; Depner, Harald; Zhang, Yong Q; Schmoranzer, Jan; Sigrist, Stephan J; Haucke, Volker

    2014-06-12

    Neurotransmission involves the exo-endocytic cycling of synaptic vesicles (SVs) within nerve terminals. Exocytosis is facilitated by a cytomatrix assembled at the active zone (AZ). The precise spatial and functional relationship between exocytic fusion of SVs at AZ membranes and endocytic SV retrieval is unknown. Here, we identify the scaffold G protein coupled receptor kinase 2 interacting (GIT) protein as a component of the AZ-associated cytomatrix and as a regulator of SV endocytosis. GIT1 and its D. melanogaster ortholog, dGIT, are shown to directly associate with the endocytic adaptor stonin 2/stoned B. In Drosophila dgit mutants, stoned B and synaptotagmin levels are reduced and stoned B is partially mislocalized. Moreover, dgit mutants show morphological and functional defects in SV recycling. These data establish a presynaptic role for GIT in SV recycling and suggest a connection between the AZ cytomatrix and the endocytic machinery. PMID:24882013

  17. Giant axonal neuropathy-associated gigaxonin mutations impair intermediate filament protein degradation.

    PubMed

    Mahammad, Saleemulla; Murthy, S N Prasanna; Didonna, Alessandro; Grin, Boris; Israeli, Eitan; Perrot, Rodolphe; Bomont, Pascale; Julien, Jean-Pierre; Kuczmarski, Edward; Opal, Puneet; Goldman, Robert D

    2013-05-01

    Giant axonal neuropathy (GAN) is an early-onset neurological disorder caused by mutations in the GAN gene (encoding for gigaxonin), which is predicted to be an E3 ligase adaptor. In GAN, aggregates of intermediate filaments (IFs) represent the main pathological feature detected in neurons and other cell types, including patients' dermal fibroblasts. The molecular mechanism by which these mutations cause IFs to aggregate is unknown. Using fibroblasts from patients and normal individuals, as well as Gan-/- mice, we demonstrated that gigaxonin was responsible for the degradation of vimentin IFs. Gigaxonin was similarly involved in the degradation of peripherin and neurofilament IF proteins in neurons. Furthermore, proteasome inhibition by MG-132 reversed the clearance of IF proteins in cells overexpressing gigaxonin, demonstrating the involvement of the proteasomal degradation pathway. Together, these findings identify gigaxonin as a major factor in the degradation of cytoskeletal IFs and provide an explanation for IF aggregate accumulation, the subcellular hallmark of this devastating human disease.

  18. Structural Basis for the Recognition of Tyrosine-based Sorting Signals by the μ3A Subunit of the AP-3 Adaptor Complex*

    PubMed Central

    Mardones, Gonzalo A.; Burgos, Patricia V.; Lin, Yimo; Kloer, Daniel P.; Magadán, Javier G.; Hurley, James H.; Bonifacino, Juan S.

    2013-01-01

    Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous μ1, μ2, μ3, and μ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on μ2 and μ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the μ family, we solved the crystal structure at 1.85 Å resolution of the C-terminal domain of the μ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The μ3A C-terminal domain consists of an immunoglobulin-like β-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on μ3A subdomain A, at a location similar to the YXXØ-binding site on μ2 but not μ4. The binding sites on μ3A and μ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the μ3A site and show that this protein binds YXXØ signals with 14–19 μm affinity. The surface electrostatic potential of μ3A is less basic than that of μ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides. PMID:23404500

  19. Triage of oxidation-prone proteins by Sqstm1/p62 within the mitochondria

    SciTech Connect

    Lee, Minjung; Shin, Jaekyoon

    2011-09-16

    Highlights: {yields} The mitochondrion contains its own protein quality control system. {yields} p62 localizes within the mitochondria and forms mega-dalton sized complexes. {yields} p62 interacts with oxidation-prone proteins and the proteins of quality control. {yields} In vitro delivery of p62 improves mitochondrial functions. {yields} p62 is implicated as a participant in mitochondrial protein quality control. -- Abstract: As the mitochondrion is vulnerable to oxidative stress, cells have evolved several strategies to maintain mitochondrial integrity, including mitochondrial protein quality control mechanisms and autophagic removal of damaged mitochondria. Involvement of an autophagy adaptor, Sqstm1/p62, in the latter process has been recently described. In the present study, we provide evidence that a portion of p62 directly localizes within the mitochondria and supports stable electron transport by forming heterogeneous protein complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of mitochondrial proteins co-purified with p62 revealed that p62 interacts with several oxidation-prone proteins, including a few components of the electron transport chain complexes, as well as multiple chaperone molecules and redox regulatory enzymes. Accordingly, p62-deficient mitochondria exhibited compromised electron transport, and the compromised function was partially restored by in vitro delivery of p62. These results suggest that p62 plays an additional role in maintaining mitochondrial integrity at the vicinity of target machineries through its function in relation to protein quality control.

  20. Characterisation of the Plasmodium falciparum Hsp70-Hsp90 organising protein (PfHop).

    PubMed

    Gitau, Grace W; Mandal, Pradipta; Blatch, Gregory L; Przyborski, Jude; Shonhai, Addmore

    2012-03-01

    Malaria is caused by Plasmodium species, whose transmission to vertebrate hosts is facilitated by mosquito vectors. The transition from the cold blooded mosquito vector to the host represents physiological stress to the parasite, and additionally malaria blood stage infection is characterised by intense fever periods. In recent years, it has become clear that heat shock proteins play an essential role during the parasite's life cycle. Plasmodium falciparum expresses two prominent heat shock proteins: heat shock protein 70 (PfHsp70) and heat shock protein 90 (PfHsp90). Both of these proteins have been implicated in the development and pathogenesis of malaria. In eukaryotes, Hsp70 and Hsp90 proteins are functionally linked by an essential adaptor protein known as the Hsp70-Hsp90 organising protein (Hop). In this study, recombinant P. falciparum Hop (PfHop) was heterologously produced in E. coli and purified by nickel affinity chromatography. Using specific anti-PfHop antisera, the expression and localisation of PfHop in P. falciparum was investigated. PfHop was shown to co-localise with PfHsp70 and PfHsp90 in parasites at the trophozoite stage. Gel filtration and co-immunoprecipitation experiments suggested that PfHop was present in a complex together with PfHsp70 and PfHsp90. The association of PfHop with both PfHsp70 and PfHsp90 suggests that this protein may mediate the functional interaction between the two chaperones.

  1. Kelch proteins: emerging roles in skeletal muscle development and diseases.

    PubMed

    Gupta, Vandana A; Beggs, Alan H

    2014-01-01

    Our understanding of genes that cause skeletal muscle disease has increased tremendously over the past three decades. Advances in approaches to genetics and genomics have aided in the identification of new pathogenic mechanisms in rare genetic disorders and have opened up new avenues for therapeutic interventions by identification of new molecular pathways in muscle disease. Recent studies have identified mutations of several Kelch proteins in skeletal muscle disorders. The Kelch superfamily is one of the largest evolutionary conserved gene families. The 66 known family members all possess a Kelch-repeat containing domain and are implicated in diverse biological functions. In skeletal muscle development, several Kelch family members regulate the processes of proliferation and/or differentiation resulting in normal functioning of mature muscles. Importantly, many Kelch proteins function as substrate-specific adaptors for Cullin E3 ubiquitin ligase (Cul3), a core component of the ubiquitin-proteasome system to regulate the protein turnover. This review discusses the emerging roles of Kelch proteins in skeletal muscle function and disease. PMID:24959344

  2. Stress conditions promote yeast Gap1 permease ubiquitylation and down-regulation via the arrestin-like Bul and Aly proteins.

    PubMed

    Crapeau, Myriam; Merhi, Ahmad; André, Bruno

    2014-08-01

    Gap1, the yeast general amino acid permease, is a convenient model for studying how the intracellular traffic of membrane transporters is regulated. Present at the plasma membrane under poor nitrogen supply conditions, it undergoes ubiquitylation, endocytosis, and degradation upon activation of the TORC1 kinase complex in response to an increase in internal amino acids. This down-regulation is stimulated by TORC1-dependent phosphoinhibition of the Npr1 kinase, resulting in activation by dephosphorylation of the arrestin-like Bul1 and Bul2 adaptors recruiting the Rsp5 ubiquitin ligase to Gap1. We report here that Gap1 is also down-regulated when cells are treated with the TORC1 inhibitor rapamycin or subjected to various stresses and that a lack of the Tco89 subunit of TORC1 causes constitutive Gap1 down-regulation. Both the Bul1 and Bul2 and the Aly1 and Aly2 arrestin-like adaptors of Rsp5 promote this down-regulation without undergoing dephosphorylation. Furthermore, they act via the C-terminal regions of Gap1 not involved in ubiquitylation in response to internal amino acids, whereas a Gap1 mutant altered in the N-terminal tail and resistant to ubiquitylation by internal amino acids is efficiently down-regulated under stress via the Bul and Aly adaptors. Although the Bul proteins mediate Gap1 ubiquitylation of two possible lysines, Lys-9 and Lys-16, the Aly proteins promote ubiquitylation of the Lys-16 residue only. This stress-induced pathway of Gap1 down-regulation targets other permeases as well, and it likely allows cells facing adverse conditions to retrieve amino acids from permease degradation.

  3. The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis.

    PubMed

    Bashline, Logan; Li, Shundai; Zhu, Xiaoyu; Gu, Ying

    2015-10-13

    Cellulose biosynthesis is performed exclusively by plasma membrane-localized cellulose synthases (CESAs). Therefore, the trafficking of CESAs to and from the plasma membrane is an important mechanism for regulating cellulose biosynthesis. CESAs were recently identified as cargo proteins of the classic adaptor protein 2 (AP2) complex of the clathrin-mediated endocytosis (CME) pathway. The AP2 complex of the CME pathway is conserved in yeast, animals, and plants, and has been well-characterized in many systems. In contrast, the recently discovered TPLATE complex (TPC), which is proposed to function as a CME adaptor complex, is only conserved in plants and a few other eukaryotes. In this study, we discovered that the TWD40-2 protein, a putative member of the TPC, is also important for the endocytosis of CESAs. Genetic analysis between TWD40-2 and AP2M of the AP2 complex revealed that the roles of TWD40-2 in CME are both distinct from and cooperative with the AP2 complex. Loss of efficient CME in twd40-2-3 resulted in the unregulated overaccumulation of CESAs at the plasma membrane. In seedlings of twd40-2-3 and other CME-deficient mutants, a direct correlation was revealed between endocytic deficiency and cellulose content deficiency, highlighting the importance of controlled CESA endocytosis in regulating cellulose biosynthesis.

  4. The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis

    PubMed Central

    Bashline, Logan; Li, Shundai; Zhu, Xiaoyu; Gu, Ying

    2015-01-01

    Cellulose biosynthesis is performed exclusively by plasma membrane-localized cellulose synthases (CESAs). Therefore, the trafficking of CESAs to and from the plasma membrane is an important mechanism for regulating cellulose biosynthesis. CESAs were recently identified as cargo proteins of the classic adaptor protein 2 (AP2) complex of the clathrin-mediated endocytosis (CME) pathway. The AP2 complex of the CME pathway is conserved in yeast, animals, and plants, and has been well-characterized in many systems. In contrast, the recently discovered TPLATE complex (TPC), which is proposed to function as a CME adaptor complex, is only conserved in plants and a few other eukaryotes. In this study, we discovered that the TWD40-2 protein, a putative member of the TPC, is also important for the endocytosis of CESAs. Genetic analysis between TWD40-2 and AP2M of the AP2 complex revealed that the roles of TWD40-2 in CME are both distinct from and cooperative with the AP2 complex. Loss of efficient CME in twd40-2-3 resulted in the unregulated overaccumulation of CESAs at the plasma membrane. In seedlings of twd40-2-3 and other CME-deficient mutants, a direct correlation was revealed between endocytic deficiency and cellulose content deficiency, highlighting the importance of controlled CESA endocytosis in regulating cellulose biosynthesis. PMID:26417106

  5. The Synaptojanin-like Protein Inp53/Sjl3 Functions with Clathrin in a Yeast TGN-to-Endosome Pathway Distinct from the GGA Protein-dependent Pathway

    PubMed Central

    Ha, Seon-Ah; Torabinejad, Javad; DeWald, Daryll B.; Wenk, Markus R.; Lucast, Louise; De Camilli, Pietro; Newitt, Richard A.; Aebersold, Ruedi; Nothwehr, Steven F.

    2003-01-01

    Yeast TGN resident proteins that frequently cycle between the TGN and endosomes are much more slowly transported to the prevacuolar/late endosomal compartment (PVC) than other proteins. However, TGN protein transport to the PVC is accelerated in mutants lacking function of Inp53p. Inp53p contains a SacI polyphosphoinositide phosphatase domain, a 5-phosphatase domain, and a proline-rich domain. Here we show that all three domains are required to mediate “slow delivery” of TGN proteins into the PVC. Although deletion of the proline-rich domain did not affect general membrane association, it caused localization to become less specific. The proline-rich domain was shown to bind to two proteins, including clathrin heavy chain, Chc1p. Unlike chc1 mutants, inp53 mutants do not mislocalize TGN proteins to the cell surface, consistent with the idea that Chc1p and Inp53p act at a common vesicular trafficking step but that Chc1p is used at other steps also. Like mutations in the AP-1 adaptor complex, mutations in INP53 exhibit synthetic growth and transport defects when combined with mutations in the GGA proteins. Taken together with other recent studies, our results suggest that Inp53p and AP-1/clathrin act together in a TGN-to-early endosome pathway distinct from the direct TGN-to-PVC pathway mediated by GGA/clathrin. PMID:12686590

  6. Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread.

    PubMed

    Kroj, Thomas; Chanclud, Emilie; Michel-Romiti, Corinne; Grand, Xavier; Morel, Jean-Benoit

    2016-04-01

    Plant immune receptors of the class of nucleotide-binding and leucine-rich repeat domain (NLR) proteins can contain additional domains besides canonical NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)) and leucine-rich repeat (LRR) domains. Recent research suggests that these additional domains act as integrated decoys recognizing effectors from pathogens. Proteins homologous to integrated decoys are suspected to be effector targets and involved in disease or resistance. Here, we scrutinized 31 entire plant genomes to identify putative integrated decoy domains in NLR proteins using the Interpro search. The involvement of the Zinc Finger-BED type (ZBED) protein containing a putative decoy domain, called BED, in rice (Oryza sativa) resistance was investigated by evaluating susceptibility to the blast fungus Magnaporthe oryzae in rice over-expression and knock-out mutants. This analysis showed that all plants tested had integrated various atypical protein domains into their NLR proteins (on average 3.5% of all NLR proteins). We also demonstrated that modifying the expression of the ZBED gene modified disease susceptibility. This study suggests that integration of decoy domains in NLR immune receptors is widespread and frequent in plants. The integrated decoy model is therefore a powerful concept to identify new proteins involved in disease resistance. Further in-depth examination of additional domains in NLR proteins promises to unravel many new proteins of the plant immune system. PMID:26848538

  7. Traffic of p24 Proteins and COPII Coat Composition Mutually Influence Membrane Scaffolding.

    PubMed

    D'Arcangelo, Jennifer G; Crissman, Jonathan; Pagant, Silvere; Čopič, Alenka; Latham, Catherine F; Snapp, Erik L; Miller, Elizabeth A

    2015-05-18

    Eukaryotic protein secretion requires efficient and accurate delivery of diverse secretory and membrane proteins. This process initiates in the ER, where vesicles are sculpted by the essential COPII coat. The Sec13p subunit of the COPII coat contributes to membrane scaffolding, which enforces curvature on the nascent vesicle. A requirement for Sec13p can be bypassed when traffic of lumenally oriented membrane proteins is abrogated. Here we sought to further explore the impact of cargo proteins on vesicle formation. We show that efficient ER export of the p24 family of proteins is a major driver of the requirement for Sec13p. The scaffolding burden presented by the p24 complex is met in part by the cargo adaptor Lst1p, which binds to a subset of cargo, including the p24 proteins. We propose that the scaffolding function of Lst1p is required to generate vesicles that can accommodate difficult cargo proteins that include large oligomeric assemblies and asymmetrically distributed membrane proteins. Vesicles that contain such cargoes are also more dependent on scaffolding by Sec13p, and may serve as a model for large carrier formation in other systems.

  8. Expression, Localization, and Binding Activity of the Ezrin/Radixin/Moesin Proteins in the Mouse Testis

    PubMed Central

    Wakayama, Tomohiko; Nakata, Hiroki; Kurobo, Miho; Sai, Yoshimichi; Iseki, Shoichi

    2009-01-01

    The ezrin, radixin, and moesin (ERM) proteins represent a family of adaptor proteins linking transmembrane proteins to the cytoskeleton. The seminiferous epithelium undergoes extensive changes in cellular composition, location, and shape, implicating roles of the membrane–cytoskeleton interaction. It remains unknown, however, whether the ERM proteins are expressed and play significant roles in the testis. In the present study, we examined the spatiotemporal expression of ERM proteins in the mouse testis by Western blotting and immunohistochemistry. Ezrin immunoreactivity was demonstrated in the cytoplasm of steps 15 and 16 spermatids from 5 weeks postpartum through adulthood, whereas radixin immunoreactivity was in the apical cytoplasm of Sertoli cells from 1 week through 2 weeks postpartum. No immunoreactivity for moesin was detected at any age. Immunoprecipitation demonstrated that ezrin was bound to the cytoskeletal component actin, whereas radixin was bound to both actin and tubulin. Of the transmembrane proteins known to interact with ERM proteins, only cystic fibrosis transmembrane conductance regulator, a chloride transporter, was bound to ezrin in elongated spermatids. These results suggest that ezrin is involved in spermiogenesis whereas radixin is involved in the maturation of Sertoli cells, through interaction with different sets of membrane proteins and cytoskeletal components. (J Histochem Cytochem 57:351–362, 2009) PMID:19064715

  9. Nuclear protein kinase CLK1 uses a non-traditional docking mechanism to select physiological substrates.

    PubMed

    Keshwani, Malik M; Hailey, Kendra L; Aubol, Brandon E; Fattet, Laurent; McGlone, Maria L; Jennings, Patricia A; Adams, Joseph A

    2015-12-15

    Phosphorylation-dependent cell communication requires enzymes that specifically recognize key proteins in a sea of similar, competing substrates. The protein kinases achieve this goal by utilizing docking grooves in the kinase domain or heterologous protein adaptors to reduce 'off pathway' targeting. We now provide evidence that the nuclear protein kinase CLK1 (cell division cycle2-like kinase 1) important for splicing regulation departs from these classic paradigms by using a novel self-association mechanism. The disordered N-terminus of CLK1 induces oligomerization, a necessary event for targeting its physiological substrates the SR protein (splicing factor containing a C-terminal RS domain) family of splicing factors. Increasing the CLK1 concentration enhances phosphorylation of the splicing regulator SRSF1 (SR protein splicing factor 1) compared with the general substrate myelin basic protein (MBP). In contrast, removal of the N-terminus or dilution of CLK1 induces monomer formation and reverses this specificity. CLK1 self-association also occurs in the nucleus, is induced by the N-terminus and is important for localization of the kinase in sub-nuclear compartments known as speckles. These findings present a new picture of substrate recognition for a protein kinase in which an intrinsically disordered domain is used to capture physiological targets with similar disordered domains in a large oligomeric complex while discriminating against non-physiological targets.

  10. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2007-09-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  11. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  12. Yeast Kre33 and human NAT10 are conserved 18S rRNA cytosine acetyltransferases that modify tRNAs assisted by the adaptor Tan1/THUMPD1

    PubMed Central

    Sharma, Sunny; Langhendries, Jean-Louis; Watzinger, Peter; Kötter, Peter; Entian, Karl-Dieter; Lafontaine, Denis L.J.

    2015-01-01

    The function of RNA is subtly modulated by post-transcriptional modifications. Here, we report an important crosstalk in the covalent modification of two classes of RNAs. We demonstrate that yeast Kre33 and human NAT10 are RNA cytosine acetyltransferases with, surprisingly, specificity toward both 18S rRNA and tRNAs. tRNA acetylation requires the intervention of a specific and conserved adaptor: yeast Tan1/human THUMPD1. In budding and fission yeasts, and in human cells, we found two acetylated cytosines on 18S rRNA, one in helix 34 important for translation accuracy and another in helix 45 near the decoding site. Efficient 18S rRNA acetylation in helix 45 involves, in human cells, the vertebrate-specific box C/D snoRNA U13, which, we suggest, exposes the substrate cytosine to modification through Watson–Crick base pairing with 18S rRNA precursors during small subunit biogenesis. Finally, while Kre33 and NAT10 are essential for pre-rRNA processing reactions leading to 18S rRNA synthesis, we demonstrate that rRNA acetylation is dispensable to yeast cells growth. The inactivation of NAT10 was suggested to suppress nuclear morphological defects observed in laminopathic patient cells through loss of microtubules modification and cytoskeleton reorganization. We rather propose the effects of NAT10 on laminopathic cells are due to reduced ribosome biogenesis or function. PMID:25653167

  13. Transient Fcho1/2⋅Eps15/R⋅AP-2 Nanoclusters Prime the AP-2 Clathrin Adaptor for Cargo Binding.

    PubMed

    Ma, Li; Umasankar, Perunthottathu K; Wrobel, Antoni G; Lymar, Anastasia; McCoy, Airlie J; Holkar, Sachin S; Jha, Anupma; Pradhan-Sundd, Tirthadipa; Watkins, Simon C; Owen, David J; Traub, Linton M

    2016-06-01

    Clathrin-coated vesicles form by rapid assembly of discrete coat constituents into a cargo-sorting lattice. How the sequential phases of coat construction are choreographed is unclear, but transient protein-protein interactions mediated by short interaction motifs are pivotal. We show that arrayed Asp-Pro-Phe (DPF) motifs within the early-arriving endocytic pioneers Eps15/R are differentially decoded by other endocytic pioneers Fcho1/2 and AP-2. The structure of an Eps15/R⋅Fcho1 μ-homology domain complex reveals a spacing-dependent DPF triad, bound in a mechanistically distinct way from the mode of single DPF binding to AP-2. Using cells lacking FCHO1/2 and with Eps15 sequestered from the plasma membrane, we establish that without these two endocytic pioneers, AP-2 assemblies are fleeting and endocytosis stalls. Thus, distinct DPF-based codes within the unstructured Eps15/R C terminus direct the assembly of temporary Fcho1/2⋅Eps15/R⋅AP-2 ternary complexes to facilitate conformational activation of AP-2 by the Fcho1/2 interdomain linker to promote AP-2 cargo engagement.

  14. Transient Fcho1/2⋅Eps15/R⋅AP-2 Nanoclusters Prime the AP-2 Clathrin Adaptor for Cargo Binding.

    PubMed

    Ma, Li; Umasankar, Perunthottathu K; Wrobel, Antoni G; Lymar, Anastasia; McCoy, Airlie J; Holkar, Sachin S; Jha, Anupma; Pradhan-Sundd, Tirthadipa; Watkins, Simon C; Owen, David J; Traub, Linton M

    2016-06-01

    Clathrin-coated vesicles form by rapid assembly of discrete coat constituents into a cargo-sorting lattice. How the sequential phases of coat construction are choreographed is unclear, but transient protein-protein interactions mediated by short interaction motifs are pivotal. We show that arrayed Asp-Pro-Phe (DPF) motifs within the early-arriving endocytic pioneers Eps15/R are differentially decoded by other endocytic pioneers Fcho1/2 and AP-2. The structure of an Eps15/R⋅Fcho1 μ-homology domain complex reveals a spacing-dependent DPF triad, bound in a mechanistically distinct way from the mode of single DPF binding to AP-2. Using cells lacking FCHO1/2 and with Eps15 sequestered from the plasma membrane, we establish that without these two endocytic pioneers, AP-2 assemblies are fleeting and endocytosis stalls. Thus, distinct DPF-based codes within the unstructured Eps15/R C terminus direct the assembly of temporary Fcho1/2⋅Eps15/R⋅AP-2 ternary complexes to facilitate conformational activation of AP-2 by the Fcho1/2 interdomain linker to promote AP-2 cargo engagement. PMID:27237791

  15. Direct Ubiquitin Independent Recognition and Degradation of a Folded Protein by the Eukaryotic Proteasomes-Origin of Intrinsic Degradation Signals

    PubMed Central

    Singh Gautam, Amit Kumar; Balakrishnan, Satish; Venkatraman, Prasanna

    2012-01-01

    Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a) origin and identification of an intrinsic degradation signal in the substrate, b) identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c) identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably catalyzed by the ATPases

  16. Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.

    PubMed

    Holland, S J; Liao, X C; Mendenhall, M K; Zhou, X; Pardo, J; Chu, P; Spencer, C; Fu, A; Sheng, N; Yu, P; Pali, E; Nagin, A; Shen, M; Yu, S; Chan, E; Wu, X; Li, C; Woisetschlager, M; Aversa, G; Kolbinger, F; Bennett, M K; Molineaux, S; Luo, Y; Payan, D G; Mancebo, H S; Wu, J

    2001-11-01

    In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

  17. Functional Cloning of Src-like Adapter Protein-2 (SLAP-2), a Novel Inhibitor of Antigen Receptor Signaling

    PubMed Central

    Holland, Sacha J.; Liao, X. Charlene; Mendenhall, Marcy K.; Zhou, Xiulan; Pardo, Jorge; Chu, Peter; Spencer, Collin; Fu, Alan; Sheng, Ning; Yu, Peiwen; Pali, Erlina; Nagin, Anup; Shen, Mary; Yu, Simon; Chan, Eva; Wu, Xian; Li, Connie; Woisetschlager, Max; Aversa, Gregorio; Kolbinger, Frank; Bennett, Mark K.; Molineaux, Susan; Luo, Ying; Payan, Donald G.; Mancebo, Helena S.Y.; Wu, Jun

    2001-01-01

    In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor–stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems. PMID:11696592

  18. A Toll/interleukin (IL)-1 receptor domain protein from Yersinia pestis interacts with mammalian IL-1/Toll-like receptor pathways but does not play a central role in the virulence of Y. pestis in a mouse model of bubonic plague.

    PubMed

    Spear, Abigail M; Rana, Rohini R; Jenner, Dominic C; Flick-Smith, Helen C; Oyston, Petra C F; Simpson, Peter; Matthews, Stephen J; Byrne, Bernadette; Atkins, Helen S

    2012-06-01

    The Toll/interleukin (IL)-1 receptor (TIR) domain is an essential component of eukaryotic innate immune signalling pathways. Interaction between TIR domains present in Toll-like receptors and associated adaptors initiates and propagates an immune signalling cascade. Proteins containing TIR domains have also been discovered in bacteria. Studies have subsequently shown that these proteins are able to modulate mammalian immune signalling pathways dependent on TIR interactions and that this may represent an evasion strategy for bacterial pathogens. Here, we investigate a TIR domain protein from the highly virulent bacterium Yersinia pestis, the causative agent of plague. When overexpressed in vitro this protein is able to downregulate IL-1β- and LPS-dependent signalling to NFκB and to interact with the TIR adaptor protein MyD88. This interaction is dependent on a single proline residue. However, a Y. pestis knockout mutant lacking the TIR domain protein was not attenuated in virulence in a mouse model of bubonic plague. Minor alterations in the host cytokine response to the mutant were indicated, suggesting a potential subtle role in pathogenesis. The Y. pestis mutant also showed increased auto-aggregation and reduced survival in high-salinity conditions, phenotypes which may contribute to pathogenesis or survival.

  19. A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis.

    PubMed

    Gonzalez, Nathalie; Pauwels, Laurens; Baekelandt, Alexandra; De Milde, Liesbeth; Van Leene, Jelle; Besbrugge, Nienke; Heyndrickx, Ken S; Cuéllar Pérez, Amparo; Durand, Astrid Nagels; De Clercq, Rebecca; Van De Slijke, Eveline; Vanden Bossche, Robin; Eeckhout, Dominique; Gevaert, Kris; Vandepoele, Klaas; De Jaeger, Geert; Goossens, Alain; Inzé, Dirk

    2015-08-01

    Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls. PMID:26232487

  20. A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis

    PubMed Central

    Gonzalez, Nathalie; Pauwels, Laurens; Baekelandt, Alexandra; De Milde, Liesbeth; Van Leene, Jelle; Besbrugge, Nienke; Heyndrickx, Ken S.; Pérez, Amparo Cuéllar; Durand, Astrid Nagels; De Clercq, Rebecca; Van De Slijke, Eveline; Vanden Bossche, Robin; Eeckhout, Dominique; Gevaert, Kris; Vandepoele, Klaas; De Jaeger, Geert; Goossens, Alain; Inzé, Dirk

    2015-01-01

    Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls. PMID:26232487

  1. Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins

    PubMed Central

    Saritas-Yildirim, Banu; Pliner, Hannah A.; Ochoa, Angelica; Silva, Elena M.

    2015-01-01

    Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development. PMID:26327321

  2. Solution structure of the human signaling protein RACK1

    PubMed Central

    2010-01-01

    Background The adaptor protein RACK1 (receptor of activated kinase 1) was originally identified as an anchoring protein for protein kinase C. RACK1 is a 36 kDa protein, and is composed of seven WD repeats which mediate its protein-protein interactions. RACK1 is ubiquitously expressed and has been implicated in diverse cellular processes involving: protein translation regulation, neuropathological processes, cellular stress, and tissue development. Results In this study we performed a biophysical analysis of human RACK1 with the aim of obtaining low resolution structural information. Small angle X-ray scattering (SAXS) experiments demonstrated that human RACK1 is globular and monomeric in solution and its low resolution structure is strikingly similar to that of an homology model previously calculated by us and to the crystallographic structure of RACK1 isoform A from Arabidopsis thaliana. Both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation techniques showed that RACK1 is predominantly a monomer of around 37 kDa in solution, but also presents small amounts of oligomeric species. Moreover, hydrodynamic data suggested that RACK1 has a slightly asymmetric shape. The interaction of RACK1 and Ki-1/57 was tested by sedimentation equilibrium. The results suggested that the association between RACK1 and Ki-1/57(122-413) follows a stoichiometry of 1:1. The binding constant (KB) observed for RACK1-Ki-1/57(122-413) interaction was of around (1.5 ± 0.2) × 106 M-1 and resulted in a dissociation constant (KD) of (0.7 ± 0.1) × 10-6 M. Moreover, the fluorescence data also suggests that the interaction may occur in a cooperative fashion. Conclusion Our SAXS and analytical ultracentrifugation experiments indicated that RACK1 is predominantly a monomer in solution. RACK1 and Ki-1/57(122-413) interact strongly under the tested conditions. PMID:20529362

  3. HIV-1 Vpr Protein Inhibits Telomerase Activity via the EDD-DDB1-VPRBP E3 Ligase Complex*

    PubMed Central

    Wang, Xin; Singh, Shailbala; Jung, Hae-Yun; Yang, Guojun; Jun, Sohee; Sastry, K. Jagannadha; Park, Jae-Il

    2013-01-01

    Viral pathogens utilize host cell machinery for their benefits. Herein, we identify that HIV-1 Vpr (viral protein R) negatively modulates telomerase activity. Telomerase enables stem and cancer cells to evade cell senescence by adding telomeric sequences to the ends of chromosomes. We found that Vpr inhibited telomerase activity by down-regulating TERT protein, a catalytic subunit of telomerase. As a molecular adaptor, Vpr enhanced the interaction between TERT and the VPRBP substrate receptor of the DYRK2-associated EDD-DDB1-VPRBP E3 ligase complex, resulting in increased ubiquitination of TERT. In contrast, the Vpr mutant identified in HIV-1-infected long-term nonprogressors failed to promote TERT destabilization. Our results suggest that Vpr inhibits telomerase activity by hijacking the host E3 ligase complex, and we propose the novel molecular mechanism of telomerase deregulation in possibly HIV-1 pathogenesis. PMID:23612978

  4. Diverse pore loops of the AAA+ ClpX machine mediate unassisted and adaptor-dependent recognition of ssrA-tagged substrates.

    PubMed

    Martin, Andreas; Baker, Tania A; Sauer, Robert T

    2008-02-29

    ClpX, an archetypal proteolytic AAA+ unfoldase, must engage the ssrA tags of appropriate substrates prior to ATP-dependent unfolding and translocation of the denatured polypeptide into ClpP for degradation. Here, specificity-transplant and disulfide-crosslinking experiments reveal that the ssrA tag interacts with different loops that form the top, middle, and lower portions of the central channel of the ClpX hexamer. Our results support a two-step binding mechanism, in which the top loop serves as a specificity filter and the remaining loops form a binding site for the peptide tag relatively deep within the pore. Crosslinking experiments suggest a staggered arrangement of pore loops in the hexamer and nucleotide-dependent changes in pore-loop conformations. This mechanism of initial tag binding would allow ATP-dependent conformational changes in one or more pore loops to drive peptide translocation, force unfolding, and mediate threading of the denatured protein through the ClpX pore.

  5. Nuclear import of the thyroid hormone receptor α1 is mediated by importin 7, importin β1, and adaptor importin α1.

    PubMed

    Roggero, Vincent R; Zhang, Jibo; Parente, Laura E; Doshi, Yazdi; Dziedzic, Rose C; McGregor, Emma L; Varjabedian, Arev D; Schad, Sara E; Bondzi, Cornelius; Allison, Lizabeth A

    2016-01-01

    The thyroid hormone receptor α1 (TRα1) is a nuclear receptor for thyroid hormone that shuttles rapidly between the nucleus and cytoplasm. Our prior studies showed that nuclear import of TRα1 is directed by two nuclear localization signals, one in the N-terminal A/B domain and the other in the hinge domain. Here, we showed using in vitro nuclear import assays that TRα1 nuclear localization is temperature and energy-dependent and can be reconstituted by the addition of cytosol. In HeLa cells expressing green fluorescent protein (GFP)-tagged TRα1, knockdown of importin 7, importin β1 and importin α1 by RNA interference, or treatment with an importin β1-specific inhibitor, significantly reduced nuclear localization of TRα1, while knockdown of other importins had no effect. Coimmunoprecipitation assays confirmed that TRα1 interacts with importin 7, as well as importin β1 and the adapter importin α1, suggesting that TRα1 trafficking into the nucleus is mediated by two distinct pathways.

  6. The proteolysis adaptor, NblA, is essential for degradation of the core pigment of the cyanobacterial light-harvesting complex.

    PubMed

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

    2015-09-01

    The cyanobacterial light-harvesting complex, the phycobilisome, is degraded under nutrient limitation, allowing the cell to adjust light absorbance to its metabolic capacity. This large light-harvesting antenna comprises a core complex of the pigment allophycocyanin, and rod-shaped pigment assemblies emanating from the core. NblA, a low-molecular-weight protein, is essential for degradation of the phycobilisome. NblA mutants exhibit high absorbance of rod pigments under conditions that generally elicit phycobilisome degradation, implicating NblA in degradation of these pigments. However, the vast abundance of rod pigments and the substantial overlap between the absorbance spectra of rod and core pigments has made it difficult to directly associate NblA with proteolysis of the phycobilisome core. Furthermore, lack of allophycocyanin degradation in an NblA mutant may reflect a requirement for rod degradation preceding core degradation, and does not prove direct involvement of NblA in proteolysis of the core pigment. Therefore, in this study, we used a mutant lacking phycocyanin, the rod pigment of Synechococcus elongatusPCC7942, to examine whether NblA is required for allophycocyanin degradation. We demonstrate that NblA is essential for degradation of the core complex of the phycobilisome. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for the interaction of NblA with allophycocyanin, and indicated that NblA interacts with allophycocyanin complexes that are associated with the photosynthetic membranes. Based on these data, as well as previous observations indicating interaction of NblA with phycobilisomes attached to the photosynthetic membranes, we suggest a model for sequential phycobilisome disassembly by NblA. PMID:26173720

  7. Human Glycolipid Transfer Protein (GLTP) Expression Modulates Cell Shape

    PubMed Central

    Gao, Yongguang; Chung, Taeowan; Zou, Xianqiong; Pike, Helen M.; Brown, Rhoderick E.

    2011-01-01

    Glycolipid transfer protein (GLTP) accelerates glycosphingolipid (GSL) intermembrane transfer via a unique lipid transfer/binding fold (GLTP-fold) that defines the GLTP superfamily and is the prototype for GLTP-like domains in larger proteins, i.e. phosphoinositol 4-phosphate adaptor protein-2 (FAPP2). Although GLTP-folds are known to play roles in the nonvesicular intracellular trafficking of glycolipids, their ability to alter cell phenotype remains unexplored. In the present study, overexpression of human glycolipid transfer protein (GLTP) was found to dramatically alter cell phenotype, with cells becoming round between 24 and 48 h after transfection. By 48 h post transfection, ∼70% conversion to the markedly round shape was evident in HeLa and HEK-293 cells, but not in A549 cells. In contrast, overexpression of W96A-GLTP, a liganding-site point mutant with abrogated ability to transfer glycolipid, did not alter cell shape. The round adherent cells exhibited diminished motility in wound healing assays and an inability to endocytose cholera toxin but remained viable and showed little increase in apoptosis as assessed by poly(ADP-ribose) polymerase cleavage. A round cell phenotype also was induced by overexpression of FAPP2, which binds/transfers glycolipid via its C-terminal GLTP-like fold, but not by a plant GLTP ortholog (ACD11), which is incapable of glycolipid binding/transfer. Screening for human protein partners of GLTP by yeast two hybrid screening and by immuno-pulldown analyses revealed regulation of the GLTP-induced cell rounding response by interaction with δ-catenin. Remarkably, while δ-catenin overexpression alone induced dendritic outgrowths, coexpression of GLTP along with δ-catenin accelerated transition to the rounded phenotype. The findings represent the first known phenotypic changes triggered by GLTP overexpression and regulated by direct interaction with a p120-catenin protein family member. PMID:21625605

  8. Roles of intramolecular and intermolecular interactions in functional regulation of the Hsp70 J-protein co-chaperone Sis1.

    PubMed

    Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy; Ciesielski, Szymon J; Baranowski, Maciej; Zhou, Min; Joachimiak, Andrzej; Craig, Elizabeth A

    2015-04-10

    Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways, Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activity with Hsp70∆EEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interactions between the J-domain and glycine-rich region control co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. However, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD binding adaptor proteins. These interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively. PMID:25687964

  9. Roles of intramolecular and intermolecular interactions in functional regulation of the Hsp70 J-protein co-chaperone sis1

    SciTech Connect

    Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy; Ciesielski, Szymon J.; Baranowski, Maciej; Zhou, Min; Joachimiak, Andrzej; Craig, Elizabeth A.

    2015-02-13

    Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activity with Hsp70ΔEEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interaction(s) between the J-domain and glycine-rich region controls co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. Yet, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD-binding adaptor